INTRA - BONE HETEROGENEITY OF RECOVERABLE DNA FROM FRESH, BURIED, AND EXPOSED FEMORA By Timothy Antinick A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Forensic Scie nce Master of Science 2015 ABSTRACT INTRA - BONE HETEROGENEITY OF RECOVERABLE DNA FROM FRESH, BURIED, AND EXPOSED FEMORA By Timothy Antinick DNA recovered from skeletal m aterial is often used to establish positive identification of severely decomposed or fragmented remains. DNA is preferentially sought from long bones like the femur, usually from cortical bone of the midshaft diaphysis. Although this strategy is often successful, the femur has extensive differences in morphology , tissue co mposition, and points of articulation that may differ in DNA content. In the research presented, intra - femoral heterogeneity was assessed in a proximal/ distal manner to determine variation in DNA quantity and quality. Mitochondrial and nuclear DNA yields were compared across nine regions of the diaphysis , and the distal and proximal epiphyses , using fresh bovine and porcine femora and two tissue digestion protocols ( non - demineralization and demineral ization ). In addition , bovine femora were subjected to bur ial and surface exposure over a six month interval to assess how DNA heterogeneity was affected by environments where forensically relevant remains are often discovered. T he epiphyses had significantly more DNA than the metaphyses, which had more than the diaphy sis, DNA quality was consistent among all regions tested, and m i tochondrial and nuclear D NA had similar regional variation. Bone d emineralization resulted in more DNA recovered at the m id - diaphysis, while the non - demineralization protocol did th e same for nuclear DNA at the epiphyses . Environmental exposure affected DNA quantity and quality, and burial influenced inter - regional DNA yields over time. These findings indicate that intra - bone DNA heterogeneity can be as important as inter - bone DNA heterogeneity, and should be considered when cho osing a sampling location for DNA isolation. Copyright by TIMOTHY ANTINICK 2015 iv ACKNOWLEDGMENTS I would like to thank my advisor, Dr. David Foran, for his hard work, guid ance, and the opportunities he afforded me throughout my time here. In addition, much appreciation goes out to my committee members, Dr. Joseph Hefner and Dr. Jennifer Cobbina, for their thoughtful comments and advice . This research was funded by the Mic higan State University Graduate School and Michigan State University Forensic Science program . S keletal material was donated by the Michigan State University Meats Laboratory ; without their generosity, this project would not have been possible. Thanks to Jingyi Zhang from the Center for Statistical Training and Consulting for advice and assistance with the statistics used in this project. I would like to thank Dr. Todd Fenton and Emily Streetman for their help and advice on mac erating skeletal material, as well as Larissa Zanetti and Dom inic Bazzano for assisting with PCR . Thank you to the current students and graduates of the Michigan State University Forensic Biology Program Ashley Mottar, Ashley Doran, Mac Hopkins, Lisa Hebda, Brianna Bermudez, and Em ily Heinz for their suggestions with this research. Special thanks go to Becca Ray, Ellen Jesmok, Alyssa Badgley, and Kait Germain for assisting me throughout this project, as well as for their friendship and support. Finally, thank you to my kin and kit h for all their love, support, and encouragement. v TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ........................ vii LIST OF FIGURES ................................ ................................ ................................ ................... xxxi KEY TO ABBREVIATIONS ................................ ................................ ................................ . xxxvii INTRODUCTION ................................ ................................ ................................ .......................... 1 Environmental Influences on DNA Degra dation from Skeletal Material ................................ ... 2 The Composition of Bone ................................ ................................ ................................ ........... 4 Femoral Bone Anatomy and Forensic Analyses ................................ ................................ ......... 6 Recovering DNA from Skeletal Material ................................ ................................ .................... 8 DNA Yield by Osseous Tissue Type ................................ ................................ ....................... 8 DNA Isolation Metho ds: Preparing Bone for Digestion and Extraction ............................... 10 Tissue Digestion Methods Used for DNA Isolation ................................ .............................. 11 Inter - Bone Heterogeneity in Recoverable DNA Quantity and Quality ................................ . 13 Intra - Bone Heterogeneity in DNA Quantity and Quality ................................ ...................... 17 Research Goals ................................ ................................ ................................ .......................... 18 MATERIALS AND METHODS ................................ ................................ ................................ .. 19 Preparation of Bovine and Porcine Femora and Tarsals ................................ ........................... 19 B ovine/Porcine Intra - bone Variation and Tarsal Comparison Experiments ......................... 19 Surface/Burial Comparison Experiment of Bovine Femora and Tarsals .............................. 22 Drilling Skeletal Material ................................ ................................ ................................ .......... 24 DNA Isolation ................................ ................................ ................................ ........................... 25 Organic Extraction Method One ................................ ................................ ............................ 26 Organic Extraction Method Two ................................ ................................ ........................... 27 Quantitative PCR Assay for Bovine and Porcine DNA Extracts ................................ .............. 27 Qualitative PCR Assay for Bovine and Porcine DNA Extracts ................................ ................ 30 Ancillary Experiments ................................ ................................ ................................ ............... 33 Changes in the Recoverable Total DNA of Buried Bovine B one Segments over a One Month Time Period ................................ ................................ ................................ ................ 33 Changes in the Recoverable Total DNA of Non - Buried Bovine Bone Segments over a One Month Time Period ................................ ................................ ................................ ................ 35 Organic versus : Total DNA Yield Comparisons Over One Week ................. 36 Mass Difference between Wet and Dry Bone ................................ ................................ ....... 37 Effect of Proteinase K Concentration on Total DNA Yields ................................ ................ 38 Comparison of Total DNA Yields from Bovine Bones Macerated by MSU Forensic Anthropologist versus MSU Forensic Biologist ................................ ................................ .... 38 Statistical Analysis of Bovine/Porcine Intra - bone Variation and Tarsal Comparison Experiments ................................ ................................ ................................ ............................... 39 RESULTS ................................ ................................ ................................ ................................ ..... 40 Qualitative Observations from Processing Porcine and Bovine Femora and Tarsals ............... 40 vi Inter - Bone and Intra - Bone Variation of Recoverable Total DNA in Fresh Por cine and Bovine Femora and Tarsals ................................ ................................ ................................ ................... 42 Normality Testing of Porcine and Bovine Bones ................................ ................................ .. 42 Quantification of Total DNA from Fresh Porc ine Femora and Tarsals ................................ 43 Quality of Total DNA Recovered from Fresh Porcine Femora and Tarsals ......................... 65 Quantification of Total DNA from Fresh Bovine Femora and Tarsals ................................ . 66 Quality of Total DNA Recovered from Fresh Bovine Femora and Tarsals .......................... 88 Surface/Burial Comparis on Experiment of Bovine Femora and Tarsals ................................ .. 90 Total DNA Quantification and Quality for Surface Exposed Bovine Bones ........................ 90 Total DNA Q uantification and Quality for Buried Bovine Bones ................................ ........ 94 Ancillary Experiments ................................ ................................ ................................ ............. 102 Changes in the Recoverable Total DNA of Buried Bovi ne Bone Segments over a One Month Time Period ................................ ................................ ................................ .............. 102 Changes in the Recoverable Total DNA of Non - Buried Bovine Bone Segments over a One Month Period ................................ ................................ ................................ ....................... 105 Organic versus SoilMaster ............... 108 Mass Difference between Wet and Dry Bone ................................ ................................ ..... 109 Effect of Proteinase K Concentration on Total DNA Yields ................................ .............. 110 Comparison of Total DNA Yields from Bovine Bones Macerated by MSU Forensic Anthropologist versus MSU Forensic Biologist ................................ ................................ .. 111 DISCUSSION ................................ ................................ ................................ ............................. 113 CONCLUSION ................................ ................................ ................................ ........................... 133 APPENDICES ................................ ................................ ................................ ............................ 134 APPENDIX A: NORMALITY TEST AND PAIRWISE COMPARISON TABLES FOR PORCINE AND BOVINE QUANTIFICATION DATA ................................ ....................... 135 APPENDIX B: MITOCHONDRIAL AND NUCLEAR DNA Q UANTIFICATION DATA AND INDIVIDUAL PCR AMPLIFICATION RESULTS FROM FRESH PORCINE FEMORA AND TARSALS ................................ ................................ ................................ .... 147 APPENDIX C: MITOCHONDRIAL AND NUCLEAR DNA QUANTIFICATION DATA AND INDIVIDUAL PCR AMPLIFICAT ION RESULTS FROM FRESH BOVINE FEMORA AND TARSALS ................................ ................................ ................................ ..................... 183 APPENDIX D: MITOCHONDRIAL AND NUCLEAR DNA QUANTIFICATION DATA AND INDIVIDUAL PCR AMPLIFICATION RESULTS FROM SURFACE EXPOSED AND BURIED BOVIN E FEMORA AND TARSALS ................................ .......................... 219 APPENDIX E: ANCILLARY EXPERIMENT DATA ................................ .......................... 251 REFERENCES ................................ ................................ ................................ ........................... 263 vii LIST OF TABLES Table 1 : Summary of Results from Published Studies Evaluating Inter - Bone Variation of Successfully Obtaining Analyz able DNA Element sampled is identified in the first column. Subsequent columns represent studies in which success rates were assessed among elements. The first number denotes the percent success and the parenthetical number signifies the sample size for that element. Success was et al. (2007) defined success as obtaining genetic data from 12 or more loci using a Promega PowerPlex 16 STR kit. Mundorff et al. ( Data from Mundorff et al. (2009) used in this table only incorporate the Complete Elements Database, a subset of the World Trade Center - Human Remains Database. NT = Not Tested CILHI = Cen tral Iden tification Laboratory in Hawaii Table 2: Sidedness, Diaphysis Length, and Midshaft Circumference of Bovine and Porcine Femora Femora were labeled by species and the sequential order processed. Total diaphysis length was measured from the proximal to distal epiphyseal lines, with the range representing differences due to irregular borders. Circumference was measured at the femoral midshaft. *, **, or *** represents femora originating from a single individual Table 3: Demarcated Regions of the Femur and Tarsal s Table 4: Regions of Bovine Femora and Tarsals Tested Table 5: Dates Buried/Surface Femora and Tarsals Were Tested Table 6: Organic Extraction Method Used p er Experiment Table 7: Primer and Probe Sequences for qPCR Table 8: Real - time PCR Reaction Recipe and Concentrations Table 9: qPCR Thermal Cycler Parameters Table 10: Primer Seq uences for Qualitative PCR Assay Table 11: PCR Thermal Cycler Parameters Annealing temperature varied among primer sets. Bovine ATPase 8 and MC1R primers used 58 ° C, porcine ATPase used 60 ° C, and IGF - 1 used 56 ° C viii Table 12: Burial Dates of Bovine Femora Segments Segments identified by burial time (0D, 2D, 4D, 1W, 11D, 2W, 3W, 4W) and by replicate (1 4), where D = day(s), W = week(s). Segments denoted with (A) were retrieved, drilled, and stored at - 20 ° C. Segments deno ted with (B) were cyclically retrieved, drilled, and reburied Table 13: Exposure Dates of Bovine Femora Segments Segments identified by time exposed (0D, 2D, 4D, 1W, 10D, 2W, 3W, 4W) and by replicate (1 4), where D = day(s), W = week(s) Table 14: Environmental Conditions Influence on Bone Mass Change Table 15: Time Points Tested for Bone Mass Change nt and correspond Table 16: mtDNA Yields from Fresh Porcine Bones Digested in Tissue Lysis Buffer Regions and concomitant locations correspond to Table 3. P - (01 08) are biological replicates. MtDNA quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviation (MAD) for variation in data. Regions 1 12 reside on the femur; the calcaneus and talus (Regi ons 13 and 14) were tested as whole elements. NT = Not Tested Table 17: mtDNA Yields from Fresh Porcine Bones Digested in Demineralization Buffer Regions and concomitant locations correspond to Table 3. P - (01 08) are biological replicates. MtDNA quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviation (MAD) for variation in data. Regions 1 12 reside on the femur; the calcaneus and talus (Regions 13 and 14) were tested as whole elements Table 18: Combined mtDNA Yields from Fresh Porcine Bones Digested in Tissue Lysis and Demineraliza tion Buffers Combined regions listed in first column and correspond to Table 3. Regions combined from P - (01 08). MtDNA quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviation (MA D) for variation in data. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. TL = tissue lysis buffer DM = demineralization buffer Table 19: Nucle ar DNA Yields from Fresh Porcine Bones Digested in Tissue Lysis Buffer Regions and concomitant locations correspond to Table 3. P - (01 08) are biological replicates. Nuclear DNA quantities are rep orted in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviation (MAD) for variation in data. ix Regions 1 12 reside on the femur; the calcaneus and talus (Regions 13 and 14) were tested as whole elements. N T = Not Tested Table 20: Nuclear DNA Yields from Fresh Porcine Bones Digested in Demineralization Buffer Regions and concomitant locations correspond to Table 3. P - (01 08) are biological replicates. Nuclear DNA quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviation (MAD) for variation in data. Regions 1 12 reside on the femur; the calca neus and talus (Regions 13 and 14) were tested as whole elements Table 21: Combined Nuclear DNA Yields from Fresh Porcine Bones Digested in Tissue Lysis and Demineralization Buffers Combined regions listed in first colu mn and correspond to Table 3. Regions are combined from P - (01 08). DNA quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviation (MAD) for variation in data. Regions 1 6 represen t the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. TL = tissue lysis buffer DM = demineralization buffer Table 22: mtDNA Yields from Fresh Bovine Bones Digested in Tiss ue Lysis Buffer Regions and concomitant locations correspond to Table 3. C - (01 08) are biological replicates. MtDNA quantities are reported in ng per mg bone powder. Median was used to cha racterize central tendency and median absolute deviation (MAD) for variation in data. Regions 1 12 reside on the femur; the calcaneus and talus (Regions 13 and 14) were tested as whole elements Table 23: mtDNA Yield s from Fresh Bovine Bones Digested in Demineralization Buffer Regions and concomitant locations correspond to Table 3. C - (01 08) are biological replicates. MtDNA quantities are reported in ng pe r mg bone powder. Median was used to characterize central tendency and median absolute deviation (MAD) for variation in data. Regions 1 12 reside on the femur; the calcaneus and talus (Regions 13 and 14) were tested as whole elements Table 24: Combined mtDNA Yields from Fresh Bovine Bones Digested in Tissue Lysis and Demineralization Buffers Combined regions listed in first column and correspond to Table 3. Regions combined from C - (01 08). MtDNA quantities are r eported in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviation (MAD) for variation in data. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. TL = tissue lysis buffer DM = demineralization buffer x Table 25: Nuclear DNA Yields from Fresh Bovine Bones Digested in Tissue Lysis Buffer Regions and concomitant locations correspond to Table 3. C - (01 08) are biological replicates. Nuclear DNA quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviation (MAD) for variation in data . Regions 1 12 reside on the femur; the calcaneus and talus (Regions 13 and 14) were tested as whole elements Table 26: Nuclear DNA Yields from Fresh Bovine Bones Digested in Demineralization Buffer Regions and concomitant locations correspond to Table 3. C - (01 08) are biological replicates. Nuclear DNA quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviation (MAD) for variation in data. Regions 1 12 reside on the femur; the calcaneus and talus (Regions 13 and 14) were tested as whole elements Table 27: Combined Nuclear DNA Yields from Fresh Bovi ne Bones Digested in Tissue Lysis and Demineralization Buffers Combined regions listed in first column and correspond to Table 3. Regions combined from C - (01 08). DNA quantities are reported in ng per mg bone powder. Median was used to characterize ce ntral tendency and median absolute deviation (MAD) for variation in data. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. TL = tissue lysis buffer DM = demin eralization buffer Table 28: PCR Amplification Results for Mitochondrial DNA Recovered from Fresh Bovine Bones Regions and concomitant locations coincide with Table 3. The largest amplicon size successfully produced per region (n = 16) are listed under their respective columns. TL = tissue lysis buffer (n = 8) DM = demineralization buffer (n = 8) Table 29: PCR Amplification Results for Nuclear DNA Recovered from Fresh Bovine Bones Regions and concomitant locations coincide with Table 3. The largest amplicon size successfully produced per region (n = 16) are listed under their respective columns. TL = tissue lysis buffer (n = 8) DM = demineralization buffer (n = 8) Table 30: mtDNA Quantificat ion from Surface Exposed Bovine Bones Region bone was drilled from listed in first column. Mean mtDNA quantities (n = 2), day zero (n = 4), are reported as ng per mg bone powder. Cells highlighted in yellow had at least one replicate with inhibition xi Table 31: Nuclear DNA Quantification from Surface Exposed Bovine Bones Region bone was drilled from listed in first column. Mean nuclear DNA quantities (n = 2), day zero (n = 4), are reported as ng per mg bone powder. Cells hi ghlighted in yellow had at least one replicate with inhibition Table 32: mtDNA Quantification from Buried Bovine Bones Region bone was drilled from listed in first column. Mean mtDNA quantities (n = 2), day zero (n = 4), are reported as ng per mg bone powder. Cells highlighted in yellow had at least one replicate with inhibition Table 33: Nuclear DNA Quantification from Buried Bovine Bones Region bone was drilled from listed in first colum n. Mean nuclear DNA quantities (n = 2), day zero (n = 4), are reported as ng per mg bone powder. Cells highlighted in yellow had at least one replicate with inhibition Table 34: mtDNA Yields of Buried Bovine Femoral Di aphysis hat were retrieved, tested, and stored at - 20 ° MtDNA quantities are normalized in ng per mg of bone powder. Mean mtDNA quantities for each length of burial are bolded. NT = Not Tested Table 35: Nuclear DNA Yields of Buried Bovine Femoral Diaphysis at - 20 ° Nuclear DNA quantities are in ng per mg of bone powder. Mean DNA quantities for each length of burial are bolded. NT = Not Tested Table 36: mtDNA Yields of Non - Buried Bovine Femoral Diaphysis Bone segment identifiers are listed in the first and third colu biological replicates of femoral diaphysis tested at each time point. MtDNA quantities are in ng per mg of bone powder. Mean mtDNA quantit ies for each length of exposure are bolded Table 37: Nuclear DNA Yields of Non - Buried Bovine Femoral Diaphysis biological replicates of femoral diaphysis tested at each time point. Nuclear DNA quantities are in ng per mg of bone powder. Mean DNA quantities for each length of exposure are bol ded xii Table 38: Total DNA Yield Comparison between Organic and S oilMaster TM Extractions Normalized values for nuclear and mitochondrial DNA are report ed as ng per mg of bone .....................109 Table 39: Mass Differences of Dehydrated Bone Powder and Bone Segment Bone powder subjected to each treatment was weighed over a one week period. Bone segment ange in mass (in bone powder/segment. N/A = Not Applicable Proteina se K was added to digestion buffer by percent total reaction volume. Trials were qPCR technical replicates. Mean DNA yields for each volume of proteinase K are bolded Table 41: Total DNA Quantification Comparisons between Bovine Bones Macerate d by MSU Forensic Anthropologist and the MSU Forensic Biologist Region tested is in the first column. DNA/mtDNA quantification is reported in nanograms per mg bone. FA = DNA derived from an element macerated by MSU Forensic Anthropology FB = DNA was d erived from an element macerated by MSU Forensic Biology Table A 1: p - Nuclear DNA Variation Region number is listed in the first column, and correspond to Table 3. Bolded values represent - Darling test, SW = Shapiro - Wilk test Table A2 : p - Nuclea r DNA Variation (with C - 01 and C - 02) Region number is listed in the first column, and correspond to Table 3. Bolded values represent - Darling test, SW = Shapiro - Wilk test Table A3 : p - Nuclear DNA Variation (without C - 01 and C - 02) Region number is listed in the first column, and correspond to Table 3. Bolded values represent re - Darling test, SW = Shapiro - Wilk test Table A4 : p - Values from Pairwise Comparisons of Median mtDNA Yields Derived from Porcine Bones Digested in Ti ssue Lysis Buffer Region identifiers are listed in the first column and row, and correspond to identifiers in Table 3. - . Bolded values indicate a significant difference xiii between regions compared. P - values in red indicate disagreement in significance between tissue lysis and demineralization buffer Table A5 : p - Values from Pairwise Comparisons o f Median mtDNA Yields Derived from Porcine Bones Digested in Demineralization Buffer Region identifiers are listed in the first column and row, and correspond to identifiers in Table 3. Pairwise comparisons were generated using XLSTAT 2014.2.01 using Dunn - between regions compared. P - values in red indicate disagreement in significance between tissue lysis and demineralization buffer Table A6 : p - Values from Multiple and Individual Pairwise Comparisons of Pooled Median mtDNA and DNA Yields Derived from Fresh Porcine and Bovine Bones Digested in Tissue Lysis and Demineralization Buffer Pooled region identifiers are l isted in the first column, and correspond to identifiers in Table 3. procedure (two - tailed) wi indicate a significant difference between regions compared Table A7 : p - Values from Pairwise Comparisons of Median Nuclear DNA Yields Derived from Porcine Bones Digested i n Tissue Lysis Buffer Region identifiers are listed in the first column and row, and correspond to identifiers in Table 3. - 0.05. Bolded values indicate a significant difference between regions compared. P - values in green indicate disagreement in significance between tissue lysis and demineralization buffer Table A8 : p - Values from Pairwise Comparis ons of Median Nuclear DNA Yields Derived from Porcine Bones Digested in Demineralization Buffer Region identifiers are listed in the first column and row, and correspond to identifiers in Table 3. Pairwise comparisons were generated using XLSTAT 2014.2.01 - between regions compared. P - values in green indicate disagreement in significance between tissue lysis and demineralization bu ffer Table A9 : p - Values from Pairwise Comparisons of Median mtDNA Yields Derived from Bovine Bones Digested in Tissue Lysis Buffer Region identifiers are listed in the first column and row, and correspond to identifiers in Table 3. - between regions compared. P - values in blue indicate disagreemen t in significance between tissue lysis and demineralization buffer xiv Table A10 : p - Values from Pairwise Comparisons of Median mtDNA Yields Derived from Bovine Bones Digested in Demineralization Buffer Region identifiers are listed in the first column and row, and correspond to identifiers in Table 3. - ce between regions compared. P - values in blue indicate disagreement in significance between tissue lysis and demineralization buffer Table A11 : p - Values from Pairwise Comparisons of Median Nuclear DNA Yields Derived from Bovine Bones Digested in Tissue Lysis Buffer Region identifiers are listed in the first column and row, and correspond to identifiers in Table 3. - tailed). Significance was d between regions compared. P - values in purple indicate disagreement in significance between tissue lysis and demineralization buffer Table A12 : p - Values fro m Pairwise Comparisons of Median Nuclear DNA Yields Derived from Bovine Bones Digested in Demineralization Buffer Region identifiers are listed in the first column and row, and correspond to identifiers in Table 3. Pairwise comparisons were generated usin - between regions compared. P - values in purple indicate disagreement in significance between tissue lysis and demineralization buffer Table B 1: mtDNA Quantification of P - 01 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to norma lize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table B 2: mtDNA Quantification of P - 01 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract vol ume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per Table B3 : mtDNA Quantification of P - 02 Extracts Digested in Tissue Lysis Buf fer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table B4 : mtDN A Quantification of P - 02 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per m g of bone powder xv Table B5 : mtDNA Quantification of P - 03 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to nor malize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. ND = No Data Table B6 : mtDNA Quantification of P - 03 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table B7 : mtDNA Quantification of P - 04 Extracts Digested in Tiss ue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Ta ble B8 : mtDNA Quantification of P - 04 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table B9 : mtDNA Quantification of P - 05 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in or der to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table B10 : mtDNA Quantification of P - 05 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table B1 1: mtDNA Quantification of P - 06 Extracts Digested in Ti ssue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table B1 2: mtDNA Quantification of P - 06 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are repor ted as ng per mg of bone powder Table B13 : mtDNA Quantification of P - 07 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered i n order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder xvi Table B14 : mtDNA Quantification of P - 07 Extracts Digested in Demineralization Buffer Region and location correspond with Tabl e 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table B15 : mtDNA Quantification of P - 08 Extracts Digested i n Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder .1 61 Table B16 : mtDNA Quantification of P - 08 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are r eported as ng per mg of bone powder Table B17 : Nuclear DNA Quantification of P - 01 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was co nsidered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder Table B18 : Nuclear DNA Quantification of P - 01 Extracts Digested in Demineralization Buffer Region and location cor respond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder Table B19 : Nuclear DNA Quantification of P - 02 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder Table B20 : Nuclear DNA Quantification of P - 02 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yie lds. Normalized values are reported as ng of per Table B2 1: Nuclear DNA Quantification of P - 03 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder. ND = No Data xvii Table B2 2: Nuclear DNA Quantification of P - 03 Extracts Digested in Demineral ization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder Ta ble B23 : Nuclear DNA Quantification of P - 04 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reporte d as ng of per mg of bone powder Table B24 : Nuclear DNA Quantification of P - 04 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was c onsidered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder Table B25 : Nuclear DNA Quantification of P - 05 Extracts Digested in Tissue Lysis Buffer Region and location corres pond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder Table B26 : Nuclear DNA Quantification of P - 0 5 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder Table B27 : Nuclear DNA Quantification of P - 06 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields . Normalized values are reported as ng of per mg of bone powder Table B28 : Nuclear DNA Quantification of P - 06 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder Table B29 : Nuclear DNA Quantification of P - 07 Extracts Digested in Tissue Lysis Bu ffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder Table B30 : N uclear DNA Quantification of P - 07 Extracts Digested in Demineralization Buffer xviii Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as n g of per mg of bone powder Table B 31: Nuclear DNA Quantification of P - 08 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder Table B 32: Nuclear DNA Quantification of P - 08 Extracts Digested in Demineralization Buffer Region and location correspond w ith Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder Table B 33: Porcine Mitochondrial DNA PCR Amplifica tion Chart for Quality Assay (Tissue Lysis Buffer) Region and location correspond to Table 3. Values (1 4) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 181 bp amplicon, 2 = 414 bp amplicon, 3 = 604 b p amplicon, 4 = 1017 bp amplicon. NT = Not Tested NA = No Ampli Table B 34: Porcine Mitochondrial DNA PCR Amplification Chart for Quality Assay (Demineralization Buffer) Region and location correspond to Table 3. V alues (1 4) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 181 bp amplicon, 2 = 414 bp amplicon, 3 = 604 bp amplicon, 4 = 1017 bp amplicon. Table B 35: Porcine Nuclear DNA PCR Amplification Chart for Quality Assay (Tissue Lysis Buffer) Region and location correspond to Table 3. Values (1 3) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 257 bp am plicon, 2 = 457 b p amplicon, 3 = 642 bp amplicon. NT = Not Tested NA = No Ampl Table B 36: Porcine Nuclear DNA PCR Amplification Chart for Quality Assay (Demineralization Buffer) Region and location correspond to Ta ble 3. Values (1 3) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 257 bp amplicon, 2 = 457 b p amplicon, 3 = 642 bp amplicon. NA = No Amplification xix Table C1: mtDNA Qua ntification of C - 01 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table C2: mtDNA Quantification of C - 01 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table C3 : mtDNA Quantification of C - 02 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and m illigrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table C4 : mtDNA Quantification of C - 02 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table C5 : mtDNA Qua ntification of C - 03 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table C6 : mtDNA Quantification of C - 03 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table C7 : mtDNA Quantification of C - 04 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and m illigrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table C8 : mtDNA Quantification of C - 04 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table C9 : mtDNA Qua ntification of C - 05 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder xx Table C10 : mtDNA Quantification of C - 05 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table C1 1: mtDNA Quantification of C - 06 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table C1 2: mtDNA Quantification of C - 06 Extracts Digested in Demineralization Buffe r Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table C13 : mtDNA Quantification of C - 07 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table C14 : mtDNA Quantification of C - 07 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to norma lize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table C15 : mtDNA Quantification of C - 08 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table C16 : mtDNA Quantification of C - 08 Extracts Digested in Demineralization B uffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder Table C17 : N uclear DNA Quantification of C - 01 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng pe r mg of bone powder Table C18 : Nuclear DNA Quantification of C - 01 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder xxi Table C19 : Nuclear DNA Quantification of C - 02 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder Table C20 : Nuclear DNA Quantification of C - 02 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder Table C2 1: Nuclear DNA Quantification of C - 03 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values a re reported as ng per mg of bone powder Table C 2 2: Nuclear DNA Quantification of C - 03 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powde r was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder Table C23 : Nuclear DNA Quantification of C - 04 Extracts Digested in Tissue Lysis Buffer Region and location co rrespond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder Table C24 : Nuclear DNA Quantification of C - 04 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder Table C25 : Nuclear DNA Quantification of C - 05 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder Table C26 : Nuclear DNA Quantification of C - 05 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and m illigrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder Table C27 : Nuclear DNA Quantification of C - 06 Extracts Digested in Tissue Lysis Buffer xxii Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder Table C28 : Nuclear D NA Quantification of C - 06 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder Table C29 : Nuclear DNA Quantification of C - 07 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder Table C30 : Nuclear DNA Quantification of C - 07 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder Table C31: Nuclear DNA Quantification of C - 08 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder Table C32: Nuclear DNA Quantification of C - 08 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are r eported as ng per mg of bone powder Table C33: Bovine Mitochondrial DNA PCR Amplification Chart for Quality Assay (Tissue Lysis Buffer) Region and location correspond to Table 3. Values (1 4) correspond to the length o f PCR amplicon successfully generated for each region per replicate: 1 = 201 bp amplicon, 2 = 390 bp amplicon, 3 = 607 b p amplicon, 4 = 994 bp amplicon. Table C34: Bovine Mitochondrial DNA PCR Amplifi cation Chart for Quality Assay (Demineralization Buffer) Region and location correspond to Table 3. Values (1 4) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 201 bp amplicon, 2 = 390 bp amplicon, 3 = 607 b p amplicon, 4 = 994 bp amplicon. xxiii Table C35: Bovine Nuclear DNA PCR Amplification Chart for Quality Assay (Tissue Lysis Buffer) Region and location correspond to Table 3. Values (1 4) correspo nd to the length of PCR amplicon successfully generated for each region per replicate: 1 = 200 bp amplicon, 2 = 410 bp amplicon, 3 = 599 bp amplicon, 4 = 989 bp amplicon. Table C36: Bovine Nuclear DNA PCR Amplification Chart for Quality Assay (Demineralization Buffer) Region and location correspond to Table 3. Values (1 4) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 200 bp amplicon, 2 = 410 bp a mplicon, 3 = 599 b p amplicon, 4 = 989 bp amplicon. Table D1: mtDNA Quantification of Day 0 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized va lues are reported as ng per mg of bone Table D2: mtDNA Quantification of Week 1 Extr acts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized va lues are reported as ng per mg of bone next to r egion denotes re Table D3: mtDNA Quantification of Week 2 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in o rder to normalize mtDNA yields. Normalized va lues are reported as ng per mg of bone Table D4: mtDNA Quantification of Week 4 Extracts from Surface Exposed Bovine Skeletal Mate rial Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized va lues are reported as ng per mg of bone quantification obtained using a 1:10 dilution of extract Table D5: mtDNA Quantification of Month 3 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized va lues are reported as ng per mg of bone xxiv Table D6: mtDNA Quantification of Month 6 Extr acts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone quantification obtained using a 1:10 dilution of extract Table D7 : mtDNA Normalized Quantification of Surface Exposed Bovine Skeletal Material Region bone powde r was collected from in first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng during the course of this experiment. Cells highlighted in yellow represent extracts that had PCR represents mtDNA quantification multiplied by dilution fa ctor Table D8 : Bovine Mitochondrial DNA PCR Amplification Chart for Quality Assay (Surface Exposed Skeletal Material) 4) correspond to the length of PCR a mplicon successfully generated for each region per replicate: 1 = 201 bp amplicon, 2 = 390 bp amplicon, 3 = 607 b p amplicon, 4 = 994 bp amplicon. Table D9: Nuclear DNA Normalized Quantification of Sur face Exposed Bovine Skeletal Material Region bone powder was collected from in first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized v alues are reported as ng the course of this experiment. Cells highlighted in yellow represent extracts that had PCR i nhibition, listed value represents DNA quantification multiplied by dilution factor Table D10 : Nuclear DNA Quantification of Day 0 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and millig rams of bone powder was considered in order to normalize DNA yields. Normalized v alues are reported as ng per mg of bone powder. Table D11 : Nuclear DNA Quantification of Week 1 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng per mg of bone powder. xxv Table D12 : Nuclear DNA Quantification of Week 2 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was co nsidered in order to normalize DNA yields. Normalized values are reported as ng per mg of bone powder. egion denotes repli Table D13 : Nuclear DNA Quantification of Week 4 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized v alues are reported as ng per mg of bone powder. quantification obtained using a 1:5 or 1:10 dilution of extract respectively Table D14 : Nuclear DNA Quantification of Month 3 Extracts from Surface Exposed Bovine Skeletal Material Region l isted in the first column. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normali zed values are reported as ng per mg of bone powder. 32 Table D15 : Nuclear DNA Quantification of Month 6 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized v alues a re reported as ng per mg of bone powder. obtained using a 1:10 dilution of extract Table D16: Bovine Nuclear DNA PCR Amplificati on Chart for Quality Assay (Surface Exposed Skeletal Material) 4) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 200 bp amplicon, 2 = 410 bp amplicon, 3 = 599 bp amplicon, 4 = 989 bp amplicon, NA = No Amplification Table D17: mtDNA Quantification of Day 0 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone Table D18: mtDNA Quantific ation of Week 1 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. xxvi Table D19: mtDNA Quantification of Week 2 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered i n order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone quantification obtained us 23 7 Table D20: mtDNA Quantification of Week 4 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported a s ng per mg of bone represents quantification obtained using a 1:5 or 1:10 dilution of extract respectively Table D21: mtDNA Quantification of Month 3 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone on denotes replicate number Table D22: mtDNA Quantification of Month 6 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize m tDNA yields. Normalized va lues are reported as ng per mg of bone egion denotes re Table D23: mtDNA Normalized Quantification of Buried Bovine Skeletal Material Region bone powder was collecte d from in first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng the cou rse of this experiment. Cells highlighted in yellow represent extracts that had PCR inhibition, listed value represents DNA quantification multiplied by dil ution factor Table D24 : Bovine Mitochondrial DNA PCR Amplification Chart for Quality Assay (Buried Skeletal Material) 4) correspond to the length of PCR amplicon s uccessfully generated for each region per replicate: 1 = 201 bp amplicon, 2 = 390 bp amplicon, 3 = 607 bp amplicon, 4 = 994 bp am plicon, NA = No Table D25: Nuclear DNA Quantification of Day 0 Extracts from Buried Bovine Skeletal Material xxvii Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized v alues are reported as ng per mg of bone powder. denotes replicate number Table D26: Nuclear DNA Quantification of Week 1 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normali ze DNA yields. Normalized v alues are reported as ng per mg of bone powder. Table D27: Nuclear DNA Quantification of Week 2 Extracts from Buried Bovine Skeletal Material Region list ed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng per mg of bone powder. presents quantification obtained using a 1:10 dilution of extract Table D28: Nuclear DNA Quantification of Week 4 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng per mg of bone powder. egion denotes repli Table D29: Nuclear DNA Quantification of Month 3 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized v alues are reported as ng per mg of bone powder. denotes replicate number Table D30: Nuclear DNA Quantification of Month 6 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normal ize DNA yields. Normalized v alues are reported as ng per mg of bone powder. obtained u sing a 1:5 dilution of Table D31: Nu clear DNA Normalized Quantification of Buried Bovine Skeletal Material Region bone powder was collected from in first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized va lues are reported a s ng the course of this experiment. Cells highlighted in yellow represent extracts that had PCR r 10 respectively to alleviate inhibition, listed value represents DNA quantification multiplied by dilution factor xxviii Table D32 : Bovine Nuclear DNA PCR Amplification Chart for Quality Assay (Buried Skeletal Material) Region listed in first column. 4) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 200 bp amplicon, 2 = 410 bp amplicon, 3 = 599 bp amplicon, 4 = 989 bp amplicon, NA = No Amplification Table E1: mtDNA Quantification of Buried Femoral Diaphysis (Segment Type A) s that were retrieved, tested , and stored at - 20 ° C. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Nor malized values are reported as ng per mg of bone powder Table E2: mtDNA Quantification of Buried Femoral Diaphysis (Segment Type B) cyclically retrieved, tested , and reburied. Extract volume and milligrams of bone powder was considered in order to n ormalize mtDNA yields. Normalized values are reported as ng per mg of bone powder. ® column damage Table E3: Nuclear DNA Quantification of Buried Femoral Diaphysis (Segment Type A) Bone segmen s that were retrieved, t ested , and stored at - 20 ° C. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng per mg of bone powder Table E4: Nuclear DNA Quantification of Buried Femoral Diaphysi s (Segment Type B) segments that were cyclically retrieved, tested , and reburied. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng per mg of bone powder Table E5: mtDNA Quantificati on of Non - Buried Bovine Femoral Diaphysis replicate of femoral diaphysi s. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized values are reported as ng per mg of bone powder Table E6: Nuclear DNA Quantification of Non - Buried Bovine Femoral Diaphysis xxix replicate of femoral diaphysis. Extract volume and m illigrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng per mg of bone powder Table E7: Total Quantification of Non - Buried Bovine Femoral Diaphysis Extracted Ut ilizing Organic and SoilMaster . kit Extraction. Extract volume and milligrams of bone powd er was considered in order to normalize DNA yields. Normalized values are reported as ng per mg of bone powder Table E8: Mass of Drying Bone over Time in Four Containers plus Whole Bone Drying time in hours listed in first column. Mass o f bone powder in milligrams are listed in NT = Not Tested Table E9: mtDNA Quantification of Bovine Bones Digested with Varied Conce ntrations of Proteinase K Bone identifier listed in the first column. TL = tissue lysis buffer, DM = demineraliza tion buffer, RB = reagent blank. proteinase K added . technical replicate s per treatment. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized values are reported as ng per mg of bone powder Table E10: Nuclear DNA Quantificat ion of Bovine Bones Digested with Varied Concentrations of Proteinase K Bone identifier listed in the first column. TL = tissue lysis buffer, DM = demineraliza tion buffer, RB = reagent blank. of 20 mg/mL proteinase K added . technical replicate s per treatment. Extract volume and milligrams of bone powder was con sidered in order to normalize nuclear DNA yields. Normalized values are reported as ng per mg of bone powder Table E11: Mitochondrial DNA Quantification of Bovine Bones Macerated by MSU Forensic Biology/Anthropology Laboratories Region tested is in the first column. FA = element macerated by MSU Forensic Anthropology. FB = element macerated by MSU Forensic Biology. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized values are reported as ng per mg of bone powder Table E12: Nuclear DNA Quantification of Bo vine Bones Macerated by MSU Forensic Biology/Anthropology Laboratories Region tested is in the first column. FA = element macerated by MSU Forensic Anthropology. FB = element macerated by MSU Forensic Biology. Extract volume and milligrams of bone xxx powde r was considered in order to normalize nuclear DNA yields. Normalized values are reported as ng per mg of bone powder xxxi LIST OF FIGURES Figure 1 : Basic Anatomy of a Human Femur Sectional view of a human femur showing the diaphysis, metaphyses, and epiphyses, along with the distribution of cortical and trabecular bone. Adapted from Gray (1918). Illustration available at http://www.bartleby.com/107/59.html#i249 ..............................................7 Figure 2 : e Data b etween Non - Demineralization and Demineralization Protocols by Element Type - demineralization protocol and right skeleton using their demineralizat ion protocol. Skeletons are in anatomical position with color coded elements corresponding to their percent success of illustration Figure 3 : Published Illustrati ons of Inter - Bone Success of Obtaining Analyzable DNA Left diagram adapted from Edson et al. (2004), middle from Mundorff et al. (2013) representing data from Mundorff et al. (2009), and right from Leney (2006). D iagrams provide an illustrative Table 1 . Illustrations are color coded/shaded to correspond with percent success rates listed in each respective legend. ....... 14 Figure 4: Bovine Femur, Talus, and Calcaneus with Labeled Regions Femur on the left, talus on the upper right, and calcaneus on the lower right. Numbers correspond to regions in Table 3. Femur and tarsal bones are not to scale Figure 5: Porcine Femur, Talus, and Calcaneus wit h Labeled Regions Immature femur on the bottom with unfused femoral head and trochanter (right side), and unfused distal epiphysis (left side). Talus displayed on the top right, and calcaneus on the top left. Bolded numbers corresp ond to regions in Table 3. Numbers penciled on femoral diaphysis (visible in picture) were changed to bolded numbers. Region 9 (partially visible in picture) is located on the late Figure 6: Surface/Bu rial Set up for Bovine Skeletal Material Figure 7: Burial Site for Bovine Bone Segments Path leading to picnic area, red box oriented towards burial site (Middle). Undisturbed burial site for bone segment s within picnic area (Right)..........................................34 Figure 8: Giltner Hall Burial Site with Bovine Diaphysis Segments Buried bone segments that were exhumed, tested, and reburied on a weekly basis (left). Partitioned burial site containing bone segments exhumed, tested, and then stored (right). Burial sites between segment types were adjacent to one another xxxii Figure 9: Organic Extraction of Epiphyseal Bone Powder Picture on the left is an example of bone powder digested in tissue lysis buffer; note the undigested powder at the bottom of the microcentrifuge tube, and the interface between layers. Picture on the right is from the same stock of bone powder digested in demin eralization buffer, it contains a less defined interface and no bone powder Figure 10: Median mtDNA Yields from Fresh Porcine Bones The x - axis lists the regions tested, while the y - axis is the median mtDNA yields in ng per mg bone pow der. Green bars represent bone powder digested in tissue lysis buffer, and blue bars bone powder digested in demineralization buffer. Error bars represent median absolute deviation. Asterisk indicates significa 7 Figure 11: Combined Median mtDNA Yields from Fresh Porcine Bones The x - axis lists the regions tested, while the y - axis is the median mtDNA yields in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer, and blue bars bone powder digested in demineralization buffer. Error bars represent median absolute deviation. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. Asterisk indicates sig nificant differences between buffers Figure 12: Pairwise Comparisons of mtDNA Yields from Fresh Porcine Bones Digested in Tissue Lysis Buffer The x - axis lists the regions tested (n = 8), while the y - axis is the median mtDNA yields normalized in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer. Regions that have the same letter did not differ significantly in mtDNA yields, while regions with different letters did. *Region 2 (n = Figure 13: Pairwise Comparisons of mtDNA Yields from Fresh Porcine Bones Digested in Demineralization Buffer The x - axis lists the regions tested (n = 8), while the y - axis is the median mtDNA yields normalized in ng per mg bone powde r. Blue bars represent bone powder digested in demineralization buffer. Regions that have the same letter did not differ significantly in mtDNA Figure 14: Pairwise Comparisons o f Combined mtDNA Yields from Fresh Porcine Bones Digested in Tissue Lysis Buffer The x - axis lists the regions tested, while the y - axis is the combined mtDNA yields normalized in ng per mg bone powder. Green bars represent bone powder digested in tissue ly sis buffer. Regions that have the same letter did not differ significantly in mtDNA yields, while regions with different letters did. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals Figure 15: Pairwise Comparisons of Combined mtDNA Yields from Fresh Porcine Bones Digested in Demineralization Buffer The x - axis lists the regions tested, while the y - axis is the median mtDNA yields normalized in ng per mg bone powder. Blue bars represent bone powder digested in demineralization buffer. xxxiii Regions that have the same letter did not differ significantly in mtDNA yields, while regions with different letters did. Regions 1 6 represent the diaphysis, 7 9 the meta physes and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals Figure 16: Median Nuclear DNA Yields from Fresh Porcine Bones The x - axis lists the regions tested, while the y - axis is the median nuclear DNA yields in ng per m g of bone powder. Green bars represent bone powder digested in tissue lysis buffer, and blue bars bone powder digested in demineralization buffer. Error bars represent median absolute deviation. Centered is a nested graph for increased resolution of Reg ions 1 4. Asterisk indicates significant differences between buffers Figure 17: Combined Median Nuclear DNA Yields from Fresh Porcine Bones The x - axis lists the regions tested, while the y - axis is the median nuclear DNA yields in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer, and blue bars bone powder digested in demineralization buffer. Error bars represent median absolute deviation. Regions 1 6 represent the diaphysis, 7 9 the meta physes and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. Asterisk indicates significant differences between buffers Figure 18: Pairwise Comparisons of Nuclear DNA Yields from Fresh Porcine Bone s Digested in Tissue Lysis Buffer The x - axis lists the regions tested (n = 8), while the y - axis is the median nuclear DNA yield normalized in ng per mg of bone powder. Green bars represent bone powder digested in tissue lysis buffer. Centered is a nested graph for increased resolution of Regions 1 4. Regions that have the same letter did not differ significantly in DNA yields, while regions with different letters did. * Region 2 (n = 7) Figure 19: Pairwise Compariso ns of Nuclear DNA Yields from Fresh Porcine Bones Digested in Demineralization Buffer The x - axis lists the regions tested (n = 8), while the y - axis is the median nuclear DNA yield normalized in ng per mg of bone powder. Blue bars represent bone powder dig ested in demineralization buffer. Regions that have the same letter did not differ significantly in DNA yields, while regions with different letters did Figure 20: Pairwise Comparisons of Combined Nuclear DNA Yields from Fresh Porcin e Bones Digested in Tissue Lysis Buffer The x - axis lists the regions tested, while the y - axis is the median DNA yields normalized in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer. Regions that have the same lette r did not differ significantly in DNA yields, while regions with different letters did. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals Figure 21 : Pairwise Comparisons of Combined Nuclear DNA Yields from Fresh Porcine Bones Digested in Demineralization Buffer xxxiv The x - axis lists the regions tested, while the y - axis is the median DNA yields normalized in ng per mg bone powder. Blue bars represent bone powder digested in demineralization buffer. Regions that have the same letter did not differ significantly in DNA yields, while regions with different letters did. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals Figure 22: Median mtDNA Yields from Fresh Bovine Bones The x - axis lists the regions tested, while the y - axis is the median mtDNA yields in ng per mg bone powder. Green bars represent bone pow der digested in tissue lysis buffer, and blue bars bone powder digested in demineralization buffer. Error bars represent median absolute deviation. Asterisk indicates significant differences between buffers Figure 23: Combined Median mtDNA Yields from Fresh Bovine Bones The x - axis lists the regions tested, while the y - axis is the median mtDNA yields in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer, and blue bars bone powder digested in demineraliza tion buffer. Error bars represent median absolute deviation. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. Asterisk indicates significant differences between buffers Figure 24: Pairwise Comparisons of mtDNA Yields from Fresh Bovine Bones Digested in Tissue Lysis Buffer The x - axis lists the regions tested (n = 8), while the y - axis is the median mtDNA yield normalized in ng per mg b one powder. Green bars represent bone powder digested in tissue lysis buffer. Regions that have the same letter did not differ significantly in mtDNA yields, while regions with different letters did Figure 25: Pairwise Compari sons of mtDNA Yields from Fresh Bovine Bones Digested in Demineralization Buffer The x - axis lists the regions tested (n = 8), while the y - axis is the median mtDNA yield normalized in ng per mg bone powder. Blue bars represent bone powder digested in demin eralization buffer. Regions that have the same letter did not differ significantly in mtDNA yields, while regions with different letters did Figure 26: Pairwise Comparisons of Combined mtDNA Yields from Fresh Bovine Bones Digested in Tissue Lysis Buffer The x - axis lists the regions tested, while the y - axis is the median mtDNA yields normalized in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer. Regions that have the same letter did not diff er significantly in mtDNA yields, while regions with different letters did. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals Figure 27: Pairwise Compar isons of Combined mtDNA Yields from Fresh Bovine Bones Digested in Demineralization Buffer The x - axis lists the regions tested, while the y - axis is the median mtDNA yields normalized in ng per mg bone powder. Blue bars represent bone powder digested in de mineralization buffer. xxxv Regions that have the same letter did not differ significantly in mtDNA yields, while regions with different letters did. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals Figure 28: Median Nuclear DNA Yields from Fresh Bovine Bones The x - axis lists the regions tested, while the y - axis is the median nuclear DNA yield in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer, and blue bars bone powder digested in demineralization buffer. Error bars represent median absolute deviation. Asterisk indicates significant differences between buffers Figure 29: Combined Median Nuclear DNA Yields from Fresh Bovine Bones The x - axis lists the regions tested, while the y - axis is the median DNA yields in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer, and blue bars bone powder digested in demineralization buff er. Error bars represent median absolute deviation. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. Asterisk indicates significa nt differences between Figure 30: Pairwise Comparisons of Nuclear DNA Yields from Fresh Bovine Bones Digested in Tissue Lysis Buffer The x - axis lists the regions tested (n = 8), while the y - axis is the median nuclear DNA yield normalized in ng pe r mg bone powder. Green bars represent bone powder digested in tissue lysis buffer. Regions that have the same letter did not differ significantly in DNA yields, while Figure 31: Pairwise Com parisons of Nuclear DNA Yields from Fresh Bovine Bones Digested in Demineralization Buffer The x - axis lists the regions tested (n = 8), while the y - axis is the median nuclear DNA yield normalized in ng per mg bone powder. Blue bars represent bone powder d igested in demineralization buffer. Regions that have the same letter did not differ significantly in DNA Figure 32: Pairwise Comparisons of Combined Nuclear DNA Yields from Fresh B ovine Bones Digested in Tissue Lysis Buffer The x - axis lists the regions tested, while the y - axis is the median DNA yields normalized in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer. Regions that have the same l etter did not differ significantly in DNA yields, while regions with different letters did. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals Figur e 33: Pairwise Comparisons of Combined Nuclear DNA Yields from Fresh Bovine Bones Digested in Demineralization Buffer The x - axis lists the regions tested, while the y - axis is the median DNA yields normalized in ng per mg bone powder. Blue bars represent b one powder digested in demineralization buffer. Regions that have the same letter did not differ significantly in DNA yields, while regions with xxxvi different letters did. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals Figure 34: Mean mtDNA Yields from Bovine Bones Exposed for Six Months The x - axis lists the regions tested, while the y - axis is the mean mtDNA yield from surface exposed bovine bones i n ng per mg bone powder. Legend is color coded to its corresponding bar representing a specific time point. Day zero time point had a sample size of four (buried and surface exposed data were combined). Remaining time points had a sample size of two .92 Figure 35: Mean Nuclear DNA Yields from Bovine Bones Exposed for Six Months The x - axis lists the regions tested, while the y - axis is the mean nuclear DNA yield from bovine bones in ng per mg bone powder. Legend is color coded to its corresponding bar and represents the specific time point listed. Day zero time point had a sample size of four (buried and surface exposed data were combined). Remaining time points had a sample size of two Figure 36: Mean mtDNA Yields from Bovine Bones Buried f or Six Months with Day Zero Time Point The x - axis lists the regions tested, while the y - axis is the mean mtDNA yield from bovine bones in ng per mg bone powder. Legend is color coded to its corresponding bar and represents the specific time point. Day ze ro time point had a sample size of four (buried and surface exposed data were combined). Remaining time points had a sample size of two Figure 37: Mean mtDNA Yields from Bovine Bones Buried for Six Months without Day Zero Time Point The x - ax is lists the regions tested, while the y - axis is the mean mtDNA yield from bovine bones in ng per mg bone powder. Legend is color coded to its corresponding bar and represents the specific time point. Each region had a sample size of two for each time po int Figure 38: Mean Nuclear DNA Yields from Bovine Bones Buried for Six Months with Day Zero Time Point The x - axis lists the regions tested, while the y - axis is the mean nuclear DNA yield from bovine bones in ng per mg bone powder. Legend is col or coded to its corresponding bar and represents the specific time point. Day zero time point had a sample size of four (buried and surface exposed data were combined). Remaining time points had a sample size of two Figure 39: Mean Nuclear DNA Yields from Bovine Bones Buried for Six Months without Day Zero Time Point The x - axis lists the regions tested, while the y - axis is the mean nuclear DNA yield from bovine bones in ng per mg bone powder. Legend is color coded to its corresponding bar a nd represents the specific time point. Each region had a sample size of two for each time point xxxvii KEY TO ABBREVIATIONS AD: Anderson - Darling (statistical test) AFDIL: Armed Forces DNA Identification Laboratory BP: Base Pairs (DNA) CILHI: Centr al Identification Laboratory in Hawaii DM: Demineralization Buffer DNA: Deoxyribonucleic Acid EDTA: Ethylene Diamine Tetra - acetic Acid F/R Primer: Forward/Reverse Primer IGF - 1 : Insulin Growth Factor - 1 (gene ) IPC: Internal Positive Control MAD: Median Abso lute Deviation MC1R : Melanocortin - 1 - Receptor (gene) MSU: Michigan State University mtDNA: Mitochondrial Deoxyribonucleic Acid NT: Not Tested PCR: Polymerase Chain Reaction qPCR: Quantitative Polymerase Chain Reaction RB: Reagent Blank RO: Reve rse Osmosis (water) RPM: Rotations per Minute SDS: Sodium Dodecyl Sulfate SLS: Sodium Lauryl Sarcosine STR: Short Tandem Repeat SW: Shapiro - Wilk (statistical test) TE: Tris EDTA TL: Tissue Lysis Buffer USDA: United States Department of Agriculture UV: Ul traviolet 1 INTRODUCTION Skeletal material is an invaluable resource for forensic investigators; it can provide how the individual died. For instance, e ach year the Armed Forces DNA Identification Laboratory (AFDIL) receives and processes hundreds of bone samples often degraded and fragmented derived from civilian and military service members who died dur ing current and past conflicts, with the goal of identi fy ing and repatriating their remains (Eds on et al., 2004; Canik, 2013). Furthermore, m edical examiners and coroners receive approximately 4,400 unidentified human decedents annually, 1,000 of which are still unidentified after one year, and of those 600 u ndergo final disposition anonymously (Hickman et al., 2007). The timely identification of human remains can aid law enforcement in their criminal investigation, and in establishing a case against a suspect . Equally important is being able to notify the d family so they can make postmortem arrangements. Forensic practitioners utilize several techniques to identify skeletonized remains. Forensic anthropologists can develop a biological profile , where t and ancestry are estimated based on metric and morphoscopic traits (SWGANTH.org). This is useful in excluding in dividuals (e.g., missing persons not of the same sex as the decedent), generating leads for law enforcement, and providing tentative identification when us ed in However, to establish positive identification, individualizing information must be obtained. This can be done by comparing postmortem skeletal or dental r adiographs with antemortem records 2 (Murphy et al., 1980; Pretty and Sweet, 2001), and some orthopedic implants have individualizing characteristics (e.g., serial numbers) that can be used to positively identify the recipient (Simpson et al., 2007). Unfort unately, identification of skeletal remains using these methods is not always possible due to a lack of antemortem records, the level of skeletal preservation, or the remains being substantially incomplete or fragmented. Identification then relies upon an alyzing DNA from the skeletal material, with the likelihood of establishing identity being increased by assaying bone regions that are rich sources of DNA for recovery. Environmental Influence s on DNA Degradation from Skeletal Material Environmental expos ure and temperature affect both skeletal material and the DNA contained within. Hochmeister et al. (1991) subjected femoral bone to outdoor exposure, outdoor exposure while wrapped in plastic, water immersion, soil burial, and frozen control samples for a three month period. They reported that frozen samples had the highest mean DNA yield (12.6 µ g DNA/g bone) followed by outdoor exposure (0.9 µ g/g), while bone wrapped in plastic (0.2 µ g/g), immersed in water (0.1 µ g/g), and buried (0.05 µ g/g) had the lowe st yields. Only the control and outdoor exposed bones had sufficient high molecular weight DNA for restriction fragment length polymorphism typing, while DNA recovered from bone subjected to all treatments was amplifiable using polymerase chain reaction ( PCR). Many authors have shown that bones aged in cooler environments typically contain better preserved DNA than bones from warmer settings (H ö ss et al., 1996; Burger et al., 1999; Collins et al., 2001; Smith et al., 2003; Fu et al., 2014). Temperature a ffects DNA degradation and preservation most likely via depurination that is highly temperature dependent ( Götherström et al., 2002) . T hus, the 3 environment, and its climate, from which remains are recovered in likely influences the odds of recovering anal yzable DNA. Specific physical and chemical characteristics of soil have also been shown to influence DNA degradation in bone. Groundwater infused soil can increase bone porosity (Hedges and Millard, 1995), resulting in the physical loss of DNA, and aids p utrefaction by providing a favorable environment for hydrolytic reactions that cause depurination and subsequent DNA fragmentation (Eglinton et al., 1991). The chemical composition of soil affect s bone crystallinity over time through the dissolution and r e - precipitation of hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ) crystals in bone (Hedges and Millard, 1995; Hedges, 2002). This process may release DNA bound to hydroxyapatite , consequently reducing its preservation and potential recovery ( Götherström et al., 2002). Furthermore, incorpora ted soil solutes (e.g., humic substances) can co - extract with DNA, leading to PCR inhibition and limiting subsequent analyses (Kemp et al., 2006; Eilert and Foran, 2009; Hebda, 2013). B one dissolution and DNA degradation are also i nfluenced by pH (Hedges and Millard, 1995). Gordon and Buikstra (1981) excavated seven burial sites in Illinois, finding a significant correlation between soil acidity and bone degradation. Crow (2008) experimentally demonstrated that mineral apatite was highly susceptible to dissolution at acidic pH (5.6 3.6), and Casallas and Moore (2012) reported near - complete mineral dissolution of bones b uried 8 10 years in soils at pH 4.2 4.8. Hughes et al. (1986) were unable to isolate DNA from peat preserve d human remains that were in an aqueous environment of pH 4.5 5.5, indicating that low pH contributed to DNA degradation. Likewise, Burger et al. (1999) noted that DNA is better preserved at a slight ly alkaline to physiological pH . Though depurination occurs spontaneously at physiological pH (Lindahl, 1993), it is accelerated at low pH and high 4 temperature (Roger and Hotchkiss, 1961) , indicating that acidic and/or warmer climates are associated with increased DNA degradation. Burial has also been sho wn to affect DNA yields . R esearchers have reported that substantial DNA loss in bone can occur over a period of years (Campos et al., 2012), months (Hochmeister et al., 1991 ), or less (Hebda and Foran, 2015). How much DNA may be lost in buried bone over even shorter periods (i.e., from days to weeks ) is unclear; however, B är et al. (1988 ) demonstrated that many soft tissues ha ve pronounced DNA degradation within days after death . The authors proposed that rapid putrefaction led to the initial loss of DNA in their stu dy, as did Campos et al. (2012). Dent et al. (2004) reviewed human decomposition in soil, and noted that putrefaction is normally initiated by autolytic processes and is generally not observed until days after death. Bell et al. (1996) postu lated that microbes, from the decedent or present in the environment, can degrade bone within months after death, and Latham and Madonna (2013) discussed how microbial activity can indirectly damage DNA by degrading the bone it is bound to, or directly thr ough enzymatic activity . Environmental exposure, particularly burial, clearly affects DNA degradation, therefore, it is not only important to determine site(s) on bone that are rich sources of DNA, but also evaluate how environment alters DNA quantity and quality at those locations. The Composition of Bone Many authors have noted that b one composition facilitates function: the mineral components provide mechanical rigidity and weight bearing strength, while the organic constituents offer elasticity a nd flexibility ( e.g., White and Folkens , 2005; Clarke, 2008). Clarke (2008) reviewed the makeup of bone, characterizing it as a complex tissue composed of 50% 5 70% mineral (i.e., hydroxyapatite) , 20% 40% organic material, and less than 10% water a nd li pids. Hydroxyapatite forms approximately 200 Å long crystalline lattices (Clarke, 2008), and makes up 62% 66% Collagen constitutes 90% 96% of organic material (Rogers et al., 1952), and is primarily compri sed of type I collagen, along with small quantities of types III, V, IX, XII, XIV, XIX, and XXI collagen (Clarke, 2008). Bone develops into two general types: cortical and trabecular (Figure 1) . Cortical osseous tissue is solid, dense, and relatively nonp orous; conversely, trabecular bone consists of a porous mesh - like network of plates and rods (Clarke, 2008). The adult human skeleton is comprised of approximately 80% cortical and 20% trabecular bone by mass (Eriksen et al., 1994); however, these ratios vary greatly among bones. For example, vertebrae have a 25:75 percent ratio of cortical to trabecular bone, whereas the femoral head is closer to 50:50, and the femoral shaft is almost entirely cortica l material (Clarke, 2008). Bone contains three cell ty pes: osteoblasts, osteocytes, and osteoclasts , which are characterized by their function. Osteoblasts synthesize the collagenous matrix and regulate its mineralization (Anderson, 2003). Fifty to seventy percent of osteoblasts undergo apoptosis after bone tissue deposition (Clarke, 2008), while the remaining cells become osteocytes within the its maintenance through the exchange of nutrients and wastes (Burger et al., 2003). Cortical bone contains approximately 20,000 osteo cytes per cubic millimeter (Martin and Burr, 1989). Osteoclasts are multinucleated cells that participate in osteolysis by binding to the bony matrix, then secreting acid and collagenase to resorb tissue (Vaananen et al., 2000). These three cell types ar e responsible for bone deposition, maintenance, and resorption that characterize the bone remodeling cycle, and provide a sourc e of DNA for forensic analysis. 6 The composition of bone also means it is one of the final tissues to decompose, which leads to it s forensic importance. Osseous tissue preserves, or at least retains , DNA better than other tissues, where it is believed to act as a physical barrier against the environment (Hochmeister et al., 1991; Collins e t al., 2002), and DNA bound to hydroxyapatit e is resistant to proteolytic digestion (Brundin et al., 2013). However, bone is a challenging substrate from which to extract and process DNA; many of its components have been shown to inhibit PCR , including collagen ( Scholz et al., 1998; Opel et al., 20 10 ), calcium ( Bickley et al., 1996; Opel et al., 2010 ), and potentially hydroxyapatite, which can bind double stranded DNA (Martinson, 1973). Femoral Bone Anatomy and Forensic Analyses The femur is the longest, heaviest, and strongest bone in the human b ody, tasked with nes like the femur (Figure 1 ) contain an elongated hollow shaft of cortical bone (diaphysis) that widens towards the growth plates (metaphyses), with rounded ends made of a robust trabecular meshwork encased in a thin layer of cortical bone (epiphy ses) (Gray, 1918 ; Clarke, 2008). The femur is one of the most commonly utilized skeletal elements in physical/forensic anthropological analyses, and many components of a biological profile have benefited from its study. For instance, a maximum length (Tr otter and Gleser, 1952, 1958), and sex can be estimated with about 90% accuracy by measuring the ve rtical head diameter (Pearson, 1917; Stewart, 1979), and/or midshaft diaphysis circumference (Black, 1978; Di Bennardo and Taylor, 1979). Likewise, investigators exploit predictable patterns in femoral development, such as diaphyseal length and 7 epiphyseal closure schedules , for sub - adult age estimation (Bass, 1995). In contrast, discriminating among major ancestral groups (e.g., European vs. African) using fe moral metric traits has had only limited success (Stewart, 1962; Craig, 1995). Figure 1 : Basic Anatomy of a Human Femur Sectional view of a human femur showing the diaphysis, metaphyses, and epiphyses, along with the distribution of cortical and trabecular bone. A dapted from Gray (1918). Illustration available at http://www.bartleby.com/107/59.html#i249. Cortical bone Metaphysis Metaphysis Epiphysis Diaphysis Epiphysis Trabecular bone 8 Investigators who recover DNA from skeletal material have relied heavily upon femoral sampling . In t he first published forensic case based o n skel etal DNA , a femur was used to positively identify a murdered adolescent eight years postmo rtem (Hagelberg et al., 1991). That same year, t he feasibility of obtaining high molecular weight DNA from femoral cortical bone for restriction fragment lengt h polymorphism and PCR based analyses was demonstrated (Hochmeister et al., 1991) . Similarly, this element is often used in ancient DNA analysis, such as that recovered from 45,000 year old human remains found in Siberia , isolated from the distal femur (F u et al., 2014). Approximately 19% of bones tested at AFDIL through 2006 were femora , the highest percentage of any single element (Edson et al., 2004; Leney, 2006). Femora comprised 46% of samples (11356/24656) tested by the International Commission on Missing Persons to identify remains from mass graves sites after the Yugoslavian conflict ( 2007). Overall, DNA from the femur has been commonly utilized in research (e.g., Cattaneo et al., 1995; Misner et al., 2009; Caputo et al., 2013; Fu e t al., 2014) as well as forensic casework (e.g., Edson et al., 2004; Leney, 2006; ). Recovering DNA from Skeletal Material DNA Yield by Osseous Tissue Type DNA yield d ifferences between cortical and trabecular bone were established in t he early bone types from unspecified human remains and found that trabecular bone contained 10 20 times more DNA than cortical bone on a per milligram basis when quantified using spectrophotofluorometry. In contrast, in a review of postmortem DNA preservation and recovery, Parsons and Weedn (1996) reported that cortical bone retains DNA better than trabecular bone over long periods, based upon unpublished observations from 9 AFDIL. Sampling cortical bone became standard operating procedure fo r AFDIL, which often reject s degraded or ancient remains consisting primarily of trabecular osseous tissue (Edson et al., 2004). The International Society for Forensic Genetics (Prinz et al., 2006) and the International Committee of the Red Cross (2009) advocate sampling from dense cortical bone preferably from weight bearing long bones for disaster victim identification. Recent investigations into DNA quantity and quality between osseous tissue types have resulted in mixed finding s. Misner et al. (2009) examined 86 bones derived from 36 skeletons exhumed from the Voegtly Cemetery in Pittsburg, PA to determine if skeletal weathering stages are predictive of DNA quantity and quality . The authors found no correlation between skeleta l weathering and recovered DNA; however, osseous tissue type significantly influenced DNA yield and amplification success. Cortical bone of the femur resulted in mtDNA amplicons of 107 bp or larger 79.3% of the time , with a mean yield of 103.2 copies mtDN A/µL, while the rib and pelvis, primarily trabecular bone, had DNA that amplified 63.6% and 36 .0 % of the time with mean yields of 87.4 and 77.6 copies of mtDNA/µL respec tively. Mundorff et al. (2009) reported similar results, where DNA recovered from the femur and tibia led to the identification of the decedent more often than the ribs or pelvis; however, they also noted that commonly overlooked elements such as the patella, tarsals, and foot phalanges were similarly successful . Mundorff and Davoren (2014 ) followed up by testing every element type from three skeletonized individual s, reporting that elements composed primarily of trabecular bone had better quality and greater quantit ies of DNA than did the traditionally accepted dense cortical weight bearin g long bones. The reason(s) for these discrepancies remain unclear; however, osseous tissue type likely influences the quantity and quality of DNA recovered and should be considered when sampling skeletal remains . 10 DNA Isolation Methods: Preparing Bone fo r Digestion and Extraction Strategies for obtaining DNA from a bone sample can be divided into two general categories: pulverizing bone into a fine powder, or excising a segment and processing it whole. Several techniques are utilized to turn whole bone i nto powder: freezing the bone usually in liquid nitrogen prior to pulverization (Hagelberg and Clegg, 1991; Cattaneo et al. , 1995; Hochmeister et al., 1991 ; Loreille et al., 2007), grinding bone using nonfreezing milling apparatuses (Edson et al., 2004; Lo reille et al., 2007; Misner et al., 2009, Adler et al., 2011), and collecting powder produced by repurposed drilling tools (Rennick et al., 2005; Adler et al., 2011; Caputo et al., 2013; Mundorff and Davoren 2014; Hebda and Foran, 2015) are the most common . Loreille et al. (2007) had inconsistent results when comparing DNA yields from bone pulverized in a freezer mill versus a nonfreezing blender cup, concluding that neither method pr oduced substantially higher yields. Adler et al. (2011) compared grindin g versus drilling of cortical bone, resulting in 5 30 fold more mtDNA from bones that were g round; however, when the drilling speed was reduced from 1,000 to 100 RPM , both drilling and grinding methods produced similar yields. The authors attributed thi s to high - contributing to mtDNA damage, and t he use of sharp drill bit s that are in good condition has been advocated by some authors to reduce the heat generated by drilling (Matthews and Hirsch, 1972; Adler et al., 2011). Caputo et al. (2013) compared DNA quantity and quality of powdered bone from low - speed drilling and thinly sliced bone segments . The drilling method resulted in 1.5 times more DNA on average; however, the bone slice method produced a greater perce ntage of full STR profiles (54.8%) compared to drilled bone powder (10%). Drilling was used in this 11 thesis research because of its ease of use, flexibility, and relatively non - destructive sampl ing of bone. Tissue Digestion Methods Used for DNA Isolation Isolating DNA from bone involves strategies that either do or do not demineralize it prior to DNA extraction. Particulars vary , but digestion buffers generally include a detergent, buffer, chelating agent, and proteinase that are incubated for some amoun t of time to lyse cells and release DNA (Butler, 2012). The chelating agent commonly used in digestion buffers is ethylene diamine tetra - acetic acid (EDTA), which b inds divalent cations . EDTA complexes with the Ca 2+ in hydroxyapatite , facilitating bone d issolution . Calcium also helps to protect DNA nucleases from proteolytic digestion (Price et al., 1969), thus its removal may be further beneficial during DNA extraction . Furthermore, EDTA bind s Mg 2+ , required by nucleases for DNA hydrolysis (Kunitz, 195 0; Price, 1975) . Overall, EDTA may have multiple important role s in liberating DNA from bone and preserving it for analyses. Demineralization protocols differ , but can be classified into procedures where demineralization occurs before the organic componen ts are digested (Hochmeister et al., 1991; Hagelberg and Clegg, 1991), or in a single process where organic and inorganic components are digested simultaneously (Loreille et al., 2007). Hochmeister et al. (199 1) incubated bone powder in a 0.5 M EDTA solut ion routinely replenishing it over three to five days followed by three washes in deionized water prior to digestion in extraction buffer and proteinase K . Hagelberg and Clegg (1991) used a similar procedure, decalcifying bone in 0.5 M EDTA for 72 hours p receding incubation in lysis buffer and proteinase K . This method of demineralization was time consuming, DNA was likely lost from the discarded EDTA solution, and some researchers 12 reported that full demineralization, compared to non - demineralization, red uced DNA yields (Hagelberg and Clegg, 1991; Fisher et al., 1993). Owing to this, AFDIL opted to use a non - demineralizing digestion solution in their casework consisting of Tris, EDTA (not in high enough concentration to appreciably demineralize bone) and sodium dodecyl sulfate (SDS) (Edson et al., 2004). This method was faster; however, according to Loreille et al. (2007) , when the supernatant was recovered, undissolved bone containing un - extracted DNA was discarded. This led AFDIL to develop and validat e a single step demineralization protocol for casework (Loreille et al., 2007), which was tested side - by - side with samples processed using the casework protocol detailed in Edson et al. (2004). The demineralization protocol resulted in significantly highe r DNA yields, improved typing success, and a reduction of starting skeletal material required for analysis. Figure 2 - demineralization and demin eralization protocols through August 2013 (Canik, 2013). Side - by - sid e comparisons of these data reinforce a conclusion made by Loreille et al. (2007): single step demineralization increases the likelihood of recovering DNA that will produce reportable gen etic information. 13 Figure 2 : - Demineralization and Demineralization Protocols by Element Type - de mineralization protocol and right skeleton using their demineralization protocol. Skeletons are in anatomical position with color coded elements corresponding to their percent success of l is shown below each illustration. Inter - Bone Heterogeneity in Recoverable DNA Quantity and Quality short tandem repeat typing , and/ or identification from skeletal DNA was influe nced by the element used for analysis ( Edson et al. , 2004; Leney, 2006; , 2007; Mundorff et al., 2009). This included large data sets that covered various postmortem intervals , environm ental conditions, and taphonomy. Results from these studi es are summarized in Table 1 and graphically depicted in Figure 3 . The Central Iden tification Laboratory and AFDIL reported mtDNA amplification success from skeletal spanning 14 World War II, the Ko rean War, and the Vietnam War (Edson et al., 2004; Leney, 2006). et al. (2007) evaluated DNA typing success among elements from more than 25,000 skeletal remains retrieved from mass grave sites in Kosovo, Bosnia, and Herzegovina. Simi larly, Mundorf f et al. (2009) compared D NA identification success among bones based on over 3,600 skeletal elements from the World Trad e Center - Human Remains Database. Figure 3 : Published Illustrati ons of Inter - Bone Success of Obtaining Analyzable DNA Left diagram adapted from Edson et al. (2004), middle from Mundorff et al. (2013) representing data from Mundorff et al. (200 9), and right from Leney (2006). Diagrams provide an illustrative Table 1 . Illustrations are color coded/shaded to correspond with percent success rates listed in each respective legend. 15 Table 1 : Summary of Results from Published Studies Evaluating Inter - Bone Variation of Successfully Obtaining Analyzable DNA Element sampled is identified in the first column. Subsequent columns represent studies in which success rates were assessed among elements. The first number denotes the percent success and the parenthetical number signifies the sample size for that element. Success was et al. (2007) defined succe ss as obtaining genetic data from 12 or more loci using a Promega Data from Mundorff et al. (2009) used in this table only incorporate the Complete E lements Database, a subset of the World Trade Center - Human Remains Database. NT = Not Tested CILHI = Central Identification Laboratory in Hawaii Element Sampled % Success of Sampled Element (Sample Size) Edson et al. 2004 mtDNA Leney 2006 mtDNA et al. 2007 Nuclear Mundorff et al. 2009 mtDNA/Nuclear Femur 95 (192) 87 (477) 87 (11356) 71 (143) Tibia 89 (145) 83 (306) 76 (1329) 70 (125) Fibula 67 (21) 63 (87) 63 (160) 60 (159) Metatarsal 81 (21) 74 (23) 1 st Metatarsal Only 33 (120) 72 (257) In nominate 63 (19) 74 (87) 53 (185) Ilium Only 63 (62) Teeth 80 (184) 72 (442) 83 (6963) NT Radius 70 (37) 61 (101) 25 (469) 60 (120) Ulna 67 (54) 57 (111) 23 (444) 61 (87) Humerus 79 (149) 71 (339) 46 (2415) 61 (110) Scapula 79 (19) 69 (39) 57 (35) 54 (92) Clavicle 77 (30) 58 (56) 26 (128) 54 (97) Vertebra 86 (14) 59 (21) 62 (146) 61 (72) Mandible 76 (34) 72 (50) 56 (131) 65 (46) Skull 51 (76) 47 (197) 41 (757) 47 (494) Rib 96 (26) Single CILHI Case 63 (43) NT 64 (1301) Metacarpal NT NT 61 (18) 44 (211) Patella NT NT NT 80 (83) Foot Phalanx NT NT NT 80 (25) Hand Phalanx NT NT NT 57 (83) Sacrum NT NT NT 59 (27) Tarsal NT NT NT 51 (37) 16 These researchers used different methods and objectives, the remains tested had varied postmortem intervals, and they were subjected to different environmental conditions and trauma; however, there were consistent findings among studies . In all instances dense cortical weight bearing long bones like the femur and tibia were more likely to retain DNA that result ed in successful downstream analyses. In contrast , bones with a greater proportion of trabecular tissue (e.g., axial skel eton) were less likely to produce useful genetic information. These studies shared several limitations however : they were retrospect ive analyses, remains wer e subjected to myriad environmental infl uences, and element types within a single individual were not assessed . Those factors led Mundorff et al. (2013; 2014) to conduct prospective research testing each element type from three in dividuals that were allowed to naturally decompose lying prone on the ground at the same plot of land in the University of Utilizing the three individuals , along with separate remains from successively longer postmortem intervals, the authors ranked elements based on the quantity and quality of DNA obtained from each. Their results demonstrated a high degree of variability in DNA among elements from a single individual. Counter to some previous findings , small predomina tely cancellous bones such as the phalanx, patella, and tarsals had higher DNA yields than weight bearing long bones like the femur and tibia. Furthermore, the results remained fairly consistent when the same element types were tested across postmortem in tervals ranging from 0 to more than 20 years. However, this research led to several unanswered questions that will need to be resolved in future investigations. First, the remains were skeletonized above ground , and as a result the authors noted that dif ferent postmortem conditions (e.g., burial) may produce different outcomes . Also, most elements w ere tested at a single location to preserve bones for future anthropological investigations ; u tilizing single site 17 sampli ng to represent an content assumes homogeneity throughout the bone. Furthermore, bone powder from many elements contained a substantial mix ture of cortical and trabecular tissue , confounding contributions by osseous tissue type. Finall y, only nuclear DNA was tested, thus i t remained unknown if similar results would be obtained based on mtDNA. Intra - Bone Heterogeneity in DNA Quantity and Quality While researchers have established that DNA heterogeneity exists among bones , very few have examined variation within a single el ement. Adler et al. (2011) tested different components of teeth and reported that cementum contained up to five times more mtDNA (copy number per 100 mg substrate) than the more commonly sampled dentin. Yamaguchi and Yamaguchi (1986) measured DNA content in femora from weanling rats, and recovered about twice as much DNA from the epiphyses than the diaphysis. Further rat research by Yamaguchi et al. (2003) demonstrated that twice as much DNA was present in the metaphyses than the diaphysis. However, Yam a guchi et al. (1986 and 2003) reported highly variable DNA yields of the diaphysis between studies, 15 20 mg/g in 1986 and 1 1.5 mg/g in 2003, making cross study comparisons among the diaphysis, metaphyses, and epiphyses problematic. Finally, Hebda (2 013) noted that the locations tested near the metaphyseal en d of segmented bovine femora had relatively higher DNA yields when compared to the diaphyseal end, leading to the thought that DNA yields vary along the femur . With the exception of Adler et al. (2011) , in which only teeth were tested, all of the researchers findings were tangential in nature, as their primary emphasis was not in examining intra - element variation, necessitating the need for prospective research to systematically examine potential heterogeneity within a single bone. 18 Research Goals The previous research detailed above has shown that DNA quantity and quality recovered from skeletal remains can differ due to many factors. H owever, there is a paucity of resear ch systematically exami ning the heterogeneity of recoverable DNA withi n a single element, and how DNA quantity and quality may change when exposed to common environments where forensically relevant remains often exist . Identifying predictable variation of nuclear and mitochondr ial DNA heterogeneity in the femur , if it exists, has the potential to enhance DNA reco very and analysis success. The research presented here was designed to determine whether DNA is heterogeneously distributed in femora, one of the most commonly utilized elements in historical and forensic analyses. The first goal was to measure DNA quantity and quality throughout fresh femora derived from porcine and bovine model systems , utilizing non - demineralization and demineralization digestion protocols. The secon d goal was to determine if/how heterogeneity changed when bones were buried or left exposed in an outdoor environment over a six month period. The final goal was to compare intra - femoral variability of DNA quantity and quality to the calcaneus and talus, two tarsals concomitantly processed along with the femur, and characterized by Mundorff and Davoren (2014) as having substantially more DNA than the femur. Overall, the research presented was a systematic analysis of intra - bone DNA heterogeneity in fresh, surface exposed, and buried femora. 19 MATERIALS AND METHODS Preparation of Bovine and Porcine Femora and Tarsals Bovine/Porcine Intra - bone Variation and Tarsal Comparison Experiments Eight fresh femora and tarsal sets from Holstein dairy cows ( Bos taurus ) and domestic pigs ( Sus scrofa domesticus ) were obtained from the Michigan State University (MSU) Meats talus, calcaneus, and other foot bones articulated with muscle, tendons, and/or ligaments. Femora and tarsal sets were stored in a - 80 ° C freezer if not immediately macerated; they were thawed overnight at room temperature prior to maceration. Soft tissue was removed through maceration in boiling 1% Terg azyme (Alconox, White Plains, NY) solution. Maceration took place in a Bayou Classic 42 - quart stainless - steal stockpot (Barbour International Inc., Brandon, MS) containing 30 L of reverse osmosis (RO) water (~10 M ), and 300 g of Tergazyme Solution wa s preheated to 95 ° C using three ceramic heating plates set to 300 ° C. Plates were arranged in a pseudo - trigonal manner for adequate heat and weight distribution of the stockpot and solution. Heating elements were adjusted to maintain boiling temperature. Skeletal material was placed into the stockpot once the solution temperature reached 90 ° C to 95 ° C. Bones were macerated until soft tissues were separated: either through boiling or by physically scraping off the gelatinized tissues with a sharp instrumen t. Bovine bones were macerated one pair at a time (two femora, calcanei, and tali) in two eight - hour intervals, with fresh Tergazyme solution used per interval. Bovine femora were rotated longitudinally halfway through each interval. Porcine bones were macerated together in a single seven hour period. Halfway through maceration, they were removed from the stockpot and partially cleaned, then re - immersed for the 20 remainder of the seven hours. Cleaned bones were assigned an identifier, each replicate con sisted of a femur, calcaneus, and talus, and measurements were taken to determine the total diaphyseal length and midshaft circumference of femora (Table 2). Table 2: Sidedness, Diaphysis Length, and Midshaft Circumference of Bovine and Porcine Femora Fem ora were labeled by species and the sequential order processed. Total diaphysis length was measured from the proximal to distal epiphyseal lines, with the range representing differences due to irregular borders. Circumference was measured at the femoral midshaft. *, **, or *** represents femora originating from a single individual. Bovine Femur Sidedness Diaphysis Length Circumference at Midshaft C - 01 Right 21.6 24.1 cm 15.2 cm C - 02 Right 21.6 24.1 cm 15.2 cm C - 03 * Right 26.7 30.5 cm 16.3 cm C - 04 * Left 26.7 30.5 cm 16.3 cm C - 05 ** Right 26.7 30.5 cm 16.3 cm C - 06 ** Left 26.7 30.5 cm 16.3 cm C - 07 *** Right 29.2 30.5 cm 15.2 cm C - 08 *** Left 29.2 30.5 cm 15.2 cm Porcine Femur Sidedness Diaphysis Length Circumference at Midshaft P - 01 Left 14.0 15.2 cm 8.9 cm P - 02 Left 14.0 15.2 cm 8.9 cm P - 03 Right 15.2 16.5 cm 8.9 cm P - 04 Right 14.0 16.5 cm 8.3 cm P - 05 Left 15.2 16.5 cm 8.9 cm P - 06 Right 15.2 17.8 cm 8.9 cm P - 07 Left 16.5 18.4 cm 9.2 cm P - 08 Left 15.2 17.1 cm 8 .9 cm Demarcated regions are listed in Tab le 3 and illustrated in Figure 4 (bovine) and Figure 5 (porcine). Eight equidistant regions were marked in pencil starting at the midshaft diaphysis, then e xtended towards the proximal and distal metaphyses. Midshaft was defined as the central point between the proximal and distal epiphyseal lines. The distal epiphysis, femoral head, and trochanter were individually marked. 21 Finally, an articulating surface Fourteen regions in total were marked and given numbered identifiers: twelve from the femur, and one for the talus and calcaneus respectively. Marked bones were stored at room temperatur e until tested. Table 3: Demarcated Regions of the Femur and Tarsals Region Location 1 Midshaft Diaphysis 2 Midshaft Diaphysis (Distal to Region 1) 3 Diaphysis (Proximal to Region 1) 4 Diaphysis (Distal to Region 2) 5 Diaphysis (Proximal to Region 3) 6 Diaphysis (Distal to Region 4) 7 Proximal Metaphysis 8 Distal Metaphysis 9 Articulating Surface 10 Distal Epiphysis 11 Femoral Head 12 Trochanter 13 Calcaneus (Tarsal Bone) 14 Talus (Tarsal Bone) Figure 4: Bovine Femur, Talus, and Calcane us with Labeled Regions Femur on the left, talus on the upper right, and calcaneus on the lower right. Numbers correspond to regions in Table 3. Femur and tarsal bones are not to scale. 22 Figure 5: Porcine Femur, Talus, and Calcaneus with Labeled Region s Immature femur on the bottom with unfused femoral head and trochanter (right side), and unfused distal epiphysis (left side). Talus displayed on the top right, and calcaneus on the top left. Bolded numbers correspond to regions i n Table 3. Numbers penciled on femoral diaphysis (visible in picture) were changed to bolded numbers. Region 9 (partially visible in picture) is located on the lateral distal aspect of the femur. Surface/Burial Comparison Experiment of Bovine Femora an d Tarsals Four fresh femora and tarsal sets from two Holstein dairy cows were obtained from the MSU Meats Laboratory. Regions tested are listed in Table 4, five along the femur, as well as the calcaneus and talus. Table 4: Regions of Bovine Femora and T arsals Tested Identifier Location Equivalent Region(s) from Table 3 D Midshaft Diaphysis Regions 1 and 2 PM Proximal Metaphysis Region 7 DM Distal Metaphysis Region 8 F Femoral Head Region 11 E Distal Epiphysis Region 10 Cal Calcaneus Region 13 Tal Talus Region 14 9 23 Soft tissue/cartilage was mechanically defleshed to expose drilling sites for each region using knives and razorblades. Calcanei were fully, and tali partially, disarticulated from the tarsal set to expose bones for drilling. Regions of bone (Table 4) were initially tested to establish pretreatment DNA yields. Figure 6 depicts the experimental setup of how the bones were exposed. Two femora and tarsal sets one per individual cow were labeled and buried in approximately eight inches o f black garden soil. The other femora and tarsals were placed on the soil surface above the buried bones. Surface bones were encased by a mesh wire fence weighted down with stones to deter scavenger activity. Bones were located at the geographical coord inates 42 ° 44 43.8 N 84 ° 17 09.4 W. Figure 6: Surface/Burial Setup for Bovine Skeletal Material Bones were retrieved, tested, and returned at the time points listed in Table 5. Skeletal material was washed onsite using a garden hose to remove exc ess soil, debris, and insects; then bagged separately and transported to the MSU Forensic Biology Laboratory. There the bones were further cleaned of soil and debris using RO water and a brush, tested, re - bagged, and brought back to the experiment site fo r reburial/re - placement. 24 Table 5: Dates Buried/Surface Femora and Tarsals Were Tested Date Tested Time Point Days Exposed/Buried 9/19/2014 Day 0 0 9/26/2014 Week 1 7 10/3/2014 Week 2 14 10/17/2014 Month 1 28 12/13/2014 Month 3 85 3/21/2015 Month 6 177 Drilling Skeletal Material Bones were drilled inside a Purifier PCR Enclosure (Labconco, Kansas City, MO) an enclosed ultraviolet (UV) workstation sanitized with 10% bleach, 70% ethanol, and then the UV lamp was activated for 10 min (unknown J/cm 2 ) prior to and after drilling. Drill bits, collars, and sleeves were soaked in 10% bleach for 10 min, rinsed with Milli - Q ® filtered water (18.2 M ), rinsed with 70% ethanol, then UV irradiated in a Spectrolinker XL - 1500 UV Crosslinker (Spectronics Corporat ion, Westbury, NY) for 10 min prior to and after use (~2.5 J/cm 2 ). Other supplies used: including a Dremel 395 MultiPro ® rotary device (Robert Bosch Tool Corporation, Mount Prospect, IL), weighing paper (VWR International, Radnor, PA), microspatula, and 2 .0 mL microcentrifuge tubes were UV irradiated for 10 min in the Spectrolinker . Sanitized equipment not in use were stored in a CleanSpot PCR/UV Work Station (Coy Laboratory Products, Grass Lake, MI) with the UV lamp activated (unknown J/cm 2 ). Bones cont aining soil/debris were washed in RO water, then allowed to air dry for at least ® number 420 heavy duty cut - off wheel to remove surface contaminants. M35 7/64 inch cobalt drill bits (B& Q 25 per bone/region/segment. Drilling locations were maximally separated, so collected powders would be representative of the bone/region/segment being drill ed. Powders produced from the drillings were combined and homogenized using a microspatula. Two milliliter microcentrifuge tubes were weighed on a PB153 - S precision balance (Mettler - Toledo, Columbus, OH), then 50 +/ - 1 mg of the homogenized powder was ad ded to each tube. Microcentrifuge tubes containing powder were stored at - 20°C pending digestion and extraction. DNA Isolation Organic extraction coupled with extract concentration using 30K Amicon ® Ultra - 0.5 mL Centrifugal Filters (Millipore Corporatio n, Billerica, MA) was utilized to isolate DNA from bone powder; however, two variants were employed depending on the experiment. Method used per experiment is listed in Table 6. Reagent blanks were generated for each extraction. DNA extracts and reagent blanks were stored at - 80°C. Table 6: Organic Extraction Method Used per Experiment Experiment Organic Extraction Method Bovine/Porcine Intra - bone Variation and Tarsal Comparison Experiments Method One Surface/Burial Comparison Experiment of Bovine Fe mora and Tarsals Method One Changes in the Recoverable Total DNA of Buried Bovine Bone Segments over a One Month Time Period Method Two Changes in the Recoverable Total DNA of Non - Buried Bovine Bone Segments over a One Month Time Period Method Two Organ ic versus : Total DNA Yield Comparisons Over One Week Method Two Mass Difference between Wet and Dry Bone Not Applicable Effect of Proteinase K Concentration on Total DNA Yields Method One Comparison of Total DNA Yields from Bovine Bones Mace rated by MSU Forensic Anthropologist versus MSU Forensic Biologist Method One 26 Organic Extraction Method One Tubes, Amicon ® columns, and solutions, except organic solvents and proteinase K, were UV irradiated for 10 min (~2.5 J/cm 2 ) . Two extraction buff ers were used side by side: bone powder was digested in a 1:15 ratio, of either tissue lysis buffer ( 20 mM Tris pH 7.5; 50 mM EDTA; 0.1% SDS ) or demineralization buffer (Loreille et al., 20 07) (0.5 M EDTA pH 8.0; 1% laury l - sarcosine (SLS)). One percent by total solution volume proteinase K (20 mg/mL) was added to the microcentrifuge tubes containing bone powder and buffer, then vortexed for 15 s NJ) set to 250 RPM . The microcentrifuge tubes. Some extractions were done in batches of 30, in these instances tubes were incubated overnight at 56°C on an model 100 Rocking Platform set to six (VWR International, Radn or, PA). An equal volume of cold 1:1 phenol chloroform solution was added to the tubes, vortexed for 15 s, and centrifuged for 10 min at maximum speed. Four hundred microliters of the aqueous layers were transferred to 30K Amicon ® filters and centrifuged for 10 min at 14,000 × g. An additional 200 µL of the aqueous layers were transferred to the columns and centrifuged for 10 min at 14,000 × g. Flow - through was discarded and the columns were washed with 400 µL of TE ( 10 mM Tris pH 7.5, 1 mM EDTA) and cen trifuged for 10 min at 14,000 × g. Flow - through was discarded, and the TE wash was repeated. Flow - through was discarded, and 400 µL of low TE (10 mM Tris pH 7.5; 0.1 mM EDTA) was added to the columns, which were centrifuged for 10 min at 14,000 × g. Col umns were inverted into new Amicon ® tubes and centrifuged for 2 min at 1,000 × g to collect extracts. Extract volumes were measured and recorded prior to - 80°C storage. 27 Organic Extraction Method Two Tubes, Amicon ® columns, and solutions, except organic solvents and proteinase K, were UV irradiate for 10 min (~2.5 J/cm 2 ) . Five hundred microliters of tissue lysis buffer and 4.5 µL of proteinase K were added to the 2.0 mL microcentrifuge tubes with bone powder, vortexed for 15 s, then incubated overnight a t 56°C. An equal volume of cold, Tris - saturated phenol was added to the tubes, vortexed for 15 s, and centrifuged for 5 min at maximum speed. Aqueous layers were transferred to new 2.0 mL microcentrifuge tubes, to which 500 µL of chloroform was added. T ubes were vortexed for 15 s, and centrifuged for 5 min at maximum speed. Aqueous layers were transferred to 30K Amicon ® filters and processed as above. Quantitative PCR Assay for Bovine and Porcine DNA Extracts Real time PCR amplification was performed on an iCycler thermal cycler, and fluorescence detected using an iQ5 multi - color real - time PCR system (Bio - Rad Laboratories, Hercules, CA). Primer and probe sequences are listed in Table 7. Bovine primers and probes targeting the nuclear Melanocortin - 1 - R eceptor ( MC1R ) gene, as well as the Internal Positive Control (IPC) primers, probe, and template were designed by Lindquist et al. (2011). Primers and probe targeting the mitochondrial ATPase 8 gene were designed by Hebda (2013). Porcine primers and prob e targeting the mitochondrial ATPase, and nuclear MC1R genes were designed using Primer3 software (Rozen and Skaletsky, 2000), based on the Sus scrofa complete mtDNA (BLAST Accession NC_00845.1), and Sus scrofa mixed breed chromosome six DNA (BLAST Accessi on NC_010448.3) from the National Center for Biotechnology Information. Primers and probes were ordered from Integrated DNA Technologies (Coralville, IA), Thermo Fisher 28 Scientific (Waltham, MA), or Sigma - Aldrich (St. Louis, MO). Bovine DNA standards were created via stock bovine muscle digested in tissue lysis buffer, organically extracted (Method Two), and diluted to 200 ng/µL based off parallel qPCR data of bovine standard from Hebda (2013). Porcine DNA standards were created by serial dilution of stoc k DNA from pig muscle digested in tissue lysis buffer and organically extracted (Method Two), then quantified using a DU - 520 UV - Visible Spectrophotometer (Beckman Coulter Inc., Brea, CA). Serial dilutions (1:3) were created using low TE containing 20 µg/m L glycogen, making standard concentrations of 50, 16.67, 5.56, 1.85, 0.62, 0.21, 0.069, and 0.023 ng/µL. Real - time PCR reactions were set up in 0.2 mL optically clear flat - capped PCR strips ( USA Scientific ® , Ocala, FL) in a 15 µL volume. Concentrations of ingredients are listed in Table 8. The recipe was based on Lindquist et al. (2011), with modification by Hebda (2013) . DNA standards were ran in duplicate. Cycling parameters used are listed in Table 9. Standard curves were generated by ystem Software, and were used to calculate the DNA concentration (ng/µL) of each extract. DNA concentration was standardized by calculating real - time PCR concentration (ng/µL), multiplied by total extract volume (µL), and divided by the mass of bone powde r (mg) to form nanograms DNA recovered per milligram bone powder (ng/mg). 29 Table 7: Primer and Probe Sequences for qPCR Primer Name Sequence Amplicon Length F Cow ATPase 8 5 - CAA AAC ACC CCT TGA GAA ACA - 3 88 bp R Cow ATPase 8 5 - AGG GTT ACG AGA GGG AGA CC - 3 Cow ATPase 8 probe 5 - 6FAM - CCT CTT TTA TTA CCC CTG TAA TTT T - BHQ1 - 3 F Cow MC1R 5 - AAT AAA TCA TAA ACC AGC CTG CTC TTC ATC AC - 3 77 bp R Cow MC1R 5 - AAT AAA TCA TAA AGC TAT GAA GAG GCC AAC GA - 3 Cow MC1R probe 5 - 6FAM - CAC AAG GTC ATC CTG CTG TGC C - MGBNFQ - 3 F Pig ATPase 5 - AGC TCT GAT CCA AGC TTA TGT GT - 3 83 bp R Pig ATPase 5 - GCA TGT GTT TGG TGG GTC A - 3 Pig ATPase probe 5 - 6FAM - TGC TAG TAA GCT TAT ACC TAC ACG ACA - BHQ1 - 3 F Pig MC1R 5 - GCC CGG TTC CTA CGT G - 3 82 bp R Pig MC1R 5 - AGA GGG TCC AGC GTC CAT A - 3 Pig MC1R probe 5 - 6FAM - CGG GCC GGA CAT CTC TGA - BHQ1 - 3 F IPC 5 - AAG CGT GAT ATT GCT CTT TCG TAT AG - 3 77 bp R IPC 5 - ACA TAG CGA CAG ATT ACA ACA TTA GTA TTG - 3 IPC probe 5 - VIC - TAC CAT GGC AAT GCT - MGBNFQ - 3 IPC templa te 5 - AAG CGT GAT ATT GCT CTT TCG TAT AGT TAC CAT GGC AAT GCT TAG AAC AAT ACT AAT GTT GTA ATC TGT CGC TAT GT - 3 30 Table 8: Real - time PCR Reaction Recipe and Concentrations Ingredient Volume (WC) added per 15 µL Reaction Working Concentration (WC) F inal Concentration (Bio - Rad Laboratories) 7.5 µL 2× 1× Forward Primer ( ATPase or MC1R ) 0.9 µL 10 µM 600 nM Reverse Primer ( ATPase or MC1R ) 0.9 µL 10 µM 600 nM Target Probe ( ATPase or MC1R ) 0.25 µL 15 µM 250 nM Forward Primer IPC 0.75 µL 2 0 µM 1 µM Reverse Primer IPC 0.75 µL 20 µM 1 µM IPC Probe 0.25 µL 15 µM 250 nM IPC Template DNA 1.0 µL 1:1 billion dilution of 100 µM 1:66.7 billion dilution of 100 µM Taq DNA Polymerase (Syzygy) 0.125 µL 5 U/µL 0.625 Units Milli - Q ® Water 1.325 µL - - DNA extract / Standard DNA 1.2 µL - - Table 9: qPCR Thermal Cycler Parameters Temperature Time Cycles 95°C 3 min 1 95°C 15 s 50 60°C 1 min Qualitative PCR Assay for Bovine and Porcine DNA Extracts Primers that generate a ~200 bp amplicon (181 257 bps), ~400 bp amplicon (390 457 bps), ~600 bp amplicon (599 642 bps), and ~1,000 bp amplicon (989 1017 bps) were utilized to evaluate DNA quality of recovered bovine and porcine mitochondrial and nuclear DNAs. Primers, sequences, and amplicon s izes are summarized in Table 10. Bovine and porcine primers were designed using Primer3 targeting bovine ATPase (BLAST Accession NC_006853), porcine ATPase (BLAST Accession NC_00845.1), and bovine MC1R (BLAST Accession 31 AC_000175) gene sequences. Porcine nuclear DNA primers targeting the Insulin Growth Factor - 1 gene ( IGF - 1 ) were designed by Michaud and Foran (2011). MtDNA quality testing began with the ~1,000 bp amplicon. Failed amplification resulted in successive testing of primer sets that produce smal ler amplicons until amplification occurred. Nuclear DNA amplification began at the ~400 bp target, and was tested stepwise until successful amplification was no longer attained, or achieved when the ~400 bp target failed to amplify. Ten microliter PCR re actions consisted of: 1 µ L of GeneAMP 10 × PCR Buffer II (Applied Biosystems, Carlsbad, CA), 1 µL of 25 mM MgCl 2 (Applied Biosystems), 1 µL of 20 µM F and R primer, 1 µL of 2 mM deoxynucleotide 5 - triphosphates, 0.2 µL of AmpliTaq Gold ® DNA Polymerase (5 U/ µL, Applied Biosystems), 4 µL of Milli - Q ® filtered water, and 1 µL of template DNA diluted to ~1 ng/µL based on quantification assay data. PCR cycling parameters are summarized in Table 11. Amplified products were assessed via agarose gel electrophoresis. Five microliters of PCR products were separated on a 1% agarose gel stained in ethidium bromide (Sigma - Aldrich). Gels were photographed using an Olympus C - 4000 Zoom digital camera (Olympus, Center Valley, PA). Digital photographs were labeled using Ado using an UP - D895 thermal printer on UPP - 110S thermal print media (Sony Corporation, Tokyo, 32 Table 10: Primer Sequences for Qua litative PCR Assay Primer Name Sequence Amplicon Length F Cow MC1R 200 bp 5 - CAA GGA CTT CAT GAC CAG CA - 3 200 bp R Cow MC1R 200 bp 5 - TAC TGC TGC ACT GCT TCC TG - 3 F Cow MC1R 400 bp 5 - CTG CTG GGT TCC CTT AAC TG - 3 410 bp R Cow MC1R 400 bp 5 - ATG GAG ATG TAG CGG TCC AC - 3 F Cow MC1R 600 bp 5 - GTG GAC CGC TAC ATC TCC AT - 3 599 bp R Cow MC1R 600 bp 5 - CCT CTT TGT CAA GGG ACT GC - 3 F Cow MC1R 1 kb 5 - CTG CTG GGT TCC CTT AAC TG - 3 989 bp R Cow MC1R 1 kb 5 - CCT CTT TGT CAA GGG ACT GC - 3 F Cow ATPas e 200 bp 5 - TCG CTT TGT AAC CCT CCA AC - 3 201 bp R Cow ATPase 200 bp 5 - GGG ATG GCT ATG CCT AGG TT - 3 F Cow ATPase 400 bp 5 - CGA CAA AGC TGA CCC ATA CA - 3 390 bp R Cow ATPase 400 bp 5 - CTG GGA TTG CGT CTG TTT TT - 3 F Cow ATPase 600 bp 5 - CTG TGA GCA GGA GCC GTA AT - 3 607 bp R Cow ATPase 600 bp 5 - ATT CCA TAA CGG AGG CCT TT - 3 F Cow ATPase 1 kb 5 - CCC GCC ATC ATC TTA ATT CT - 3 994 bp R Cow ATPase 1 kb 5 - TGG TGT GAA TGA ATG GGG TA - 3 IGF - 1 Forward 5 - AAT CAT TTG CCC CTC AAG TG - 3 N/A IGF - 1 R257 5 - TGA CCC CCT CAT CCT AGT TG - 3 257 bp IGF - 1 R457 5 - GGC AGG AAG ACA CAC ACA TC - 3 457 bp IGF - 1 R642 5 - TCT CTC CCT CTT CTG GCA AA - 3 642 bp F Pig ATPase 5 - TGG ATC AAA CCA CAG CTT CA - 3 N/A R Pig ATPase 200 bp 5 - TGT GGA TGT ATC TAG TTG TGG CAT A - 3 181 bp R Pig ATPase 400 bp 5 - TGG GAA TAG TAA GCT TGG GAA T - 3 414 bp R Pig ATPase 600 bp 5 - TGG TGG GTG TGA ATG AGT GT - 3 604 bp R Pig ATPase 1 kb 5 - CAT GTG TTT GGT GGG TCA TT - 3 1017 bp Table 11: PCR Thermal Cycler Parameters Annealing temperature varied among primer sets. Bovine ATPase 8 and MC1R primers used 58 ° C, porcine ATPase used 60 ° C, and IGF - 1 used 56 ° C. Temperature Time Cycles 94° C 10 min 1 94° C 30 s 38 56°C 60°C 1 min 72° C 45 s 72° C 5 min 1 4° C Infinite Hold 33 Ancillary Experiments Changes in the Recoverable Total DNA of Buried Bovine Bone Segments over a One Month Time Period Two fresh femora from four year old Holstein dairy cows were obtained from the MSU Meats Laboratory, and segmented using an electric butchers saw a t their facility. Two segment types were derived from femoral diaphysis: one was quartered longitudinally and cut into ~1 in segments (type A), while the other was hemisected longitudinally and cut into ~3 in long segments (type B). Bone segments were st ored at - 80 ° C prior to burial. The 3 in segments underwent a weekly cycle of exhumation, testing, and reburial, while the 1 in segments were exhumed, tested, and stored at - 20 ° C. Exterior soft tissue was excised/scraped off using a razorblade, and marrow was removed. Segments were buried in 8 12 in of soil near Giltner Four bone segments were drilled per time point. Burial and retrieval dates are shown i n Table 12. Segments were cleaned using RO water and a brush to remove soil and debris prior to drilling. Bone powders from drillings were digested in tissue lysis buffer, organically extracted and quantified. DNA extracts were stored at - 80 °C. 34 Fig ure 7: Burial Site for Bovine Bone Segments Path leading to picnic area, red box oriented towards burial site (Middle). Undisturbed burial site for bone segments within picnic area (Right). Figure 8: Giltner Hall Burial Site with Bovine Diaphysis Segments Buried bone segments that were exhumed, tested, and reburied on a weekly basis (left). Partitioned burial site containing bone segments exhumed, tested, and then stored (right). Burial sites between segment types were adjacent to one another. 35 Table 12: Burial Dates of Bovine Femora Segments Segments identified by burial time (0D, 2D, 4D, 1W, 11D, 2W, 3W, 4W) and by replicate (1 4), where D = day(s), W = we ek(s). Segments denoted with (A) were retrieved, drilled, and stored at - 20 ° C. Segments denoted with (B) were cyclically retrieved, drilled, and reburied. Segment Identifier Date Buried Date Retrieved Number of Days Buried 0D - 1 4 (A) Not Buried 9/19/2 013 0 2D - 1 4 (A) 9/19/2013 9/21/2013 2 4D - 1 4 (A) 9/19/2013 9/23/2013 4 1W - 1 4 (A) & (B) 9/19/2013 9/26/2013 7 11D - 1 4 (A) 9/19/2013 9/30/2013 11 2W - 1 4 (A) & (B) 9/19/2013 10/3/2013 14 3W - 1 4 (A) & (B) 9/19/2013 10/10/2013 21 4W - 1 4 ( A) & (B) 9/19/2013 10/17/2013 28 Changes in the Recoverable Total DNA of Non - Buried Bovine Bone Segments over a One Month Time Period Four fresh hemisected segments of bovine femoral diaphysis were retrieved from - 80 ° C storage. External soft tissue was excised using a razorblade, and marrow removed. Bones were placed in labeled weigh boats, and placed in vacant office room. Bone segments were drilled for bone powder on the dates listed in Table 13. Powder from drillings was collected, digested in tiss ue lysis buffer, organically extracted, and quantified. DNA extracts were stored at - 80 °C. 36 Table 13: Exposure Dates of Bovine Femora Segments Segments identified by time exposed (0D, 2D, 4D, 1W, 10D, 2W, 3W, 4W) and by replicate (1 4), where D = da y(s), W = week(s). Segment Identifier Dates Tested & Returned Days Exposed Segment Identifier Dates Tested & Returned Days Exposed 0D - 1 11/5/2013 0 10D - 1 11/15/2013 10 0D - 2 10D - 2 0D - 3 10D - 3 0D - 4 10D - 4 2D - 1 11/7/2013 2 2W - 1 11/19/2013 14 2D - 2 2W - 2 2D - 3 2W - 3 2D - 4 2W - 4 4D - 1 11/9/2013 4 3W - 1 11/26/2013 21 4D - 2 3W - 2 4D - 3 3W - 3 4D - 4 3W - 4 1W - 1 11/12/2013 7 4W - 1 12/3/2013 28 1W - 2 4W - 2 1W - 3 4W - 3 1W - 4 4W - 4 Organic versus : Total DNA Yield C omparisons Over One Week A segment of fresh bovine diaphysis was taken from - 80°C storage, defleshed, and tested at 0, 2, 5, and 7 days. The diaphysis segment was stored in a vacant office for the one week experiment period. Two hundred milligrams of powdered bone, taken at each time point, was modifications used in Hebda (2013), with one alteration: after soil lysis buffer incubation, the microcentrifuge tube was centrifuged for 2 min at 10,000 × g, followed by transfer of all supernatant to a new tube. DNA extracts were quantified then stored at - 80°C. 37 Mass Difference between Wet and Dry Bone A segment of bovine femoral diaphysis was weighed over time to compare mass deference between hydrated versus dehydrated bone. External soft tissue was excised using a razorblade, and marrow removed. Approximately 500 milligrams of bone powder was evenly distributed among four storage containers tested in duplicate, along with the remaining bone segment, and are listed in Table 14. Materials were stored in the CleanSpot PCR/UV Work Station for the du ration of the experiment. Microcentrifuge tubes and weigh boats were weighed prior to and after the addition of bone powder, and subsequently weighed along with the remaining bone segment for a two week period at time points listed in Table 15. Table 1 4: Environmental Conditions Influence on Bone Mass Change Treatment Description 1 Closed 1.5 mL microcentrifuge tube 2 Open 1.5 mL microcentrifuge tube 3 Powder in condensed pile in weigh boat 4 Powder spread along surface of weigh boat 5 Diaphysis se gment minus extracted powder Table 15: Time Points Tested for Bone Mass Change and correspond to Table 14. Time Point Treatment Weighed Time Point Treatme nt Weighed 0 min 1 5 23.5 hours 1 5 30 min 1 5 48.5 hours 1 5 1 hour 1 5 75 hours 1 5 2 hours 1 5 100 hours 1 5 3 hours 1 5 175 hours 5 4 hours 1 5 250 hours 5 5 hours 1 5 325 hours 5 6 hours 1 5 38 Effect of Proteinase K Concentration on Total DNA Yields External soft tissue was excised using a razorblade, and morrow removed from a segment of fresh bovine diaphysis retrieved from - 80°C storage. Approximately one gram of bone powder was generated by drilling, homogenized, and then 50 mg +/ - 1 mg aliquots of powder were digested using demineralization or tissue lysis buffer with 0%, 0.5%, 1%, or 2% by total solution volume proteinase K (20 mg/mL) added. DNA was organically extracted and nuclear and mitochondrial DNAs were quantified in duplicate. Comparison of Total DNA Yields from Bovine Bones Macerated by MSU Forensic Anthropologist versus MSU Forensic Biologist C - 01 and C - 02 were immature bones based on incomplete fusion of the epiphyses; however, they were also proce ssed by the MSU Forensic Anthropology Laboratory. C - 03 C - 08 had completely fused epiphyses and were processed by the MSU Forensic Biology Laboratory. Assessing how DNA yields were affected by methodology, a pair of bovine femora and tarsals from a Hols tein steer were obtained from the MSU Meats Laboratory. One femur and tarsal set was given to the MSU Forensic Anthropology Laboratory for maceration using their standard protocols. The other femur and tarsal set was macerated at the MSU Forensic Biology - hour intervals. Bone powder (50 mg +/ - 1 mg) was recovered from post - macerated femora by drilling at the midshaft diaphysis, distal epiphysis, femoral head, and the calcanei, and tali. Powders were dig ested in tissue lysis and demineralization buffers, organically extracted, then mitochondrial and nuclear DNAs were quantified. 39 Statistical Analysis of Bovine/Porcine Intra - bone Variation and Tarsal Comparison Experiments Statistical analyses were perform ed using XLSTAT version 2014.2.01 (Addinsoft, New York, NY). Anderson - Darling and Shapiro - Wilk tests were used to determine normality. Kruskal - (two - tailed) for post hoc m ultiple pairwise comparisons of non - combined mitochondrial and nuclear DNA quantification data. Combined femoral quantification data were analyzed using Kruskal - - tailed). Regions were combined based on common features: Regions 1 6 (diaphysis), Regions 7 9 (metaphyses and articulating surface), Regions 10 12 (epiphyses), and Regions 13 14 (tarsals). Individual pairwise comparisons between combined femoral regions and tarsals, and differ ences in DNA quantity and quality between digestion buffers were done using Mann - Whitney U. DNA quality was ranked prior to utilizing Mann - Whitney U based on amplicon generated: 4 for ~1,000 bp, 3 for ~600 bp, 2 for ~400 bp, 1 for ~200 bp, and 0 for no am plification. Statistical significance 40 RESULTS Qualitative Observations from Processing Porcine and Bovine Femora and Tarsals Post maceration, differences in age were detectable among bones. Bovine C - 01 and C - 02 femora were immature, based on incomplete fusion of the distal epiphysis and non - fused femoral head and trochanter. C - 03 C - 08 were older, determined by the complete fusion of the distal and proximal epiphyses. Porcine P - 01 P - 08 were immature, containing non - fus ed distal and proximal epiphyses. As bones from both species were stored, their exterior surface became greasy, beginning at the epiphyses/metaphyses and migrating towards the midshaft diaphysis. Drilling porcine bones was easier than their bovine counter parts, which were harder and required greater force to acquire the necessary powder. Conversely, drilling the porcine epiphyses required a soft touch to obtain powder from the thin layer of cortical bone. This was achieved by taking the tip of the drill bit (while the device was on ) and lightly touching the Qualitatively, there were often region dependent differences in color and consistency of the bone powder. Regio ns 1 6 and 9, in both species, generally produced a dry fine grain white powder. Porcine Regions 7, 8, and 10 12 often produced a darker brown powder (not due to thermal damage). Bovine bone powder from Regions 7, 8, and 10 12 had a white to yellow ish coloration with larger grains and appeared somewhat greasy. Regions 13 and 14 had an intermediate appearance to Regions 1 6 and 9, and that of Regions 7, 8, and 10 12. Environmental exposure affected characteristics of the bones both visually and physically. Soft tissue and cartilage on non - buried bone dehydrated and hardened around the element and persisted beyond the six month experiment window. Soft tissue on buried bone 41 putrefied rapidly during the first month, and largely vanished by three m onths. Exposed and buried bones had insect activity present during the first month of environmental treatment. Decomposition progressed enough by week 4 to determine that replicates A2/B2 were immature bones based on incomplete fusion of the epiphyses. Buried bones had a brownish discoloration bones. Drilling non - buried bone was similar to fresh bone, while buried bone was easier to drill after one week, which w as maintained throughout. Figure 9 depicts differences between digestion buffers using bone powder from an epiphysis. Bone powder remained in tissue lysis buffer, collecting at the bottom of the aqueous/organic mixture. Conversely, demineralization buff er dissolved the bone completely. Bone powder from the epiphyses and metaphyses often formed a white interface between the aqueous and organic phases, whereas the diaphysis did not. This interphase occurred using both buffers; however, this was more pron ounced in tissue lysis extractions. 42 Figure 9: Organic Extraction of Epiphyseal Bone Powder Picture on the left is an example of bone powder digested in tissue lysis buffer; note the undigested powder at the bottom of the m icrocentrifuge tube, and the interface between layers. Picture on the right is from the same stock of bone powder digested in demi neralization buffer, it contains a less defined interface and no bone powder. Inter - Bone and Intra - Bone Variation of Recover able Total DNA in Fresh Porcine and Bovine Femora and Tarsals Normality Testing of Porcine and Bovine Bones DNA quantity data from some regions of fresh porcine and bovine bone did not follow a normal distribution. Porcine quantification data had mitoch ondrial and nuclear DNA yields that were not normally distributed, though nuclear DNA had more, and data from DNAs isolated in tissue lysis buffer were more likely than demineralization buffer to be non - normally distributed. Individual p - values from norma lity testing of DNA quantification data derived from porcine bones, per region and digestion buffer, are listed in Appendix A1. Bovine DNA quantity data were not normally distributed in about half the regions for both mitochondrial and nuclear DNA using b oth buffers. There were more non - normally Aqueous Layer (Tissue Lysis Buffer) Interphase Organic Layer Bone Powder Aqueous Layer (D emineralizati on Buffer) Interphase Organic Layer 43 dis tributed regions of nuclear than mitochondrial DNA data . Nuclear DNA data were more likely to be non - normally distributed a t the epiphyses, while mtDNA data were at the diaphysis. Combined regional quantifica tion data were not normally distributed in all but one instance, but only from the Anderson - Darling test: mtDNA from the tarsals digested in tissue lysis buffer (p = 0.069). C - 01 and C - 02 contributed substantially to these non - normal distributions; when th ey were removed from analysis only mtDNA recovered from Region 6 (p < 0.04) and nuclear DNA from Region 4 (p < 0.04) digested in demineralization buffer were not normally distributed. Combined mtDNA yields from the diaphysis digested in both buffers (p < 0.04), and DNA from the epiphyses digested in demineralization buffer (p < 0.03) were not normal. MtDNA from the metaphyses and articulating surface digested in tissue lysis buffer had inconsistent significance between Anderson - Darling (p = 0.060) and Sha piro - Wilk (p = 0.019) tests. Individual p - values from normality testing of DNA quantification data derived from bovine bones, per region and digestion buffer, are listed in Appendix A2 (with C - 01 and C - 02) and A3 (without C - 01 and C - 02). Quantification of Total DNA from Fresh Porcine Femora and Tarsals Median mtDNA yields from porcine bone powder digested in tissue lysis (Table 16) and demineralization buffer (Table 17) were lowest in Regions 1 4, with higher quantities along regions of the diaphysis c loser to the proximal and distal metaphyses. The exception to this was Region 9, which had the highest mtDNA yields from the diaphysis. Regions 10 12 had higher mtDNA yields than Regions 1 6, 13, and 14. Regions 13 and 14 had similar yields as Regio ns 7 9. These results are graphically depicted in Figure 10. Combined regional data for mtDNA 44 yields from bone digested in tissue lysis and demineralization buffers, listed in Table 18 and graphically depicted in Figure 11 were highest at the epiphyses , followed by intermediate yields from the tarsals, metaphyses and articulating surface, and the diaphysis had the lowest yields. Individual values for these data, including normalization parameters and total mtDNA recovered, are available in Appendix B1 B16. Efficiencies of qPCR ranged from 93.6 to 100.0 as calculated from the slopes of the standard curves, with R 2 - values ranging from 0.976 to 0.994. No PCR inhibition was detected via the IPC. 45 Table 16: mtDNA Yields from Fresh Porcine Bones Digested i n Tissue Lysis Buffer Regions and concomitant locations correspond to Table 3. P - (01 08) are biological replicates. MtDNA quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviation (MAD) for variation in data. Regions 1 12 reside on the femur; the calcaneus and talus (Regions 13 and 14) were tested as whole elements. NT = Not Tested Region Location on Element mtDNA Yie ld (ng/mg) P - 01 P - 02 P - 03 P - 04 P - 05 P - 06 P - 07 P - 08 Median MAD 1 Midshaft Diaphysis 5.31 11.98 16.96 6.66 7.39 16.85 10.25 18.62 11.12 5.09 2 Midshaft Diaphysis 3.74 5.06 NT* 5.30 20.55 13.09 11.52 16.05 8.41 6.34 3 Proximal Diaphysis 9.55 5.81 25.93 7.60 10.12 14.94 16.49 15.81 12.53 3.62 4 Distal Diaphysis 4.45 5.60 15.41 10.66 9.05 12.45 8.05 14.07 9.86 3.40 5 Proximal Diaphysis 18.35 9.63 40.84 35.94 40.37 29.07 41.35 28.39 32.50 8.10 6 Distal Diaphysis 21.13 6.90 25.13 17.74 16.87 23.59 19.61 2 3.14 20.37 2.99 7 Proximal Metaphysis 31.02 29.26 73.14 51.82 52.69 40.88 44.89 28.27 42.89 10.83 8 Distal Metaphysis 31.14 25.54 43.75 45.85 37.95 43.70 39.36 33.26 38.66 5.25 9 Articulating Surface 59.76 32.35 72.24 38.66 36.21 54.62 46.53 54.55 50.54 10.55 10 Distal Epiphysis 323.92 146.67 175.70 163.77 205.20 156.82 110.17 110.71 160.30 30.15 11 Femoral Head 175.06 85.60 98.67 91.27 171.20 86.59 104.39 86.94 94.97 8.88 12 Trochanter 172.04 89.80 179.59 89.85 202.88 157.48 198.45 151.96 164.76 24.2 6 13 Calcaneus 43.44 39.10 55.77 38.88 73.71 32.47 53.04 36.66 41.27 6.70 14 Talus 82.60 51.52 67.76 51.54 53.25 57.60 69.41 44.31 55.43 7.51 * Microcentrifuge Tube for P - 03 Region 2 digested in tissue lysis buffer ruptured during incubation and the sup ernatant was lost and as a result not tested. 46 Table 17: mtDNA Yields from Fresh Porcine Bones Digested in Demineralization Buffer Regions and concomitant locations correspond to Table 3. P - (01 08) are biological replicates. MtDNA quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviation (MAD) for variation in da ta. Regions 1 12 reside on the femur; the calcaneus and tal us (Regions 13 and 14) were tested as whole elements. Region Location on Element mtDNA Yield (ng/mg) P - 01 P - 02 P - 03 P - 04 P - 05 P - 06 P - 07 P - 08 Median MAD 1 Midshaft Diaphysis 13.00 16.35 23.69 11.51 18.35 17.84 17.12 23.89 17.48 2.80 2 Midshaft Diaphysis 10.53 14.99 22.73 13.73 26.62 22.13 13.26 25.16 18.56 5.06 3 Proximal Diaphysis 14.27 8.73 18.18 12.77 12.54 19.14 18.92 20.73 16.23 3.19 4 Distal Diaphysis 12.98 9.57 20.69 14.50 15.44 23.24 19.98 18.63 17.04 3.30 5 Proximal Diaphysis 22.47 12.28 46.87 27.40 48.31 29.38 32.52 23.60 28.39 5.36 6 Distal Diaphysis 18.61 10.53 19.08 14.53 19.15 24.86 21.49 19.11 19.10 1.44 7 Proximal Metaphysis 36.51 36.00 79.82 33.56 50.19 44.95 53.41 25.69 40.73 8.32 8 Distal Metaphysis 35.22 27.27 53.68 37.17 43.89 35.96 44.60 27.09 36.56 7.68 9 Articulating Surface 73.80 46.96 49.29 37.48 54.25 61.94 57.07 68.33 55.66 7.54 10 Distal Epiphysis 195.14 97.61 122.45 111.18 439.92 134.64 104.00 111.47 116.96 15.32 11 Femoral Head 242.18 131.82 141.80 84.48 150.54 90.24 72.49 103.57 117.69 30.15 12 Trochanter 216.86 209.04 115.05 60.11 398.08 70.22 179.41 90.72 147.23 65.72 13 Calcaneus 50.64 43.17 57.44 45.66 69.71 35.26 45.28 35.39 45.47 7.63 14 Talus 67.43 53.05 64.20 38.38 59.79 36.26 60. 41 27.58 56.42 9.39 47 Figure 10: Median mtDNA Yields from Fresh Porcine Bones The x - axis lists the regions tested, while the y - axis is the median mtDNA yields in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer, and blue bars bone powder digested in demineralization buffer. Error bars represent median absolute deviation. Asterisk indicates significant differences between buffers. * * * 0.00 50.00 100.00 150.00 200.00 250.00 Median mtDNA Quantification (ng/mg) Tissue Lysis Buffer Demineralization Buffer 48 Table 18: Combined mtDNA Yields from Fresh Porcine Bones Digested in Tissue Lysis and Demineralization Buffers Combined regions listed in first column and correspond to Table 3. Regions combined from P - (01 08). MtDNA quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absol ute deviation (MAD) for variation in data. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. TL = tissue lysis buffer DM = demineralization buffer Combined Region s Median MAD TL DM TL DM 1 6 14.94 18.78 5.89 4.49 7 9 42.29 44.78 9.73 8.86 10 12 154.39 118.75 44.14 30.15 13 14 52.29 48.15 11.02 11.76 Figure 11: Combined Median mtDNA Yields from Fresh Porcine Bones The x - axis lists the regions test ed, while the y - axis is the median mtDNA yields in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer, and blue bars bone powder digested in demineralization buffer. Error bars represent median absolute deviation. Re gions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. Asterisk indicates significant differences between buffers. * 0.00 50.00 100.00 150.00 200.00 250.00 Regions 1 6 Regions 7 9 Regions 10 12 Regions 13 14 Median mtDNA Quantification (ng/mg) Tissue Lysis Buffer Demineralization Buffer 49 Median mtDNA yields differed significantly among regions of por cine bone digested in tissue lysis and demineralization buffers (p < 0.0001). P - values for multiple pairwise comparisons are listed in Appendix A4 and A5. Pairwise comparisons of mtDNA yields derived from bone digested in tissue lysis and demineralizatio n buffers are depicted in Figures 12 and 13 respectively. Regions 10 12 had significantly more mtDNA than Regions 1 6, 7, and 8 (p = 0.044 0.0001). Region 9 had significantly more mtDNA than Regions 1 4, and 6 (p = 0.025 0.0001), and significan tly less than Regions 10 and 12 (p = 0.035, 0.041); however, the latter result was inconsistent between buffers. Regions 10 (p = 0.127) and 12 (p = 0.159) were not significantly different from Region 9 with bone powder digested in demineralization buffer. Regions 13 and 14 had significantly more mtDNA than Regions 1 4 and 6 (p = 0.041 0.0001). Region 13 had significantly less mtDNA than Regions 10 and 12 (p = 0.049 0.021). MtDNA recovered from Regions 13 and 14 were not significantly different fro m each other (p = 0.461, 0.817), or among Regions 7 9 (p = 0.203 0.846). Significantly more mtDNA was recovered from Regions 1, 2, and 4 digested in demineralization buffer based on individual pairwise comparisons between buffers (p = 0.050, 0.040, an d 0.007). 50 Figure 12: Pairwise Comparisons of mtDNA Yields from Fresh Porcine Bones Digested in Tissue Lysis Buffer The x - axis lists the regions tested (n = 8), while the y - axis is the median mtDNA yields normalized in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer. Regions that have the same letter did not differ significantly in mtD NA yields, while regions with different letters did. *Region 2 (n = 7) f c d a b c d e a a b* a b a b b c d c d e f f d e d e f 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00 180.00 Median mtDNA Quantification (ng/mg) Tissue Lysis Buffer 51 Figure 13: Pairwise Comparisons of mtDNA Yields from Fresh Porcine Bones Digested in Demineralization Buffer The x - axis lists the regions tested (n = 8), while the y - axis is the median mtDNA yields normalized in ng per mg bone powder. Blue bars represent bone powder digested in demineralization buffer. Re gions that have the same letter did not differ significantly in mtDNA yields, while regions with different letters did. d b a b c d a a a a a b b c d d b c b c d 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00 Median mtDNA Quantification (ng/mg) Demineralization Buffer 52 Combined regional porcine mtDNA yields differed significantly among the femoral diaphysis, metaphyses and articulating surface, and ep iphyses digested in both buffers (p < 0.0001). P - values for multiple pairwise comparisons of the femur and individual pairwise comparison between tarsals and femora are listed in Appendix A6. Pairwise comparisons of combined mtDNA yields derived from bon e digested in tissue lysis and demineralization buffers are depicted in Figures 14 and 15 respectively. Combined regions were significantly different at a Bonferroni corrected significance level of = 0.0167. The epiphyses had significantly higher mtDNA yields than the diaphysis (p < 0.0001), and metaphyses and articulating surface (p = 0.002 0.001) digested in both buffers. The metaphyses and articulating surface had significantly higher mtDNA yields than the diaphysis (p < 0.0001). The tarsals had significantly higher yields than the diaphysis (p < 0.0001), and significantly lower than the epiphyses (p < 0.0001). Comparisons between the tarsals and metaphyses and articulating surface were inc onsistent between buffers, where the tarsals had significantly higher mtDNA yields when digested in tissue lysis buffer (p = 0.041), but not in demineralization buffer (p = 0.420). Significantly more mtDNA was recovered from diaphysis digested in deminera lization buffer versus tissue lysis buffer (p = 0.008). There was no significant difference between buffers for the metaphyses and articulating surface (p = 0.533), epiphyses (p = 0.675), or tarsals (p = 0.491). 53 Figure 14: Pairwise Comparisons of Combi ned mtDNA Yields from Fresh Porcine Bones Digested in Tissue Lysis Buffer The x - axis lists the regions tested, while the y - axis is the combined mtDNA yields normalized in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buf fer. Regions that have the same letter did not differ significantly in mtDNA yields, while regions with different letters did. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the ta rsals. A B D C 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00 180.00 Regions 1 6 Regions 7 9 Regions 10 12 Regions 13 14 Median mtDNA Quantification (ng/mg) Tissue Lysis Buffer 54 Figure 15: Pairwise Comparisons of Combined mtDNA Yields from Fresh Porcine Bones Digested in Demineralization Buffer The x - axis lists the regions tested, while the y - axis is the median mtDNA yields normalized in ng per mg bone powder. Blue bars represent bone powder digested in demineralization buffer. Regions that have the same letter did not differ significantly in mtDNA yields, while regions with different letters did. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articula ting surface, 10 12 the epiphyses, and 13 14 the tarsals. A B C B 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 Regions 1 6 Regions 7 9 Regions 10 12 Regions 13 14 Median mtDNA Quantification (ng/mg) Demineralization Buffer 55 Nuclear DNA yields from porcine bone digested in tissue lysis (Table 19) and demineralization buffer (Table 20) were lowest in Regions 1 4, with increased DNA along regions of the dia physis closer to the proximal and distal metaphyses. The exception to this was Region 9, which had yields higher than the diaphysis and similar to the metaphyses. Regions 10 12 had higher yields than Regions 1 9, 13, and 14. Regions 13 and 14 had si milar yields as Regions 7 9. Tissue lysis and demineralization buffers had similar variation in quantity among regions; however, bone digested in demineralization buffer had higher DNA yields from Regions 1 4, while Regions 9 12 had higher yields fr om bone digested in tissue lysis buffer. Figure 16 graphically depicts median nuclear DNA yields for both buffers. Combined nuclear DNA yields from bone digested in tissue lysis and demineralization buffers are listed in Table 21 and graphically depicted in Figure 17. The epiphyses had the highest DNA yields, followed by intermediate yields from the metaphyses and articulating surface as well as the tarsals, and the diaphysis had the lowest. Individual values for these data, including normalization para meters and total DNA recovered are available in Appendix B17 B32. Efficiencies of qPCR ranged from 89.9 to 100.9 as calculated from the slopes of the standard curves, with R 2 - values ranging from 0.982 to 0.993. No PCR inhibition was detected via the IP C. 56 Table 19: Nuclear DNA Yields from Fresh Porcine Bones Digested in Tissue Lysis Buffer Regions and concomitant locations correspond to Table 3. P - (01 08) are biological replicates. Nuclear DN A quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviation (MAD) for variation in data. Regions 1 12 reside on the femur; the calcaneus and talus (Regions 13 and 14) were tested as whole elements. NT = Not Tested Region Location on Element Nuclear DNA Yield (ng/mg) P - 01 P - 02 P - 03 P - 04 P - 05 P - 06 P - 07 P - 08 Median MAD 1 Midshaft Diaphysis 40.06 10.71 6.26 2.44 6.27 6.86 7.86 13.62 7.36 2.22 2 Midshaft Diaphysis 34.34 6.92 NT* 1. 60 20.72 3.22 13.52 10.98 10.98 8.57 3 Proximal Diaphysis 76.41 19.58 19.14 2.01 8.80 11.17 20.01 13.07 16.10 4.42 4 Distal Diaphysis 47.94 13.29 8.88 3.06 12.65 3.12 4.43 8.51 8.70 4.43 5 Proximal Diaphysis 187.65 45.47 302.45 73.53 184.25 91.00 484.30 214.37 185.95 103.68 6 Distal Diaphysis 189.04 57.24 124.02 69.43 51.97 99.11 242.21 161.57 111.56 52.16 7 Proximal Metaphysis 384.06 225.84 720.00 237.18 132.55 110.32 672.35 163.44 231.51 110.07 8 Distal Metaphysis 236.88 161.28 325.65 71.50 71.50 96 .63 348.92 225.71 193.50 109.43 9 Articulating Surface 552.24 256.47 554.69 253.80 263.65 375.33 694.30 584.08 463.79 160.22 10 Distal Epiphysis 151.67 1482.35 3192.00 2389.71 1033.60 1859.59 2203.47 2571.43 2031.53 544.54 11 Femoral Head 1129.41 464.00 1690.39 2841.18 872.00 978.82 835.10 1028.78 1003.80 150.25 12 Trochanter 1312.40 1338.78 3193.47 2204.90 1913.60 1973.16 1720.82 2713.06 1943.38 433.06 13 Calcaneus 397.88 345.44 389.74 301.18 552.00 161.47 366.18 405.84 377.96 30.20 14 Talus 571.90 5 28.80 608.63 421.80 365.59 384.00 416.47 440.22 431.01 56.22 * See Table 16 57 Table 20: Nuclear DNA Yields from Fresh Porcine Bones Digested in Demineralization Buffer Regions and concomitant loc ations correspond to Table 3. P - (01 08) are biological replicates. Nuclear DNA quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviation (MAD) for variation in data. Regions 1 1 2 reside on the femur; the calcaneus and talus (Regions 13 and 14) were tested as whole elements. Region Location on Element Nuclear DNA Yield (ng/mg) P - 01 P - 02 P - 03 P - 04 P - 05 P - 06 P - 07 P - 08 Median MAD 1 Midshaft Diaphysis 145.50 148.96 130.21 82.60 113.53 86.20 165.00 126.46 128.33 18.90 2 Midshaft Diaphysis 142.29 95.33 161.41 124.87 131.45 88.94 119.08 128.70 126.79 11.61 3 Proximal Diaphysis 149.06 105.85 115.71 98.61 124.39 84.70 163.71 108.31 112.01 12.89 4 Distal Diaphysis 150.45 111.98 176. 16 126.72 116.20 91.84 161.40 118.80 122.76 19.24 5 Proximal Diaphysis 225.88 114.29 319.00 157.04 254.04 106.41 225.76 161.15 193.46 48.50 6 Distal Diaphysis 136.53 110.00 171.60 130.00 120.50 110.88 250.29 131.29 130.65 14.96 7 Proximal Metaphysis 281 .10 224.12 520.01 261.00 252.71 184.44 361.80 131.39 256.86 52.58 8 Distal Metaphysis 289.52 262.09 245.16 202.04 277.54 153.18 343.53 182.86 253.62 43.74 9 Articulating Surface 486.00 325.10 238.76 257.29 207.08 167.29 348.58 182.39 248.03 71.36 10 Dis tal Epiphysis 1029.02 968.63 783.67 1050.00 479.47 508.86 702.00 629.06 742.84 229.88 11 Femoral Head 1156.76 951.60 852.24 772.20 712.92 577.16 624.98 789.80 781.00 113.63 12 Trochanter 1207.84 850.20 1209.50 1147.14 864.00 561.16 931.70 519.84 897.85 2 79.64 13 Calcaneus 457.14 295.10 482.99 423.03 323.43 218.10 335.57 274.90 329.50 74.07 14 Talus 432.27 513.59 416.40 352.78 491.14 219.26 457.14 315.06 424.33 69.18 58 Figure 16: Median Nuclear DNA Yields from Fresh Porcine Bones The x - axis lists the regions tested, while the y - axis is the median nuclear DNA yields in ng per mg of bone powder. Green bars represent bone powder digested in tissue lysis buffer, and blue bars bone powder digested in demineralization buffer. Error bars represent median ab solute deviation. Centered is a nested graph for increased resolution of Regions 1 4. Asterisk indicates significant differences between buffers. * * * * * * 0.00 500.00 1000.00 1500.00 2000.00 2500.00 3000.00 Median DNA Quantification (ng/mg) Tissue Lysis Buffer Demineralization Buffer * * * * 0.00 50.00 100.00 150.00 200.00 59 Table 21: Combined Nuclear DNA Yields from Fresh Porcine Bones Digested in Tissue Lysis and Demineralizat ion Buffers Combined regions listed in first column and correspond to Table 3. Regions are combined from P - (01 08). DNA quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviation (M AD) for variation in data. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. TL = tissue lysis buffer DM = demineralization buffer Combined Regions Median MAD TL DM TL DM 1 6 19.14 127.71 16.02 21.27 7 9 255.14 255.00 121.39 61.53 10 12 1705.60 820.00 674.42 192.98 13 14 401.86 384.59 37.32 72.55 Figure 17: Combined Median Nuclear DNA Yields from Fresh Porcine Bones The x - axis lists the regions tes ted, while the y - axis is the median nuclear DNA yields in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer, and blue bars bone powder digested in demineralization buffer. Error bars represent median absolute deviati on. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. Asterisk indicates significant differences between buffers. * * 0.00 500.00 1000.00 1500.00 2000.00 2500.00 Regions 1 6 Regions 7 9 Regions 10 12 Regions 13 14 Median DNA Quantification (ng/mg) Tissue Lysis Buffer Demineralization Buffer 60 Median porcine nuclear DNA yields differed significantl y among regions of porcine bone digested in tissue lysis and demineralization buffers (p < 0.0001). P - values for multiple pairwise comparisons are listed in Appendix A7 and A8. Pairwise comparisons of nuclear DNA yields derived from bone powder digested in tissue lysis and demineralization buffers are depicted in Figures 18 and 19 respectively. Regions 10 12 had significantly higher nuclear DNA yields than Regions 1 6 (p = 0.007 0.0001), and Regions 7 and 8 (p = 0.049 0.001). Region 9 had signif icantly higher yields than Regions 1 4 (p = 0.022 0.0001), and significantly lower than Regions 10, 11, and 12 (p = 0.027, 0.020, and 0.012); however, the latter result was only present in demineralization extracts. Regions 10, 11, and 12 were not sig nificantly different from Region 9 (p = 0.167, 0.214, 0.068) with bone digested in tissue lysis buffer. Regions 13 and 14 had significantly higher DNA yields than Regions 1 4 (p = 0.004 0.0001). Region 13 had significantly lower DNA yields than Regio n 12 (p = 0.030) for bone digested in tissue lysis buffer. DNA yields from Regions 13 and 14 were not significantly different from each other (p = 0.571), and neither differed significantly when compared to Regions 7 12 (p = 0.062 0.709). Utilizing i ndividual pairwise comparisons between buffers, significantly more DNA was recovered from Regions 1 4 digested in demineralization buffer (p = 0.0003 0.0001), and significantly more DNA was recovered from Regions 10 and 12 digested in tissue lysis buff er (p = 0.015, 0.0001). 61 Figure 18: Pairwise Comparisons of Nuclear DNA Yields from Fresh Porcine Bones Digested in Tissue Lysis Buffer The x - axis lists the regions tested (n = 8), while the y - axis is the median nuclear DNA yield normalized in ng per mg of bone powder. Green bars represent bone powder digested in tissue lysis buffer. Centered is a nested graph for increased resolution of Reg ions 1 4. Regions that have the same letter did not differ significantly in DNA yields, while regions with diff erent letters did. * Region 2 (n = 7) e f b c d a b c c d e f a a* a a b b c d c d e f f c d e d e f 0.00 500.00 1000.00 1500.00 2000.00 2500.00 Median DNA Quantification (ng/mg) Tissue Lysis Buffer a a* a a b 0.00 5.00 10.00 15.00 20.00 62 Figure 19: Pairwise Comparisons of Nuclear DNA Yields from Fresh Porcine Bones Digested in Demineralization Buffer The x - axis lists the regions tested (n = 8), while the y - axis is the median nuclear DNA yield norma lized in ng per mg of bone powder. Blue bars represent bone powder digested in demineralization buffer. Regions that have the same letter did not differ signif icantly in DNA yields, while regions with different letters did. e b c d a b c d a a a a a b c c d e e c d e d e 0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.00 Median DNA Quantification (ng/mg) Demineralization Buffer 63 Combined porcine nuclear DN A yields differed significantly among the femoral diaphysis, metaphyses and articulating surface, and epiphyses digested in both buffers (p < 0.0001). P - values for multiple pairwise comparisons of the femur , and individual pairwise comparisons between the tarsals and femora are listed in Appendix A6. Pairwise comparisons of combined nuclear DNA yields derived from bone digested in tissue lysis and demineralization buffers are depicted in Figures 20 and 21 respectively. Combined regions were significantly different at a Bonferroni corrected significance level of = 0.0167. The epiphyses had significantly higher DNA yields, for both buffers, than the diaphysis (p < 0.0001), and the metaphyses and articulating surface (p = 0.001). The metaphyses and articulating surface had significantly higher yields than the di aphysis (p < 0.0001) digested in both buffers. The tarsals had significantly higher DNA yields than the diaphysis (p < 0.0001), and the metaphyses and articulating surface (p = 0.035 0.002), but significantly lower than the epiphyses (p < 0.0001) for bo ne powder digested with both buffers. Comparing yields between buffers, significantly more nuclear DNA was recovered from the diaphysis digested in demineralization buffer (p < 0.0001), and the epiphyses digested in tissue lysis buffer (p < 0.0001). Ther e was no significant difference in DNA yields between buffers for the metaphyses and articulating surface (p = 0.688), or the tarsals (p = 0.376). 64 Figure 20: Pairwise Comparisons of Combined Nuclear DNA Yields from Fresh Porcine Bones Digested in Tissu e Lysis Buffer The x - axis lists the regions tested, while the y - axis is the median DNA yields normalized in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer. Regions that have the same letter did not differ signific antly in DNA yields, while regions with different letters did. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. A B D C 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 1400.00 1600.00 1800.00 Regions 1 6 Regions 7 9 Regions 10 12 Regions 13 14 Median DNA Quantification (ng/mg) Tissue Lysis Buffer 65 Figure 21: Pairwise Comparisons of Combined Nuclear DN A Yields from Fresh Porcine Bones Digested in Demineralization Buffer The x - axis lists the regions tested, while the y - axis is the median DNA yields normalized in ng per mg bone powder. Blue bars represent bone powder digested in demineralization buffer. Regions that have the same letter did not differ significantly in DNA yields, while regions with different letters did. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. Quality of Total DNA Recovered from Fresh Porcine Femora and Tarsals Mitochondrial and nuclear DNA PCR amplification results are individually listed in Appendix B33 B36. MtDNA isolated from bone digested in tissue lysis and demineralization buffers p roduced the 1017 bp amplicon, the largest tested, for all regions across all eight replicates. Nuclear DNA isolated from bone digested in tissue lysis and demineralization buffers produced the 642 bp amplicon, the largest tested, in all but nine instances . Extracts that failed to amplify the 642 bp amplicon, amplified the 457 bp amplicon. Seven of nine extracts that failed to produce the 642 bp amplicon were from P - 01: Regions 5, 6, and 12 from bone digested in A B D C 0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 Regions 1 6 Regions 7 9 Regions 10 12 Regions 13 14 Median DNA Quantification (ng/mg) Demineralization Buffer 66 tissue lysis buffer, and Regions 2, 6, 7, a nd 12 from bone digested in demineralization buffer. P - 08 Region 3 digested in demineralization buffer and P - 03 Region 7 digested in tissue lysis buffer also failed to produce the 642 bp amplicon. Digestion buffers did not significantly affect the qualit y of the mitochondrial or nuclear DNA recovered from porcine bone (p > 0.05). Reagent blanks and negative controls had no amplification. Quantification of Total DNA from Fresh Bovine Femora and Tarsals Median bovine mtDNA yields from bone powder digeste d in tissue lysis buffer (Table 22) and demineralization buffer (Table 23) were lowest at Regions 1 4, with increasing quantities along regions of the diaphysis closer to the proximal and distal epiphyses. The exception to this was Region 9 that had the highest yields from the diaphysis. Regions 10 12 had higher yields than Regions 1 9, 13, and 14. Regions 13 and 14 had slightly higher yields than Regions 7 9. Figure 22 graphically depicts median mtDNA yields for both buffers. Combined mtDNA yi elds from bone powder digested in tissue lysis and demineralization buffers, listed in Table 24 and graphically depicted in Figure 23 were highest at the epiphyses , followed by intermediate yields from metaphyses and articulating surface as well as the tar sals, and the diaphysis had the lowest yields. Individual values for these data, including normalization parameters and total mtDNA recovered are available in Appendix C1 C16. Efficiencies of qPCR ranged from 83.9 to 98.5 as calculated from the slopes of the standard curves, with R 2 - values ranging from 0.974 to 0.995. No PCR inhibition was detected via the IPC. 67 Table 22: mtDNA Yields from Fresh Bovine Bones Digested in Tissue Lysis Buffer Regions and concomitant locations correspond to Table 3. C - (01 08) are biological replicates. MtDNA quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviation (MAD) for variation in data. Regions 1 12 reside on the femur; the calcaneus and talus (Regions 13 and 14) were tested as whole elements. Region Location on Element mtDNA Yield (ng/mg) C - 01 C - 02 C - 03 C - 04 C - 05 C - 06 C - 07 C - 08 Median MAD 1 Midshaft Diaphysis 7.59 11.38 1.80 3.63 2.90 1.82 2.68 2.24 2.79 0.91 2 Midshaft Diaphysis 5.30 16.35 2.37 1.44 3.86 3.48 4.63 2.66 3.67 1.16 3 Proximal Diaphysis 8.30 22.84 2.36 2.58 4.01 1.92 5.60 3.64 3.83 1.62 4 Distal Diaphysis 7.63 11.69 2.73 2.15 5.88 4. 33 4.53 2.20 4.43 1.96 5 Proximal Diaphysis 5.92 16.04 1.89 2.40 4.83 4.27 4.59 4.04 4.43 0.94 6 Distal Diaphysis 5.78 11.54 2.60 3.04 5.72 5.55 6.29 2.94 5.63 1.63 7 Proximal Metaphysis 13.42 43.20 4.81 3.29 8.34 7.04 20.19 11.72 10.03 4.30 8 Distal M etaphysis 14.56 38.27 3.11 2.71 8.82 13.67 5.65 10.26 9.54 4.58 9 Articulating Surface 28.53 52.56 5.93 7.68 27.78 11.94 11.36 12.84 12.39 5.59 10 Distal Epiphysis 62.40 102.90 12.89 20.36 23.02 37.41 28.64 38.45 33.03 11.34 11 Femoral Head 50.89 85.96 11.51 9.48 26.59 26.22 6.82 24.36 25.29 14.79 12 Trochanter 28.77 56.23 11.40 5.23 39.71 23.84 30.35 24.94 26.85 8.18 13 Calcaneus 17.69 39.37 2.89 2.99 30.68 19.72 17.91 11.26 17.80 9.71 14 Talus 57.71 59.77 3.73 2.29 27.34 23.57 14.99 9.52 19.28 12.66 68 Table 23: mtDNA Yields from Fresh Bovine Bones Digested in Demineralization Buffer Regions and concomitant locations correspond to Table 3. C - (01 08) are biological replicates. MtDN A quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviation (MAD) for variation in da ta. Regions 1 12 reside on the femur; the calcaneus and talus (Regions 13 and 14) were tested as whole elements. Region Location on Element mtDNA Yield (ng/mg) C - 01 C - 02 C - 03 C - 04 C - 05 C - 06 C - 07 C - 08 Median MAD 1 Midshaft Diaphysis 18.63 18.54 3.31 2.01 5.70 4.34 4.22 2.44 4.28 1.63 2 Midshaft Diaphysis 11.17 18.88 2.22 2.98 7.63 4.34 3.65 3.29 3.99 1.39 3 Proximal Diaphysis 13.08 19.96 2.62 3.16 6.23 2.64 5.82 4.75 5.29 2.39 4 Distal Diaphysis 10.82 15.89 2.85 3.15 4.83 6.29 3.09 2.72 3.99 1.21 5 Proximal Diaphysis 12.31 22.74 2.51 3.38 11.14 10.74 7.39 4.58 9.26 3.86 6 Distal Diaphysis 12.24 16.28 2.93 2.98 6.42 13.44 2.96 5.16 5.79 2.85 7 Proximal Metaphysis 19.56 49.40 4.10 4.30 15.41 13.16 20.80 13.96 14.68 5.50 8 Distal Metaphysis 31.56 46.05 4.08 2.89 12.66 18.90 7.04 10.70 11.68 7.41 9 Articulating Surface 3 8.88 47.11 4.59 10.54 20.86 18.37 14.27 10.53 16.32 5.78 10 Distal Epiphysis 78.24 156.16 13.16 18.78 25.37 30.60 26.02 35.31 28.31 8.27 11 Femoral Head 84.00 75.92 11.89 14.47 31.06 42.38 23.56 30.03 30.55 13.96 12 Trochanter 40.35 45.60 11.04 5.18 24. 35 30.86 25.93 18.31 25.14 10.47 13 Calcaneus 26.39 41.29 3.19 3.09 18.06 16.65 13.20 13.61 15.13 7.10 14 Talus 55.23 52.87 3.31 2.34 15.47 17.97 8.91 11.99 13.73 7.62 69 Figure 22: Median mtDNA Yields from Fresh Bovine Bones The x - axis lists the regions tested, while the y - axis is the median mtDNA yields in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer, and blue bars bone powder digested in demineralization buffer. Error bars represent median absolute deviation . Asterisk indicates significant differences between buffers. 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 Median mtDNA Quantification (ng/mg) Tissue Lysis Buffer Demineralization Buffer 70 Table 24: Combined mtDNA Yields from Fresh Bovine Bones Digested in Tissue Lysis and Demineralization Buffers Combined regions listed in first column and correspond to Table 3. Regions combine d from C - (01 08). MtDNA quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviation (MAD) for variation in data. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articu lating surface, 10 12 the epiphyses, and 13 14 the tarsals. TL = tissue lysis buffer DM = demineralization buffer Combined Regions Median MAD TL DM TL DM 1 6 4.02 4.79 1.64 2.01 7 9 11.54 14.11 5.06 6.72 10 12 26.40 28.03 12.68 11. 02 13 14 17.80 14.54 11.21 8.43 Figure 23: Combined Median mtDNA Yields from Fresh Bovine Bones The x - axis lists the regions tested, while the y - axis is the median mtDNA yields in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer, and blue bars bone powder digested in demineralization buffer. Error bars represent median absolute deviation. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 t he tarsals. Asterisk indicates significant differences between buffers. * 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 Regions 1 6 Regions 7 9 Regions 10 12 Regions 13 14 Median mtDNA Quantification (ng/mg) Tissue Lysis Buffer Demineralization Buffer 71 Median bovine mtDNA yields differed significantly among regions of bone digested in tissue lysis and demineralization buffers (p < 0.0001). P - values for multiple pairwise comparison s are listed in Appendix A9 and A10. Pairwise comparisons of mtDNA yields from bone digested in tissue lysis and demineralization buffers are depicted in Figures 24 and 25 respectively. Regions 10 12 had significantly higher mtDNA yields than Regions 1 6 (p = 0.007 0.0001); however, were not significantly different from Region 9 (p = 0.644 0.171). Region 9 had significantly higher mtDNA yields than Regions 1 4 (p = 0.036 0.002). Region 8 had significantly lower mtDNA yields than Region 10 (p = 0.038, 0.019), but was not significantly different from Region 11 (p = 0.109, 0.051) or Region 12 (p = 0.075, 0.183). Region 7 had significantly higher mtDNA yields than Region 1 (p = 0.018, 0.031). Regions 13 and 14 had inconsistent results between b uffers. Regions 13 and 14 digested in tissue lysis buffer had significantly higher mtDNA yields than Regions 1 5 (p = 0.044 0.004); however, these were not statistically significant in bone digested in demineralization buffer (p = 0.067 0.239). MtD NA yields from Regions 13 and 14 were not significantly different from each other (p = 0.841, 0.869), and neither were significantly different from Regions 7 12 (p = 0.896 0.070). There were no significant differences between buffers based on individu al pairwise comparisons (p = 0.234 0.959). 72 Figure 24: Pairwise Comparisons of mtDNA Yields from Fresh Bovine Bones Digested in Tissue Lysis Buffer The x - axis lists the regions tested (n = 8), while the y - axis is the median mtDNA yield normalized in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer. Regions that have the same letter did not differ significantly i n mtDNA yields, while regions with different letters did. f b c d e a b c d e f a b c a b a a b c a b c c d e e f e f d e f e f 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Median mtDNA Quantification (ng/mg) Tissue Lysis Buffer 73 Figure 25: Pairwise Comparisons of mtDNA Y ields from Fresh Bovine Bones Digested in Demineralization Buffer The x - axis lists the regions tested (n = 8), while the y - axis is the median mtDNA yield normalized in ng per mg bone powder. Blue bars represent bone powder digested in demineralization buf fer. Regions that have the same letter did not differ significantly in mtDNA yields, while regions with different letters did. e a b c d a b c c d e a a b a b a b a b c b c d e d e d e b c d e a b c d e 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Median mtDNA Quantification (ng/mg) Demineralization Buffer 74 Combined bovine mtDNA yields differed significantly among the femoral diaphysis, metaphyses and articulating surface, and epip hyses digested in both buffers (p < 0.0001). P - values for multiple pairwise comparisons of the femur and individual pairwise comparisons between tarsals and femora are listed in Appendix A6. Pairwise comparisons of combined mtDNA yields derived from bone powder digested in tissue lysis and demineralization buffers are depicted in Figures 26 and 27 respectively. Combined regions were significantly different at a Bonferroni corrected significance level of = 0.0167. The epiphyses had significantly higher mtDNA yields than the diaphysis (p < 0.0001) digested in both buffers. Comparisons between the epiphyses and the metaphyses and articulating surface were inconsistent between buffers, where the epiphyses had significantly higher yields from bone digested in demineralization buffer (p = 0.0004), but were not significantly different when digested in tissue lysis buffer (p = 0.020). The metaphyses and articulating surface had significantly higher yields tha n the diaphysis for both buffers (p < 0.0001). The tarsals had significantly higher mtDNA yields than the diaphysis digested in both buffers (p = 0.006 0.0001), and significantly lower yields than the epiphyses digested in demineralization buffer (p = 0 .012). MtDNA yields from the tarsals were not significantly different from the metaphyses and articulating surface digested in both buffers (p = 0.838 0.345), or the epiphyses digested in tissue lysis buffer (p = 0.070). The diaphysis digested in demin eralization buffer had significantly higher mtDNA yields than tissue lysis buffer (p = 0.037); however, there were no differences between buffers for the metaphyses and articulating surface (p = 0.310), the epiphyses (p = 0.690), or the tarsals (p = 0.696) . 75 Figure 26: Pairwise Comparisons of Combined mtDNA Yields from Fresh Bovine Bones Digested in Tissue Lysis Buffer The x - axis lists the regions tested, while the y - axis is the median mtDNA yields normalized in ng per mg bone powder. Green bars represen t bone powder digested in tissue lysis buffer. Regions that have the same letter did not differ significantly in mtDNA yields, while regions with different letters did. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. A B B B 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Regions 1 6 Regions 7 9 Regions 10 12 Regions 13 14 Median mtDNA Quantification (ng/mg) Tissue Lysis Buffer 76 Figure 27: Pairwise Comparisons of Combined mtDNA Yields from Fresh Bovine Bones Digested in Demineralization Buffer The x - axis lists the regions tested, while the y - axis is the median mtDNA yields normali zed in ng per mg bone powder. Blue bars represent bone powder digested in demineralization buffer. Regions that have the same letter did not differ significantly in mtDNA yields, while regions with different letters did. Regions 1 6 represent the diap hysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. A B C B 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Regions 1 6 Regions 7 9 Regions 10 12 Regions 13 14 Median mtDNA Quantification (ng/mg) Demineralization Buffer 77 Nuclear bovine DNA yields from extracts digested in tissue lysis buffer (Table 25) and demineralization buffer (Table 26) were lowest in Regions 1 6, with increased DNA yields at the metaphyses. The exception was Region 9 that had marginally higher yields than Regions 1 6, and similar to Regions 7 and 8. Regions 10 12 had higher yields than Regions 1 9, 13 and 14. Regions 13 and 14 had s imilar yields to Regions 7 9. Both buffers had similar overall variation in quantity; however, demineralization buffer extracts had higher yields at Regions 1 4 while Regions 9 14 had higher yields from tissue lysis buffer extracts. Combined nuclea r DNA yields from bone digested in tissue lysis and demineralization buffers are listed in Table 27 and graphically depicted in Figure 29. The highest yields were at the epiphyses, followed by the metaphyses and articulating surface as well as the tarsals , with the diaphysis having the lowest yields. Individual values for these data, including normalization parameters and total DNA recovered are available in Appendix C17 C32 (even numbers ). Efficiencies of qPCR ranged from 88.4 to 105.6 as calculated f rom the slopes of the standard curves, with R 2 - values ranging from 0.977 to 0.992. No PCR inhibition was detected via the IPC. 78 Table 25: Nuclear DNA Yields from Fresh Bovine Bones Digested in Tissue Lysis Buffer Regions and concomitant locations correspond to Table 3. C - (01 08) are biological replicates. Nuclear DNA quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absolute deviati on (MAD) for variation in data. Regions 1 12 reside on the femur; the calcaneus and talus (Regions 13 and 14) were tested as whole elements. Region Location on Element Nuclear DNA Yield (ng/mg) C - 01 C - 02 C - 03 C - 04 C - 05 C - 06 C - 07 C - 08 Median MAD 1 Midshaft Diaphysis 180.00 33.44 39.85 60.55 27.78 16.46 65.54 57.60 48.72 16.05 2 Midshaft Diaphysis 193.00 147.06 91.80 50.98 50.49 46.31 93.60 84.36 88.08 37.60 3 Proximal Diaphysis 160.35 87.55 77.40 52.80 28.84 7.89 87.58 68.12 72.76 17.39 4 Distal Diaphysis 170.45 90.61 100.44 79.38 40.26 57.35 89.29 76.19 84.33 12.12 5 Proximal Diaphysis 164.40 154.67 95.70 71.65 30.49 50.40 88.33 88.71 88.52 27.50 6 Distal Diaphysis 92.35 104.00 113.88 105.50 73.60 116.33 83.53 104.00 104. 00 10.76 7 Proximal Metaphysis 350.20 678.40 234.24 155.52 93.60 83.05 223.47 186.30 204.89 80.33 8 Distal Metaphysis 289.88 295.04 194.40 123.53 90.00 144.24 137.25 165.87 155.06 35.44 9 Articulating Surface 251.02 852.00 211.53 198.45 214.40 259.80 16 4.16 152.82 212.96 42.45 10 Distal Epiphysis 720.00 1913.73 375.65 352.06 246.96 269.36 285.20 257.04 318.63 59.30 11 Femoral Head 462.29 1167.06 324.49 207.60 275.29 239.40 205.29 220.40 257.35 50.90 12 Trochanter 830.20 1051.57 379.00 220.20 307.92 28 5.36 376.88 256.24 342.40 71.60 13 Calcaneus 279.56 679.59 214.80 156.12 255.36 275.28 233.20 190.49 244.28 33.14 14 Talus 656.47 1109.80 171.60 130.41 275.55 275.00 268.24 171.05 271.62 100.30 79 Table 26: Nuclear DNA Yields from Fresh Bovine Bo nes Digested in Demineralization Buffer Regions and concomitant locations correspond to Table 3. C - (01 08) are biological replicates. Nuclear DNA quantities are reported in ng per mg bone powder . Median was used to characterize central tendency and median absolute deviation (MAD) for variation in data. Regions 1 12 reside on the femur; the calcaneus and talus (Regions 13 and 14) were tested as whole elements. Region Location on Element Nuclea r DNA Yield (ng/mg) C - 01 C - 02 C - 03 C - 04 C - 05 C - 06 C - 07 C - 08 Median MAD 1 Midshaft Diaphysis 224.00 297.60 81.18 52.51 93.38 137.67 85.28 63.15 89.33 31.50 2 Midshaft Diaphysis 220.92 244.71 89.41 95.40 143.29 111.51 92.08 57.71 103.46 26.94 3 Proxima l Diaphysis 210.00 296.25 87.35 99.33 106.29 113.06 92.56 77.38 102.81 12.85 4 Distal Diaphysis 231.76 197.78 114.29 110.35 108.00 113.37 74.12 60.35 111.86 20.80 5 Proximal Diaphysis 183.67 328.28 94.12 97.73 131.20 100.65 94.22 55.02 99.19 18.54 6 Dis tal Diaphysis 216.73 264.29 116.00 122.82 106.80 168.00 73.76 74.36 119.41 45.35 7 Proximal Metaphysis 279.76 328.64 153.44 128.18 159.22 104.92 190.93 144.53 156.33 31.38 8 Distal Metaphysis 274.08 359.60 122.25 100.90 146.16 187.80 86.06 114.24 134.21 40.73 9 Articulating Surface 346.82 120.14 158.04 165.18 182.82 168.56 154.86 106.53 161.61 14.08 10 Distal Epiphysis 541.18 915.20 228.39 266.40 223.02 216.33 288.32 245.30 255.85 32.65 11 Femoral Head 816.00 1059.18 249.27 226.12 244.35 206.98 200.34 174.04 235.24 31.57 12 Trochanter 395.29 882.00 241.43 113.65 240.84 205.32 259.29 101.78 241.13 81.65 13 Calcaneus 218.57 752.94 136.32 97.34 231.14 210.54 174.59 115.73 192.57 47.41 14 Talus 555.47 1047.76 116.47 78.51 189.41 191.63 137.47 107.46 163. 44 51.48 80 Figure 28: Median Nuclear DNA Yields from Fresh Bovine Bones The x - axis lists the regions tested, while the y - axis is the median nuclear DNA yield in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer, and blue bars bone powder digested in demineralization buffer. Error bars represent median absolute deviation. Asterisk indicates significant differences between buffers. * * 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00 450.00 Median DNA Quantification (ng/mg) Tissue Lysis Buffer Demineralization Buffer 81 Table 27: Combined Nuclear DNA Yields from Fresh Bovine Bones Digested in Tissue Lys is and Demineralization Buffers Combined regions listed in first column and correspond to Table 3. Regions combined from C - (01 08). DNA quantities are reported in ng per mg bone powder. Median was used to characterize central tendency and median absol ute deviation (MAD) for variation in data. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. TL = tissue lysis buffer DM = demineralization buffer Combined Reg ions Median MAD TL DM TL DM 1 6 83.94 107.40 24.87 28.12 7 9 196.43 156.45 53.39 34.34 10 12 296.64 242.89 76.34 36.74 13 14 261.80 182.00 59.16 57.33 Figure 29: Combined Median Nuclear DNA Yields from Fresh Bovine Bones The x - axis lists the regions tested, while the y - axis is the median DNA yields in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer, and blue bars bone powder digested in demineralization buffer. Error bars represent median absolute deviation. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. Asterisk indicates significant differences between buffers. * * * 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00 Regions 1 6 Regions 7 9 Regions 10 12 Regions 13 14 Median DNA Quantification (ng/mg) Tissue Lysis Buffer Demineralization Buffer 82 Median bovine nuclear DNA yields differed signif icantly among regions of bone digested in tissue lysis and demineralization buffers (p < 0.0001). P - values from multiple pairwise comparisons are available in Appendix A11 and A12. Pairwise comparisons of nuclear DNA yields from bone digested in tissue l ysis and demineralization buffers are depicted in Figures 30 and 31 respectively. Regions 10 12 had significantly higher DNA yields than Regions 1 6 (p = 0.013 0.0001), except for Region 12 digested in demineralization buffer was not significantly d ifferent from Region 6 (p = 0.052). DNA yields among Regions 1 9, 13, and 14 digested in demineralization buffer were not significantly different (p = 0.994 0.064). Bone powder digested in tissue lysis buffer had a greater number of regions that dif fered significantly. Region 7 had significantly more DNA than Regions 1 4 (p = 0.029 0.002). Region 8 had significantly more DNA than Region 1 (p = 0.006) and Region 3 (p = 0.016). Region 9 had significantly more DNA than Regions 1 6 (p = 0.026 0.0001), but not Region 7 (p = 0.675) or Region 8 (p = 0.446). Similarly, Regions 13 and 14 had significantly more DNA than Regions 1 6 (p = 0.003 0.0001), but not significant to Regions 7 12 or to each other (p = 0.920 0.271). Significantly more nuclear DNA was recovered from Region 1 (p = 0.021) and Region 3 (p = 0.028) digested in demineralization buffer than tissue lysis buffer, other regions were not statistically different between buffers (p = 0.846 0.104). 83 Figure 30: Pairwise Comparison s of Nuclear DNA Yields from Fresh Bovine Bones Digested in Tissue Lysis Buffer The x - axis lists the regions tested (n = 8), while the y - axis is the median nuclear DNA yield normalized in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer. Regions that have the same letter did not differ significantly in DNA yields, while regions with different letters did. e b c d a b c d e a b a b a a a b c d e d e e d e d e 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00 Median DNA Quantification (ng/mg) Tissue Lysis Buffer 84 Figure 31: Pairwise Comparisons of Nuclear DNA Yields from Fresh Bovine Bones Digested in Demineralization Buff er The x - axis lists the regions tested (n = 8), while the y - axis is the median nuclear DNA yield normalized in ng per mg bone powder. Blue bars represent bone powder digested in demineralization buffer. Regions that have the same letter did not differ si gnificantly in DNA yields, while regions with different letters did. d a b c a b a b c a a a a a a b c d c d c d a b c d a b c d 0.00 50.00 100.00 150.00 200.00 250.00 300.00 Median DNA Quantification (ng/mg) Demineralization Buffer 85 Combined bovine nuclear DNA yields differed significantly among the femoral diaphysis, the metaphyses and articulating surface, and the epiphyses digested in both buffers (p < 0.0001). P - values for multiple pairwise comparisons of the femur and individual pairwise comparison between tarsals and femora are available in Appendix A6. Pairwise comparisons of combined nuclear DNA yields derived from bone digested in tissue lysis and deminer alization buffers are depicted in Figures 32 and 33 respectively. Combined regions were significantly different at a Bonferroni corrected significance level of = 0.0167. The epiphyses had significantly higher DNA yields, for both buffers, than the diap hysis (p < 0.0001), and the metaphyses and articulating surface (p = 0.0158 0.008). The metaphyses and articulating surface had significantly higher yields than the diaphysis (p = 0.011 0.0001) digested in both buffers. The tarsals had significantly higher yields than the diaphysis (p = 0.008 0.0001), were not significantly different from the metaphyses and articulating surface (p = 0.576 0.079), and were significantly lower than the epiphyses (p = 0.041 0.014) for bone powder digested in both b uffers. Comparing yields between buffers, significantly more nuclear DNA was recovered from the diaphysis digested in demineralization buffer (p < 0.0001), and the epiphyses and tarsals digested in tissue lysis buffer respectively (p = 0.046, 0.047). The re was no significant difference between buffers for the metaphyses and articulating surface (p = 0.153). 86 Figure 32: Pairwise Comparisons of Combined Nuclear DNA Yields from Fresh Bovine Bones Digested in Tissue Lysis Buffer The x - axis lists the regions tested, while the y - axis is the median DNA yields normalized in ng per mg bone powder. Green bars represent bone powder digested in tissue lysis buffer. Regions that have the same letter did not differ significantly in DNA yields, while regions with diff erent letters did. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. A B C B 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 Regions 1 6 Regions 7 9 Regions 10 12 Regions 13 14 Median DNA Quantification (ng/mg) Tissue Lysis Buffer 87 Figure 33: Pairwise Comparisons of Combined Nuclear DNA Yields from Fresh Bovine Bones Digested in D emineralization Buffer The x - axis lists the regions tested, while the y - axis is the median DNA yields normalized in ng per mg bone powder. Blue bars represent bone powder digested in demineralization buffer. Regions that have the same letter did not diff er significantly in DNA yields, while regions with different letters did. Regions 1 6 represent the diaphysis, 7 9 the metaphyses and articulating surface, 10 12 the epiphyses, and 13 14 the tarsals. A B C B 0.00 50.00 100.00 150.00 200.00 250.00 300.00 Regions 1 6 Regions 7 9 Regions 10 12 Regions 13 14 Median DNA Quantification (ng/mg) Demineralization Buffer 88 Quality of Total DNA Recovered from Fre sh Bovine Femora and Tarsals MtDNA PCR amplification results are individually listed in Appendix C33 and C34, and are listed by the maximum amplicon generated for each region per digestion buffer in Table 28. The vast majority of regions for each replicat e produced the 994 bp mtDNA amplicon from bone digested in tissue lysis buffer (78.6%) and demineralization buffer (85.7%). Remaining mtDNA extracts derived from digestion in demineralization buffer amplified the 607 bp amplicon (14.3%). Remaining tissue lysis buffer digested extracts produced the 607 bp amplicon (16.1%), except six extracts that amplified the 390 bp amplicon (5.3%). Eighteen of twenty - four tissue lysis buffer, and eleven of sixteen demineralization buffer extracts that were unable to pr oduce the 994 bp mtDNA amplicon, including the six extracts that produced the 390 bp amplicon, came from C - 03 and C - 04, bones derived from a single cow. Region 2 did not generate the 994 bp amplicon in half of the extracts tested. The remaining extracts that did not produce the 994 bp amplicon were sporadically distributed among the other regions. Overall, there was more variability in mtDNA amplicon size recovered from the diaphysis, particularly regions 1, 2, and 9, than the epiphyses or tarsals. Demi neralized bone powder had better quality mtDNA than powder digested in tissue lysis buffer (p = 0.011). Reagent blanks for C - 01 and C - 02 amplified the 994 bp amplicon in both buffers; remaining reagent blanks and negative controls had no amplification. 89 Table 28: PCR Amplification Results for Mitochondrial DNA Recovered from Fresh Bovine Bones Regions and concomitant locations coincide with Table 3. The largest amplicon size successfully produced per region (n = 16) are listed under their respective columns. TL = tissue lysis buffer (n = 8) DM = demineralization buffer (n = 8) Region Location on Element Mitochondrial DNA ( ATPase ) 201 bp 390 bp 607 bp 994 bp Total TL DM TL DM TL DM TL DM 1 Midshaft Diaphysis 0 0 0 0 2 2 6 6 16 2 M idshaft Diaphysis 0 0 0 0 5 3 3 5 16 3 Proximal Diaphysis 0 0 1 0 0 1 7 7 16 4 Distal Diaphysis 0 0 1 0 1 1 6 7 16 5 Proximal Diaphysis 0 0 0 0 1 1 7 7 16 6 Distal Diaphysis 0 0 1 0 1 3 6 5 16 7 Proximal Metaphysis 0 0 1 0 1 0 6 8 16 8 Distal Metaphy sis 0 0 1 0 0 0 7 8 16 9 Articulating Surface 0 0 0 0 3 2 5 6 16 10 Distal Epiphysis 0 0 1 0 1 1 6 7 16 11 Femoral Head 0 0 0 0 0 0 8 8 16 12 Trochanter 0 0 0 0 1 1 7 7 16 13 Calcaneus 0 0 0 0 1 1 7 7 16 14 Talus 0 0 0 0 1 0 7 8 16 Total 0 0 6 0 18 16 88 96 224 Nuclear DNA PCR amplification results are individually listed in Appendix C35 and C36, and are listed by the maximum amplicon generated for each region per digestion buffer in Table 29. Nuclear DNA extracts from bone powder digested in tiss ue lysis and demineralization buffers amplified the 410 bp amplicon 79.5% and 92.9% of the time respectively. There were two extracts, C - 01 Region 3 digested in demineralization buffer and C - 08 Region 3 digested in tissue lysis buffer, which generated the 599 bp amplicon; remaining extracts that did not amplify the 410 bp amplicon produced the 200 bp amplicon. Similar to mtDNA, demineralized bone powder had better quality nuclear DNA than bone digested in tissue lysis buffer (p = 0.004). More variability in amplicon size was present at the diaphysis than the epiphyses or tarsals. Reagent blanks C - 02 and C - 05 for tissue lysis buffer amplified 410 bps, while remaining reagent blanks and negative controls had no amplification. 90 Table 29: PCR Amplification Re sults for Nuclear DNA Recovered from Fresh Bovine Bones Regions and concomitant locations coincide with Table 3. The largest amplicon size successfully produced per region (n = 16) are listed under their respective columns. TL = tissue lysis buffer (n = 8) DM = demineralization buffer (n = 8) Region Location on Element Nuclear DNA ( MC1R ) 200 bp 410 bp 599 bp 989 bp Total TL DM TL DM TL DM TL DM 1 Midshaft Diaphysis 1 0 7 8 0 0 0 0 16 2 Midshaft Diaphysis 3 0 5 8 0 0 0 0 16 3 Proximal D iaphysis 2 2 5 5 1 1 0 0 16 4 Distal Diaphysis 3 0 5 8 0 0 0 0 16 5 Proximal Diaphysis 3 0 5 8 0 0 0 0 16 6 Distal Diaphysis 1 1 7 7 0 0 0 0 16 7 Proximal Metaphysis 2 0 6 8 0 0 0 0 16 8 Distal Metaphysis 2 0 6 8 0 0 0 0 16 9 Articulating Surface 0 1 8 7 0 0 0 0 16 10 Distal Epiphysis 1 0 7 8 0 0 0 0 16 11 Femoral Head 0 0 8 8 0 0 0 0 16 12 Trochanter 0 0 8 8 0 0 0 0 16 13 Calcaneus 2 2 6 6 0 0 0 0 16 14 Talus 2 1 6 7 0 0 0 0 16 Total 22 7 89 104 1 1 0 0 224 Surface/Burial Comparison Experimen t of Bovine Femora and Tarsals Total DNA Quantification and Quality for Surface Exposed Bovine Bones Mean mtDNA yields from surface exposed bovine femora and tarsals over a six month period are listed in Table 30 and graphically depicted in Figure 34. MtD NA yields declined over the six month period; however, variation among the five regions of the femur, the calcaneus, and the talus remained consistent throughout the six months. The femoral head and distal epiphysis had the highest mtDNA yields (~16 ng/mg ) followed by the calcaneus and talus (~11 ng/mg), with the diaphysis and the proximal and distal metaphyses having the lowest yields (~4 ng/mg) at six months. Individual values for these data, including normalization parameters and total mtDNA recovered are available in Appendix D1 D6, and final normalized quantifications for mtDNA recovered at each region and time point are listed in Appendix D7. Efficiencies of 91 qPCR ranged from 92.1 to 100.4 as calculated from the slopes of the standard curves, with R 2 - values ranging from 0.993 to 0.996. There was no relationship between the quantity and quality of recoverable mtDNA, nor was quality affected over the six months based on amplification results. The 994 bp amplicon was successfully produced from the se ven regions at each time point. The week one reagent blank amplified 994 bps, while remaining reagent blanks and all negative controls had no amplification. Individual PCR amplification results are listed in Appendix D8. Real time PCR inhibition occurr ed (detected by IPC) in the femoral head week two replicate A1 and week four replicate A2, distal epiphysis week four replicates A1 and A2, calcaneus week two and week four replicate A2, and talus week four replicates A1 and A2 as well as month six replica te A2. Inhibited extracts were diluted and re - quantified to determine uninhibited yields listed in Appendix D7, and subsequently used in Table 30 and Figure 34. Table 30: mtDNA Quantification from Surface Exposed Bovine Bones Region bone was drilled from listed in first column. Mean mtDNA quantities (n = 2), day zero (n = 4), are reported as ng per mg bone powder. Cells highlighted in yellow had at least one replicate with inhibition. Region Mean mtDNA (ng/mg) Day 0 Week 1 Week 2 Week 4 Month 3 Month 6 Diaphysis 24.29 1.70 9.58 11.72 4.91 3.03 Proximal Metaphysis 16.79 6.02 12.34 14.59 5.60 3.98 Distal Metaphysis 25.73 8.55 10.13 15.67 4.91 4.58 Femoral Head 56.37 42.55 38.58 38.77 32.80 16.70 Distal Epiphysis 64.05 39.85 28.39 34.45 16.28 16.06 Calcaneus 30.91 20.41 21.27 19.01 13.60 11.08 Talus 41.57 24.15 38.62 11.01 9.78 10.93 92 Figure 34: Mean mtDNA Yields from Bovine Bones Exposed for Six Months The x - axis lists the regions tested, while the y - axis is the mean mtDNA yield from surface exp osed bovine bones in ng per mg bone powder. Legend is color coded to its corresponding bar representing a specific time point. Day zero time point had a sample size of four (buried and surface exposed data were combined). Remaining time points had a sam ple size of two. Mean nuclear DNA yields from surface exposed bovine femora and tarsals over a six month period are listed in Table 31 and graphically depicted in Figure 35. Mean nuclear DNA yields varied substantially throughout the six months. Week tw o, week four, and month three extracts often contained higher DNA yields than extracts from the initial time point. Extracts derived from replicate A2 generally had higher DNA quantification than A1 extracts (Appendix D9). The femoral head had higher mea n yield at month six (385.65 ng/mg) than day zero (281.53 ng/mg). The other regions had lower mean month six yields than day zero. Mean DNA yield variation among the five regions of the femur, the calcaneus, and the talus remained generally consistent ov er the six month interval. The femoral head had the highest yields 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Diaphysis Proximal Metaphysis Distal Metaphysis Femoral Head Distal Epiphysis Calcaneus Talus Mean mtDNA Quantification (ng/mg) Day 0 Week 1 Week 2 Week 4 Month 3 Month 6 93 (385.65 ng/mg), followed by the talus (231.18 ng/mg), distal metaphysis (217.14 ng/mg), and calcaneus (202.92 ng/mg); with the distal epiphysis (169.43 ng/mg), proximal metaphysis (155.11 ng/mg), and diaphysis (130.86 ng/mg) having the lowest yields. Individual values for these data, including normalization parameters and total DNA recovered are available in Appendix D10 D15, and final normalized quantifications for DNA recovered at each region and time point are listed in Appendix D9. Efficiencies of qPCR ranged from 99.4 to 105.3 as calculated from the slopes of the standard curves, with R 2 - values ranging from 0.981 to 1.000. Table 31: Nuclear DNA Quantification from Surface Exposed B ovine Bones Region bone was drilled from listed in first column. Mean nuclear DNA quantities (n = 2), day zero (n = 4), are reported as ng per mg bone powder. Cells highlighted in yellow had at least one replicate with inhibition. Region Mean Nuclear DN A (ng/mg) Day 0 Week 1 Week 2 Week 4 Month 3 Month 6 Diaphysis 204.48 56.20 156.35 165.36 166.18 130.86 Proximal Metaphysis 203.96 123.21 216.04 221.06 255.54 155.11 Distal Metaphysis 245.80 225.07 181.54 265.19 299.31 217.14 Femoral Head 281.53 313. 12 613.03 456.54 399.34 385.65 Distal Epiphysis 305.99 250.64 274.93 367.94 370.98 169.43 Calcaneus 228.49 222.74 383.83 361.20 385.86 202.92 Talus 287.32 229.90 386.38 345.66 279.38 231.18 There was no relationship between the quantity and quality o f recoverable nuclear DNA, nor was quality affected over the six months based on amplification results. The 410 bp amplicon was successfully produced from the seven regions at each time point. Reagent blanks and negative controls had no amplification. I ndividual PCR amplification results are listed in Appendix D16. Real time PCR inhibition occurred in week four and month six replicate A2 from the femoral head and talus, and week four replicate A2 distal epiphysis and calcaneus. 94 Inhibited extracts were diluted and re - quantified to determine uninhibited yields listed in Appendix D9, and subsequently used in Table 31 and Figure 35. Figure 35: Mean Nuclear DNA Yields from Bovine Bones Exposed for Six Months The x - axis lists the regions tested, while the y - axis is the mean nuclear DNA yield from bovine bones in ng per mg bone powder. Legend is color coded to its corresponding bar and represents the specific time point listed. Day zero time point had a sample size of four (buried and surface exposed data were combined). Remaining time points had a sample size of two. Total DNA Quantification and Quality for Buried Bovine Bones Mean mtDNA yields from buried bovine femora and tarsals over a six month period are listed in Table 32 and graphically depicted in Figures 36 and 37. Mean mtDNA yields from each region decreased by 90% or more during the first week of burial. Additionally, variation among regions present at day zero decreased by month six, except the talus, which maintained a yield of greater tha n 1 ng/mg at six months. Individual values for these data, including normalization 0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 Diaphysis Proximal Metaphysis Distal Metaphysis Femoral Head Distal Epiphysis Calcaneus Talus Mean DNA Quantification (ng/mg) Day 0 Week 1 Week 2 Week 4 Month 3 Month 6 95 parameters and total mtDNA recovered, are available in Appendix D17 D22, and final normalized quantifications for mtDNA recovered at each region and time point are listed in Appendix D23. Efficiencies of qPCR ranged from 92.1 to 100.4 as calculated from the slopes of the standard curves, with R 2 - values ranging from 0.993 to 0.996. There was no relationship between the quantity and quality of recoverable mtDNA, nor was qua lity affected over the six months based on amplification results. The 994 bp amplicon was successfully produced from the seven regions at each time point. The day zero reagent blank produced the 994 bp amplicon, and remaining reagent blanks and negative controls had no amplification. Individual PCR amplification results are listed in Appendix D24. Real time PCR inhibition occurred in week two replicate B2 distal epiphysis and calcaneus, week four replicate B1 distal metaphysis distal epiphysis calcaneus and talus, and month six replicate B2 femoral head. Inhibited extracts were diluted then re - quantified to determine uninhibited yields listed in Appendix D23, and subsequently used in Table 32 and Figures 36 and 37. 96 Table 32: mtDNA Quantification from Buried Bovine Bones Region bone was drilled from listed in first column. Mean mtDNA quantities (n = 2), day zero (n = 4), are reported as ng per mg bone powder. Cells highlighted in yellow had at least one replicate with inhibition. Region Mean mt DNA (ng/mg) Day 0 Week 1 Week 2 Week 4 Month 3 Month 6 Diaphysis 24.29 0.69 0.47 0.40 0.58 0.46 Proximal Metaphysis 16.79 0.86 0.60 0.51 0.34 0.86 Distal Metaphysis 25.73 0.62 0.47 0.88 0.16 0.18 Femoral Head 56.37 3.37 2.97 1.85 0.61 0.93 Distal Ep iphysis 64.05 1.55 0.82 0.77 0.50 0.64 Calcaneus 30.91 2.43 0.99 0.72 0.46 0.70 Talus 41.57 1.73 1.77 0.94 1.24 1.52 97 Figure 36: Mean mtDNA Yields from Bovine Bones Buried for Six Months with Day Zero Time Point The x - axis lists the regions tested, while the y - axis is the mean mtDNA yield from bovine bones in ng per mg bone powder. Legend is color coded to its corresponding bar and represents the specific time point. Day zero time point had a sample size of four (buried and surface exposed data wer e combined). Remaining time points had a sample size of two. 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Diaphysis Proximal Metaphysis Distal Metaphysis Femoral Head Distal Epiphysis Calcaneus Talus Mean mtDNA Quantification (ng/mg) Day 0 Week 1 Week 2 Week 4 Month 3 Month 6 98 Figure 37: Mean mtDNA Yields from Bovine Bones Buried for Six Months without Day Zero Time Point The x - axis lists the regions tested, while the y - axis is the mean mtDNA yield from bovine bones in ng per mg bone powder. Legend is color coded to its corresponding bar and represents the specific time point. Each region had a sample size of two for each time point. Mean nuclear DNA yields from buried bovine bones over a six month period are list ed in Table 33 and graphically depicted in Figures 38 and 39 . Mean nuclear DNA yields decreased by 85% or more during the first week of burial. The diaphysis and metaphyses had the largest decrease in yields from day zero to week one, then remained relat ively stable for the remainder of the six months. Mean yields from the distal epiphysis, femoral head, calcaneus, and talus decreased further from week one to week four, and increased from week four to month six. Overall, at six months the femoral head a nd talus had the highest nuclear DNA yields (~16 ng/mg), followed by the distal epiphysis and calcaneus (~7 ng/mg), and the lowest yields were from the diaphysis and metaphyses (~1 ng/mg). Individual values for these data, including 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Diaphysis Proximal Metaphysis Distal Metaphysis Femoral Head Distal Epiphysis Calcaneus Talus Mean mtDNA Quantification (ng/mg) Week 1 Week 2 Week 4 Month 3 Month 6 99 normalization paramete rs and total DNA recovered are available in Appendix D25 D30, and final normalized quantifications for DNA recovered at each region and time point are listed in Appendix D31. Efficiencies of qPCR ranged from 99.4 to 105.3 as calculated from the slopes o f the standard curves, with R 2 - values ranging from 0.981 to 1.000. A total of 84 DNA extracts were tested over the six months: 32% amplified the 599 bp amplicon (27/84), 51% amplified the 410 bp amplicon (43/84), 8% amplified the 200 bp amplicon (7/84), an d 8% failed to amplify (7/84). DNA extracts that failed to amplify had PCR inhibition during qPCR except week four replicate B1 distal metaphysis. Extracts where the 200 bp amplicon was produced came primarily from week two replicate B2 and week four rep licate B1, with two exceptions: week one replicate B1 diaphysis and calcaneus. Twenty five of twenty seven extracts that produced the 599 bp amplicon came from bones that were buried for at least a week. Three were from week one, six from week two, two f rom week four, ten from month three, and four from month six sporadically distributed among the seven regions. Reagent blanks and negative controls had no amplification. Individual PCR amplification results are listed in Appendix D32. Inhibited extracts were diluted and re - quantified to determine uninhibited yields listed in Appendix D31, and subsequently used in Table 33 and Figures 38 and 39. 100 Table 33: Nuclear DNA Quantification from Buried Bovine Bones Region bone was drilled from listed i n first column. Mean nuclear DNA quantities (n = 2), day zero (n = 4), are reported as ng per mg bone powder. Cells highlighted in yellow had at least one replicate with inhibition. Region Mean mtDNA (ng/mg) Day 0 Week 1 Week 2 Week 4 Month 3 Month 6 Diaphysis 204.48 3.88 1.48 2.05 1.31 1.71 Proximal Metaphysis 203.96 0.88 0.72 0.80 0.43 0.93 Distal Metaphysis 245.80 0.48 0.49 0.71 0.21 0.67 Femoral Head 281.53 35.19 17.76 5.43 8.19 16.48 Distal Epiphysis 305.99 7.27 14.00 4.23 7.74 7.58 Calcaneus 228.48 34.38 6.83 0.70 2.70 7.66 Talus 287.32 25.74 11.93 2.18 8.11 16.19 101 Figure 38: Mean Nuclear DNA Yields from Bovine Bones Buried for Six Months with Day Zero Time Point The x - axis lists the regions tested, while the y - axis is the mean nucle ar DNA yield from bovine bones in ng per mg bone powder. Legend is color coded to its corresponding bar and represents the specific time point. Day zero time point had a sample size of four (buried and surface exposed data were combined). Remaining time points had a sample size of two. 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 Diaphysis Proximal Metaphysis Distal Metaphysis Femoral Head Distal Epiphysis Calcaneus Talus Mean DNA Quantification (ng/mg) Day 0 Week 1 Week 2 Week 4 Month 3 Month 6 102 Figure 39: Mean Nuclear DNA Yields from Bovine Bones Buried for Six Months without Day Zero Time Point The x - axis lists the regions tested, while the y - axis is the mean nuclear DNA yield from bovine bones in ng per mg b one powder. Legend is color coded to its corresponding bar and represents the specific time point. Each region had a sample size of two for each time point. Ancillary Experiments Changes in the Recoverable Total DNA of Buried Bovine Bone Segments ove r a One Month Time Period The mitochondrial and nuclear DNA quantification results for buried bovine femoral diaphysis are summarized in Tables 34 and 35 respectively. Mean mtDNA yields decreased as burial length increased. Mean day zero yield of 0.43 ng /mg was obtained from the larger segments (type B) that were buried, tested, and reburied on a weekly basis under the assumption similar yields would be obtained from the smaller fragments (type A); however, mean day two (2.16 ng/mg) and day four (1.12 ng/ mg) yields from type A were substantially higher than day 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 Diaphysis Proximal Metaphysis Distal Metaphysis Femoral Head Distal Epiphysis Calcaneus Talus Mean DNA Quantification (ng/mg) Week 1 Week 2 Week 4 Month 3 Month 6 103 zero type B (0.15 ng/mg). Mean mtDNA yields rapidly decreased over the first eleven days, not including mean day zero, then stabilized until the end of the four weeks. Segment types A and B had si milar decreases in mtDNA yields over time, and at concomitant time points, both segment types had similar yields at week one (0.32 ng/mg, 0.33 ng/mg), week two (0.05 ng/mg, 0.02 ng/mg), week three (0.03 ng/mg, 0.01 ng/mg), and week four (0.02 ng/mg, 0.02 n g/mg). Retrieving, testing, and reburying segments to be retested at later time points did not affect mean mtDNA recovery when compared to segments that were retrieved and tested once. Individual values for these data, including normalization parameters and total mtDNA recovered, are available in Appendix E1 (type A) and E2 (type B). Efficiencies of qPCR ranged from 100.6 to 104.2 as calculated from the slopes of the standard curves, with R 2 - values ranging from 0.998 to 0.999. Mean nuclear DNA yields of segment type B decreased as burial length increased. Mean day zero yield of 25.77 ng/mg was similar to week one (27.83 ng/mg), but decreased at week two (0.59 ng/mg), and remained around the same through week four (0.69 ng/mg). Day two mean DNA yield of 93.18 ng/mg (type A) was higher than day zero (type B); however, segment type A yields decreased at week one (8.47 ng/mg) and week two (0.50 ng/mg), remaining about the same at week three (0.25 ng/mg) and week four (0.22 ng/mg). Mean nuclear DNA yields b etween segment types were similar at concurrent time points, except week one segment type A had a lower mean (8.47 ng/mg) than type B (27.83 ng/mg). Retrieving, testing, and reburying segments to be retested later did not appreciably affect mean nuclear D NA recovery when compared to segments that were retrieved and tested once. Individual values for these data, including normalization parameters and total mtDNA recovered are available in Appendix E3 104 (type A) and E4 (type B). Efficiencies of qPCR ranged f rom 174.3 to 993.2 as calculated from the slopes of the standard curves, with R 2 - values ranging from 0.998 to 0.999. Table 34: mtDNA Yields of Buried Bovine Femoral Diaphysis , 1W, 11D, 2W, at - 20 ° cyclically retrieved, tested, and reburied. MtDNA quantities are normalized in ng per mg of bone powder. Mean mtDNA quantities for each length of burial are bolded. NT = Not Tested Segment Identifier A B Segment Identifier A B 0D - 1 NT 0.15 11D - 1 0.06 N T 0D - 2 NT 0.82 11D - 2 0.02 NT 0D - 3 NT NT * 11D - 3 0.04 NT 0D - 4 NT 0.36 11D - 4 0.02 NT 0D Mean NT 0.44 11D Mean 0.03 NT 2D - 1 1.27 NT 2W - 1 0.01 0.01 2D - 2 2.24 NT 2W - 2 0.09 0.02 2D - 3 2.38 NT 2W - 3 0.05 0.03 2D - 4 2.74 NT 2W - 4 0.03 0.02 2D Mean 2.16 NT 2W Mean 0.05 0.02 4D - 1 1.35 NT 3W - 1 0.02 0.01 4D - 2 1.09 NT 3W - 2 0.02 0.01 4D - 3 0.97 NT 3W - 3 0.05 0.02 4D - 4 1.09 NT 3W - 4 0.01 0.01 4D Mean 1.12 NT 3W Mean 0.03 0.01 1W - 1 0.19 0.30 4W - 1 0.02 0.01 1W - 2 0.03 0.29 4W - 2 0.01 0.02 1W - 3 0. 99 0.23 4W - 3 0.02 0.04 1W - 4 0.08 0.48 4W - 4 0.02 0.02 1W Mean 0.32 0.33 4W Mean 0.02 0.02 *0D - 3 (B) failed to produce a testable extract due to Amicon ® column damage. 105 Table 35: Nuclear DNA Yields of Buried Bovine Femoral Diaphysis Bone segment ident , and stored at - 20 ° Nuclear DNA quantities are in ng per mg of bone powder. Mean DNA quantities for each length of burial are bolded. NT = Not Tested Segment Identifie r A B Segment Identifier A B 0D - 1 NT 0.14 11D - 1 0.14 NT 0D - 2 NT 72.50 11D - 2 0.02 NT 0D - 3 NT NT* 11D - 3 0.76 NT 0D - 4 NT 4.66 11D - 4 0.22 NT 0D Mean NT 25.77 11D Mean 0.29 NT 2D - 1 39.81 NT 2W - 1 0.32 0.006 2D - 2 36.01 NT 2W - 2 1.11 0.48 2D - 3 62.53 NT 2W - 3 0.40 1.38 2D - 4 234.37 NT 2W - 4 0.17 0.49 2D Mean 93.18 NT 2W Mean 0.50 0.59 4D - 1 2.51 NT 3W - 1 0.00 1.40 4D - 2 77.78 NT 3W - 2 0.002 0.48 4D - 3 99.48 NT 3W - 3 0.94 0.59 4D - 4 75.71 NT 3W - 4 0.04 0.04 4D Mean 75.75 NT 3W Mean 0.25 0.63 1W - 1 0.24 7.22 4W - 1 0.60 0.001 1W - 2 19.32 10.98 4W - 2 0.04 0.09 1W - 3 13.22 13.09 4W - 3 0.20 2.16 1W - 4 1.11 80.03 4W - 4 0.02 0.52 1W Mean 8.47 27.83 4W Mean 0.22 0.69 *0D - 3 (B) failed to produce a testable extract due to Amicon ® column damage Changes in the Recoverable Total DNA of Non - Buried Bovine Bone Segments over a One Month Period The mitochondrial and nuclear DNA quantification results for non - buried bovine femoral diaphysis are summarized in Tables 36 and 37 respectively. Overall, mean mtDNA yiel ds did not appreciably change from the initial time point and week four; however, there were 106 substantial increases in yields during the first week of exposure. Mean mtDNA yield at day zero (1.94 ng/mg) was lower than day two (4.17 ng.mg), day four (4.39 n g/mg), and week one (3.12 ng/mg). MtDNA quantification at day ten (1.32 ng/mg), week two (1.55 ng.mg), week three (1.61 ng/mg), and week four (1.90 ng/mg) had similar yields as day zero. Individual values for these data, including normalization parameter s and total mtDNA recovered, are available in Appendix E5. Efficiency of qPCR was 105.5 as calculated from the slope of the standard curve, with a R 2 - value of 0.998. Mean nuclear DNA yields changed between the initial time point and one month. DNA yield from day zero (85.82 ng/mg) was lower than day two (235.13 ng/mg), day four (146.47 ng/mg), and week one (153.03 ng/mg). The initial time point was similar to means from day ten (82.35 ng/mg) and week two (88.70 ng/mg); however, means increased at week th ree (108.83 ng/mg) and week four (152.23 ng/mg). Individual values for these data, including normalization parameters and total DNA recovered, are available in Appendix E6. Efficiency of qPCR was 181.0 as calculated from the slope of the standard curve, with a R 2 - value of 0.999. There was a difference in total DNA yields between buried and non - buried bone. Non - buried bones did not show an appreciable difference in DNA yields over the one month time period, except for the increase during the first week th at disappeared by the second. Buried bones on the other hand showed a rapid decline in DNA yields over the same period. 107 Table 36: mtDNA Yields of Non - Buried Bovine Femoral Diaphysis Bone segment identifiers are listed in the first and third column. biological replicates of femoral diaphysis tested at each time point. MtDNA quantities are in ng per mg of bone powder. Mean mtDNA quantities f or each length of exposure are bolded. Segment Identifier Quantification Segment Identifier Quantification 0D - 1 3.20 10D - 1 1.68 0D - 2 1.75 10D - 2 0.91 0D - 3 0.97 10D - 3 1.26 0D - 4 1.85 10D - 4 1.43 0D Mean 1.94 10D Mean 1.32 2D - 1 5.64 2W - 1 1.53 2D - 2 3 .34 2W - 2 1.66 2D - 3 3.39 2W - 3 1.82 2D - 4 4.32 2W - 4 1.19 2D Mean 4.17 2W Mean 1.55 4D - 1 5.56 3W - 1 2.92 4D - 2 3.43 3W - 2 0.64 4D - 3 4.67 3W - 3 1.45 4D - 4 3.79 3W - 4 1.45 4D Mean 4.39 3W Mean 1.61 1W - 1 3.36 4W - 1 2.70 1W - 2 2.18 4W - 2 1.23 1W - 3 3.70 4W - 3 1.92 1W - 4 3.23 4W - 4 1.74 1W Mean 3.12 4W Mean 1.90 108 Table 37: Nuclear DNA Yields of Non - Buried Bovine Femoral Diaphysis biological replicates of femoral diaphysis tested at each time point. Nuclear DNA quantities are in ng per mg of bone powder. Mean DNA quantities for each length of exposure are bolded. Segment Id entifier Quantification Segment Identifier Quantification 0D - 1 107.52 10D - 1 74.62 0D - 2 77.84 10D - 2 71.61 0D - 3 68.99 10D - 3 91.51 0D - 4 88.94 10D - 4 91.66 0D Mean 85.82 10D Mean 82.35 2D - 1 288.60 2W - 1 54.29 2D - 2 110.84 2W - 2 106.80 2D - 3 192.29 2W - 3 110.73 2D - 4 348.23 2W - 4 82.98 2D Mean 235.13 2W Mean 88.70 4D - 1 211.64 3W - 1 192.68 4D - 2 113.68 3W - 2 51.50 4D - 3 138.00 3W - 3 91.96 4D - 4 122.55 3W - 4 99.20 4D Mean 146.47 3W Mean 108.83 1W - 1 227.57 4W - 1 231.16 1W - 2 141.69 4W - 2 89.14 1W - 3 1 27.57 4W - 3 161.67 1W - 4 115.27 4W - 4 126.93 1W Mean 153.03 4W Mean 152.23 Organic versus SoilMaster DNA Yield Comparisons Over One Week Table 38 summarizes nuclear and mitochondrial DNA yields from bone powder extracted utilizing either organic o r SoilMaster TM . Overall, SoilMaster TM led to higher DNA yields than organic extraction. Furthermore, organically extracted nuclear DNA (0.63 ng/mg) and mtDNA (0.19 ng/mg) yields at day zero were lower than day two nuclear (12.70 ng/mg) and mitochondrial DNA (0.46 ng/mg). SoilMaster TM extraction of bone led to lower day zero yields of nuclear DNA (22.52 ng/mg) than day two (30.50 ng/mg); however, day zero SoilMaster TM 109 mtDNA extract had the highest yield (2.38 ng/mg) and the day seven extract had the lowes t (0.42 ng/mg). MtDNA efficiency of qPCR was 96.0 as calculated from the slope of the standard curve, with a R 2 - value of 0.999. Nuclear DNA efficiency of qPCR was 132.0 as calculated from the slope of the standard curve, with a R 2 - value of 0.987. Indivi dual values for these data, including normalization parameters and total DNA recovered are available in Appendix E7. Table 38: Total DNA Yield Comparison between Organic and S oilMaster TM Extractions Normalized values for nuclear and mitochondri al DNA are reported as ng per mg of bone powder. mtDNA (ng/mg) Nuclear DNA (ng/mg) Time Point Organic SoilMaster TM Time Point Organic SoilMaster TM Day 0 0.19 2.38 Day 0 0.63 22.52 Day 2 0.46 1.80 Day 2 12.70 30.50 Day 5 0.22 1.25 Day 5 11.00 21.16 Day 7 0.12 0.42 Day 7 4.64 13.28 Mass Difference between Wet and Dry Bone Table 39 depicts changes in bone powder and whole bone mases subjected to storage treatments over time. Bone powder in closed microcentrifuge tubes retained its mass over a one week period. Bone powder in open microcentrifuge tubes or weigh boats resulted in a loss of mass, with mass stabilization occurring after several days. The bone segment continually lost mass for the span of the experiment: 4% mass was lost in the first week, and another 1% the following week. Individual values for the change in mass over time are listed in Appendix E8. 110 Table 39: Mass Differences of Dehydrated Bone Powder and Bone Segment Bone powder subjected to each treatment was weighed over a one week period. Bone segment bone powder/segment. N/A = Not Applicable Mass of Bone Powder/Bone Segment (mg) Condition 1 Closed Tube 2 Open Tube 3 Piled Powder in Weigh Boat 4 Powder Spread in Weigh Boat 5 Bone Segment Trial 1 2 1 2 1 2 1 2 1 Mass (mg) 0 0 - 2 - 2 0 - 2 - 1 - 2 - 2,669 % Diff. 0 0 4 4 0 4 2 4 5 Mean % Diff. 0 4 2 3 N/A Effect of Proteinase K Concentration on Total DNA Yields Nuclear and mitochondrial DNA yields from bovine diaphysis digested in tissue lysis and demineralization buffers with varied volumes of proteinase K are listed in Table 40. Proteinase K incr eased nuclear and mitochondrial DNA yields from bone powder digested in both buffers; however, increasing the amount proteinase K did not have an appreciable effect on DNA yields for these samples. MtDNA efficiency of qPCR was 84.9 as calculated from the slope of the standard curve, with a R 2 - value of 0.993. Nuclear DNA efficiency of qPCR was 195.0 as calculated from the slope of the standard curve, with a R 2 - value of 0.990. Individual values for these data, including normalization parameters and total m tDNA and DNA recovered are available in Appendix E9 and E10 respectively. 111 Proteinase K was added to digestion buffer by percent total reaction volume. Trials were qPCR technical replic ates. Mean DNA yields for each volume of proteinase K are bolded. mtDNA (ng/mg) Tissue Lysis Buffer Demineralization Buffer Proteinase K (20 mg/mL) 0% 0.5% 1% 2% 0% 0.5% 1% 2% Trial 1 5.17 9.43 7.90 9.92 5.33 11.20 10.4 12.18 Trial 2 4.50 8.79 9.8 8 9.43 5.56 10.96 12.42 10.26 Mean 4.84 9.11 8.89 9.67 5.45 11.08 11.43 11.22 Nuclear DNA (ng/mg) Tissue Lysis Buffer Demineralization Buffer Proteinase K (20 mg/mL) 0% 0.5% 1% 2% 0% 0.5% 1% 2% Trial 1 284.67 533.96 503.84 679.59 202.20 295.10 261.6 0 333.00 Trial 2 259.22 668.78 769.60 648.98 253.80 336.73 416.40 229.20 Mean 271.95 601.37 636.22 664.29 228.00 315.92 339.00 281.10 Comparison of Total DNA Yields from Bovine Bones Macerated by MSU Forensic Anthropologist versus MSU Forensic Biologis t Nuclear and mitochondrial DNA quantification from bovine bones that were macerated at both sites are listed in Table 41. Elements processed at the MSU Forensic Biology Laboratory resulted in higher total DNA yields utilizing both tissue lysis and dem ineralization buffers. MtDNA efficiency of qPCR was 99.5 as calculated from the slope of the standard curve, with a R 2 - value of 0.997. Nuclear DNA efficiency of qPCR was 99.7 as calculated from the slope of the standard curve, with a R 2 - value of 0.985. Individual values for these data, including normalization parameters and total mtDNA and nuclear DNA recovered are available in Appendix E11 and E12 respectively. 112 Table 41: Total DNA Quantification Comparisons between Bovine Bones Macerated by MSU Foren sic Anthropologist and the MSU Forensic Biologist Region tested is in the first column. DNA/mtDNA quantification is reported in nanograms per mg bone. FA = DNA derived from an element macerated by MSU Forensic Anthropology FB = DNA was derived from an element macerated by MSU Forensic Biology. Region Mitochondrial DNA Yields (ng/mg) Nuclear DNA Yields (ng/mg) Tissue Lysis Buffer Demineralization Buffer Tissue Lysis Buffer Demineralization Buffer FA FB FA FB FA FB FA FB Diaphysis 2.30 6.40 5.33 10. 71 80.07 120.00 184.59 236.08 Distal Epiphysis 31.73 55.12 19.65 45.08 411.22 495.56 244.52 318.24 Femoral Head 26.74 51.06 20.55 36.76 570.72 611.14 299.20 340.00 Calcaneus 4.24 41.29 12.13 24.21 128.12 647.45 220.54 257.40 Talus 6.47 16.00 8.30 14.00 192.71 390.00 261.52 298.29 113 DISCUSSION Establishing positive identification of human remains has vital importance to the legal system, family and friends of the decedent, and society in general. DNA identification from skeletal material c an be very effective even when remains are highly decomposed, largely incomplete, substantially damaged and/or fragmented, or other forms of positive identification are unavailable. Maximizing DNA recovery from skeletal material relies on how bone is hand led from discovery to testing, the element assayed, and the processes used to isolate DNA from it. Many studies have been undertaken to compare how skeletal elements vary in recoverable DNA and successful downstream analyses; however, little research has focused on differences in DNA quantity and quality within a single element. Therefore, a systematic investigation of intra - femoral heterogeneity of DNA content was conducted. The first step in this process was to establish DNA heterogeneity in fresh bone , while the second was to determine how it was affected by burial and outdoor exposure, environments in which forensically relevant remains are commonly found. An ancillary goal was to compare DNA content within the femur to the calcaneus and talus, two t arsals that Mundorff and Davoren (2014) found harbor more DNA. The most interesting finding of the research presented here was that DNA quantity varied significantly along the femur, and the distribution of DNA was highly similar between porcine and bov ine bones, wherein the epiphyses harbored more DNA than the metaphyses, and both had more than the diaphysis. This was also consistent with results from previous studies that utilized weanling rat femora (Yamaguchi and Yamaguchi 1986; Yamaguchi et al., 20 03), indicating that common factors are likely influencing DNA heterogeneity. DNA quantity may vary between the 114 diaphysis and epiphyses because they originate as separate bones. The femur is often thought of in its mature form, as a single element; howev er, it is the sum total of primary and secondary ossification centers fused together. Epiphyseal closure schedules have been well characterized in primary ossificat ion center begins at the mid - diaphysis and grows longitudinally to fuse with the distal and proximal epiphyses. The femoral head and trochanter (i.e., proximal epiphysis) of porcine and bovine femora fuse to the diaphysis at 3 3.5 years of age, while th e distal epiphysis fuses to the diaphysis at 3 3.5 years in porcines and 3.5 4 years in bovines (Silver, 1963). DNA quantity differed among femoral ossification centers in this study, which is consistent with bones from the same body varied greatly in DNA yield. If bone development affects DNA quantity among unlike elements (e.g., femur versus cranium), then separate ossifications centers of the same bone will likely vary in quantity as well. Comparing DNA y ields from primary and secondary ossification sites from other long bones, or elements like vertebrae that have a different morphology, could provide more evidence that ossification centers affect DNA heterogeneity within a single element. Animals used thr oughout this research varied in antemortem age, likely influencing DNA heterogeneity. Longitudinal growth of the femoral diaphysis takes place at the epiphyseal plates and between the epiphyses and articular cartilage (Bisgard and Bisgard, 1935). The gro wing ends of the diaphysis (i.e., metaphyses) and the epiphyses may harbor more cells, resulting in a richer source of DNA. Bovine femora C - 01 and C - 02 came from cows slaughtered around 36 months of age, while C - 03 C - 08 originated from dairy cows 48+ mo nths old (Ryan Varner, MSU Meats Laboratory, personal communication). This age difference likely led to the higher DNA yields from the younger bovine specimens. Porcine femora also came from younger 115 animals, as the pigs were slaughtered at 3 7 months o ld. The disparity in bovine and porcine femoral development might have contributed to the more pronounced difference in DNA yields at the metaphyses and epiphyses in porcine bone. Old age could affect DNA quantity as well; Atkinson et al. (1962) characte rized changes in human femoral density with age, and reported that it decreased as the age of the individual increased and that the metaphyses decreased in density at a faster rate than the diaphysis in individuals over fifty. Additionally, emerging evide nce links femoral osteocyte lacunar density a proxy measure of osteocyte density with age. In a poster presentation, Hunter and Agnew (2014) described the osteocytic lacunae density in 20 cross sections of midshaft diaphysis from deceased human males rang ing in ages from 29 to 79 years with no known skeletal pathology. The authors reported a significantly higher density in males aged 29 59 than those aged 60 79, as well as a significant negative correlation between age and density. Once an individual becomes physically mature, cell density (and presumably DNA yield) in bone could decline. This postulate is consistent with the current research where younger specimens had more DNA than their older counterparts. Another potential cause of intra - femoral DNA heterogeneity involves bone remodeling. Regions of the femur with high DNA yields, like the femoral head and distal epiphysis, may undergo more remodeling because they distribute static body weight and facilitate locomotion (Carter, 1984). Similarly, the trochanter and Region 9 are points of muscle attachment that undergo stress during muscle contraction, and thus might require more remodeling. Ruff et al. (2006) reviewed bone functional adaptation, and concluded that bone cells respond to local mech Furthermore, bone remodeling involves the continuous removal and replacement of old osseous tissue to maintain bone strength and mineral homeostasis (Clarke, 2008) . Although most 116 remodeling sites are thought to arise spontaneously (Clarke, 2008), Mashiba et al. (2001) showed that generating micro - damage in dog radii increased remodeling at damaged sites, and in a review of bone remodeling mechanisms Burr (2002) con cluded that about 30% of it occurs in a targeted manner. Leney (2006) postulated that bone remodeling of the femur and tibia was why those bones were more likely to contain mtDNA that led to reportable sequence data. The author also proposed that the man dible was a good source of mtDNA because remodeling at points of muscle attachment related to mastication increased bone density. A similar hypothesis could be extended to partially explain why the patella, carpals, and tarsals were found to be a rich sou rce of DNA in previous research (Mundorff et al., 2009; Mundorff and Davoren, 2014), in which entire elements that undergo more remodeling may also contain more cells. Overall, separate ossification sites, antemortem age, and remodeling of the femur, as w ell as factors not yet considered, likely influenced DNA yield throughout the present research. The above - mentioned results came from fresh (macerated) bone, which would not be recovered during a forensic investigation. Therefore, non - macerated bovine fem ora and tarsals remains are found. The burial and surface exposure of bone led to several interesting findings throughout this research. For instance, many regions of the bones exposed on the soil surface (most notably from the epiphyses and tarsals) had higher nuclear DNA yields at the week two, four, and month three time points than the initial time point. The reason for this is unclear; however, environmental expos ure may have softened the bone, making it easier to drill and caused less DNA degradation (discussed below). In contrast, decreased DNA yields at the six - month time point likely resulted from sustained environmental exposure. Why nuclear DNA yields incre ased more at certain time points than mtDNA did is currently unknown, but the 117 increase might have been a result of nuclear DNA being more sensitive to damage during drilling, something to keep in mind for future investigations. Researchers have shown that burying bone can have a drastic effect on DNA quantity over time, but never in such a short period as demonstrated in the current research. Campos et al. (2012) reported that mtDNA quantity was reduced by more than 90% in buried bovine bone compared to c ontrol bone at one year. Hebda and Foran (2015) established that nuclear and mitochondrial DNA from bovine femoral diaphyses declined substantially between one week and one month of burial; however, pre - burial yields were not assessed, so the level of DNA loss during the first week was unclear. Based on the present study, the rapid decline in DNA quantity likely occurs within days or weeks of burial, although the exact cause of this is unclear. Soil pH was approximately 5.5 at the burial site, which is c onsidered moderately acidic (Horneck et al., 2011). Lange (2008) determined that pH was the only chemical factor to correlate with skeletal weathering, in which lower pHs (~5.5) were associated with higher weathering stages of skeletons recovered from the Voegtly cemetery. Acidic soil is known to deleteriously affect DNA and bone ( Turner - Walker, 2008; Latham and Madonna, 2013); however, the amount of time it takes to degrade DNA to the extent observed in the present research is unclear, and degradation se ems unlikely to occur over the course of only a few weeks. Campos et al. (2012) proposed that skeletal DNA quantity declines due to the rapid putrefaction of osteoblasts, osteoclasts, bone - lining cells, and blood cells. Additionally, microbial attack on bone can influence DNA degradation (Latham and Madonna, 2013), and Bell et al. (1996) reported that postmortem alteration of bone, visualized through scanning electron microscopy, was likely due to microbial attack and can occur in as little as three month s, the shortest postmortem interval tested. In the current research, burial may have facilitated an anaerobic environment that 118 allowed the bones to putrefy rapidly, aiding in the steep decline of DNA by one week. Additionally, microbes intrinsic to bone and/or soil could have contributed to DNA loss by degrading both bone and DNA. Further research will be required to determine the cause(s) of short - term DNA loss resulting from burial, paying close attention to bone tissue decomposition, microbial communi ties, pH, temperature, and soil chemistry. Another interesting finding from the present study was that DNA quantity in buried femora declined faster at the epiphyses than the diaphysis over six months, which brought DNA yields among regions closer over tim e. However, it is noteworthy that throughout the period the epiphyses still had as much or more DNA as the diaphysis. Femoral epiphyses contain less cortical bone than the mid - diaphysis (Clarke, 2008), potentially making them more susceptible to factors that deleteriously affect DNA, resulting in differential rates of DNA loss among bone regions. The extension of burial interval from months to years or decades might invert regional DNA variation if dense cortical bone can better withstand environmental a ttack over time. This could help explain why weight bearing long bones were more likely to contain analyzable DNA when extended postmortem periods were a factor (Edson et al., 2004; Leney, 2006; 2007), while researchers using non - buried rema ins with shorter postmortem intervals reported that elements like the carpals, tarsals, and patella had better quantity and quality DNA than long bones (Mundorff et al., 2009; Mundorff and Davoren, 2014). An interesting, albeit unexpected, result in the cu rrent research was that 27 of 84 DNA extracts from bovine buried bones generated the 599 bp nuclear DNA amplicon, a much higher percentage than from surface exposed (0 of 84) or macerated (2 of 224) samples. DNAs that were able to amplify out to 599 bps p rimarily came from week two (6 of 27) and month three (10 of 27) time points, in which an interaction between burial and drilling might have influenced the 119 results. Starting at week one, the bones were noticeable softer and easier to drill. Burial likely led to collagen and hydroxyapatite breakdown, weakening bone structure and reducing DNA damage during drilling. After the three month time point when amplification of the 599 bp amplicon was most successful, environmental degradation of DNA could have re duced DNA quality. Finally, several DNAs tested from the week four time point exhibited PCR inhibition (discussed below), potentially lowering the frequency of generating the 599 bp amplicon. A final interesting finding from the environmental exposure stu dy was that buried and surface exposed bones that were only eight vertical inches apart had drastic differences in total DNA yields, showing that local environment can have a profound effect on DNA. DNA yields of buried bones plummeted over a short period many instances nuclear DNA quality was higher in buried bones than in surface exposed samples (as noted above). This discrepancy could have resulted from an interaction between environment and DNA amplifi cation methodology. For example, DNA degradation in buried bone may have been largely complete; however, a small amount of high quality DNA was protected within hydroxyapatite, allowing the 599bp fragment to amplify. This would also be true for surface e xposed bone, but the higher overall DNA quantity in them indicates that less complete degradation took place. The bovine DNA quantitative assay used in this research was based on ~80 bp amplicons. If the majority of DNA from surface exposed bones was deg raded below 600 bp but not below 80 bp, its abundance might swamp out any high molecular weight DNA bound to hydroxyapatite, resulting in the observation that DNA from surface exposed bones had lower quality than buried ones. Creating real - time PCR amplic ons of increasing size (e.g., 100 600 bps) and testing DNA extracts from buried and surface exposed bones with them would help determine how degraded the DNAs actually are. If DNA bound within hydroxyapatite and 120 subsequently released through demineraliz ation is less degraded, it would provide further evidence that demineralization of bone leads to higher quality DNA for analysis, an obvious boon for forensic casework and research alike. The research on environmental exposure and burial of bone was design ed to generate baseline knowledge of how intra - femoral DNA heterogeneity identified in macerated bone differs under more forensically relevant conditions; however, it had several limitations. First, low sample size per treatment (n = 2) made statistical a nalysis impractical. Second, an age discrepancy between replicates was a potential confound: one set came from an adult, the other a juvenile. Of course in a real world scenario the age of the decedent would not be controlled for, so future investigation s that utilize different age groups may be helpful in determining how environment influences DNA yield over time. Third, the fact that environmentally exposed bones were not macerated limited drilling to areas that were amenable to mechanical defleshing. For instance, articulating surfaces often had tendon or ligament attached that could not be completely removed, and areas of bone with rough surfaces retained soft tissue in the crevices, even after vigorous defleshing. Sampling at these locations while other tissues are present would have resulted in soft tissue comingled with bone powder, likely creating a substantial confound, whereas macerating bones prior to environmental exposure would have allowed for more locations to sample. Finally, these bones were exposed to a single environment, a mid - Michigan garden near deciduous woods; how intra - femoral DNA heterogeneity is influenced by different soil types (e.g., sand, silt, clay), climates (e.g., dry vs. humid), or environments (e.g., marsh, desert) rem ains unanswered. Substantial inter - replicate variation in DNA quantity was evident throughout this research, which, combined with low sample size (n = 8), contributed to some non - parametric 121 results, thus all data sets were treated as such. While not ideal , normality tests like Shapiro - Wilk can assess non - normal distributions with sample sizes of around ten (Shapiro et al., 1968), and were employed in this research because normality could not be visually determined (e.g., via histograms or boxplots). Inter - replicate variance was more than 100% in some instances, which is consistent with previous quantitative DNA research on ancient seal ribs (Barta et al., 2014) and skeletonized human remains (Amory et al., 2011; Mundorff and Davoren, 2014). Bovine and por cine bones came from livestock raised for human consumption, therefore individuals of each species lived in a similar habitat, shared the same diet, were brought to slaughter around the same age, and were monitored for disease and other pathologies as requ ired by the USDA, meaning that environment, age, and disease were largely controlled for. The exception was the younger bovine replicates C - 01 and C - 02, which confounded bovine DNA quantitation data and contributed to overall variance. The removal of C - 0 1 and C - 02 data decreased variance and resulted in a normal distribution in all but two samples; however, all data were included for analysis so as to not further limit sample size. Furthermore, the non - parametric data sets for porcine DNA were mostly con fined to nuclear DNA (5/7) and more specifically to regions of the mid - diaphysis (3/7). Replicate P - 01 DNA yields were much higher at the mid - diaphysis and P - 04 yields were lower, otherwise outliers that potentially contributed to non - normally distributed data sets were random. Another possible cause of inter - replicate variation involved a limitation of the real - time PCR technique used for quantifying DNA. DNA concentrations double every cycle under optimal conditions during the exponential phase of PCR , when quantity measurements take place. Therefore, a deviation of just one PCR cycle between replicates causes a twofold difference in DNA concentration estimates. Further, technical replicates were not conducted in 122 the current research, so qPCR precisi on was not assessed. Running the same sample in duplicate or more is common practice (Kline et al., 2005; Nielsen et al., 2008), and would likely help determine if outliers in quantification data resulted from qPCR error. The identification of outliers f ollowed by re - quantification of those samples, and/or the inclusion of technical replicates, could reduce non - parametric data sets in future research. Inherent biological differences in femora may have affected inter - replicate DNA yields from both species; however, with the exception of C - 01 and C - 02 having overall higher yields, there was no noticeable pattern based upon the region of bone assayed. For instance, DNA yields from a diaphysis were not consistently more or less variable than the corresponding epiphyses. Interestingly, DNA yields often differed substantially between the left and right femora of the same individual, although not in a consistent manner. Though great care was taken to obtain bone powder from equivalent sites among replicates, th e identical spatial distribution of osteocytes and DNA bound to hydroxyapatite between paired femora is highly unlikely, potentially affecting inter - replicate yields. Furthermore, DNA quantity probably varies at locations adjacent to one another. Hebda a nd Foran (2015) discovered that when DNA was isolated from bone drillings 1 2 cm apart, highly variable amounts of DNA were often obtained. They found that homogenizing the bone powder prior to distribution among extraction techniques resulted in more u niform DNA yields, a strategy adopted for inter - regional comparisons in the current research. DNA quantity likely differs throughout an element; however, DNA yield heterogeneity in this study was only assayed in a proximal/distal orientation (diaphysis to epiphysis); thus, potential medial/lateral intra - regional DNA variation was not considered, and its detection was prevented methodologically because bone powder was purposefully homogenized among drilling 123 sites. Hunter and Agnew (2014) reported that human medial and lateral femoral midshaft diaphysis had higher osteocyte lacunae density than the anterior or posterior aspects; however, these differences were not significant. In contrast, Carter et al. (2013) characterized the mean osteocyte lacunar density of human proximal femoral diaphyses, and found that the medial/lateral portions had a significantly greater density (up to 30%) than the anterior/posterior aspects. The authors postulated that the regional heterogeneity in osteocyte density within the fe moral cortex was caused by its growth/development and remodeling, similar to reasons proposed for intra - femoral DNA heterogeneity in the current research. Similarly, DNA yield might vary at adjacent locations due to discrete pockets of bone remodeling. B eyond this, it is important to note that a 30% difference in osteocyte lacunae density (and presumably in DNA yield), though found to be significant by Carter et al. (2013), equates to one - third of a PCR cycle, which may not be detectable through the real - time assay used in this research. Investigations into DNA heterogeneity at adjacent locations, and assaying DNA quantity in a medial/lateral orientation, are ongoing in our laboratory, and should give a more complete picture of how DNA is distributed thro ughout the femur. Increasing DNA yield is irrelevant if the DNA recovered is of insufficient quality for analysis. DNA quality assessment in this research produced unexpected results in both macerated and environmentally exposed bone. For example, one mi ght expect to obtain similar nuclear and mitochondrial DNA quality from macerated bone; however, that was not the case. A couple of factors may have influenced why mtDNA generated larger amplicons than nuclear DNA, as well as the higher quality nuclear DN A recovered from porcine versus bovine bone. Foran (2006) demonstrated that nuclear DNA degraded faster than mtDNA in whole tissues, but when cells were homogenized, mtDNA degradation was similar to nuclear DNA, indicating that 124 mitochondria provide better physical protection from DNA degradation relative to the nuclear envelope. Furthermore, a single cell can harbor hundreds to thousands of mitochondria, each containing 2 7 mtDNA molecules (Robin and Wong 1988; Iborra et al., 2004), with estimated 500 10,000 copies per cell (Satoh and Kuroiwa, 1991; Iborra et al., 2004). It is possible that hydroxyapatite or some other aspect of bone preserves a fraction of the mtDNA during the maceration process, increasing the odds of generating larger amplicons. M tDNA amplification in the present study was potentially more successful due to these factors, either singly or in combination. Maceration in boiling detergent may have itself influenced DNA quantity and quality, as preliminary research on maceration times at the MSU Forensic Laboratory has shown that bovine femoral segments lose DNA as maceration time increases. Furthermore, DNA yields in the current research were approximately two - fold higher at most locations in non - macerated versus macerated bone. This differed from results of Steadman et al. (2006), who compared sampling approximately 0.7 g of trabecular bone. The authors found that the detergent/carbonate maceration technique similar to what was used in the present study resulted in higher DNA yields (1.60 µ g/ µ L) than mechanical defleshing (0.82 µ g/ µ L). This discrepancy potentially ure of DNA yield gels showed less high molecular weight DNA present in mechanically defleshed than detergent macerated samples. How much time passed between mechanically defleshing the ribs and DNA isolation was not reported, but if the bones were not sam pled immediately, bacteria and nucleases could have resulted in DNA degradation. In contrast, the boiling detergent method would denature nucleases and kill bacteria, better preserving DNA. Since very little lag time 125 (hours) occurred between mechanically defleshing elements and starting DNA isolation procedures in the research presented here, this may have resulted in higher DNA yields from non - macerated bone. The duration of maceration could have also influenced DNA quality in this research; however, t his was only detected in nuclear DNA, as the largest mtDNA amplicon almost always amplified. Porcine bones were smaller and contained less soft tissue, requiring half the maceration time as bovine bones, which might have led to better nuclear DNA quality from porcine samples. Steadman et al. (2006) associated maceration time in aqueous solution with nuclear DNA degradation, because techniques that took the longest to macerate bone (room temperature water, 0.3% hydrogen peroxide, 10% bleach (sodium hypochl orite concentration unknown), and EDTA/papain) resulted in low - quality DNA. However, bleach degrades DNA, likely contributing to the low DNA quality for that method, and the rest of the techniques took weeks to remove soft tissue from bone, potentially le ading to degraded DNA. Maceration could also affect mitochondrial and nuclear DNA degradation differently. Steadman et al. (2006) reported that mtDNA amplification was successful with any of the maceration techniques used; nuclear DNA amplification was n ot. Similarly, Rennick et al. (2005) macerated skeletal remains for 4 hours in either boiling water, 3% bleach (~0.2% sodium hypochlorite), or 1% 1:1 detergent/carbonate. Nuclear DNA amplification was unsuccessful following these treatments; however, mtD NA amplified for all, and the detergent/carbonate method resulted in higher quality mtDNA than the other techniques. These findings are consistent with the current research; larger amplicons were generated from mtDNA than nuclear DNA in both species, indi cating that nuclear DNA may have been more susceptible to degradation during maceration. Steadman et al. (2006) demonstrated that techniques employing high temperatures (boiling) decreased 126 maceration time from weeks to hours, and increased DNA recovery, b ut there is a paucity of research to determine if DNA quantity and/or quality decline during maceration using a single technique, or to what extent this causes a disparity between nuclear and mitochondrial DNA recovery. As noted, drilling could also have i nfluenced DNA quantity and quality throughout this research. The harder portions of the diaphysis were more difficult to drill and took longer to produce bone powder than the metaphyses and epiphyses, potentially affecting DNA recovery in both species. F urthermore, bovine bones were harder to drill than their porcine counterparts, likely resulting from the fact that bovine cortical bone in denser than porcine bone (Aerssens et al., 1998). As described in the Introduction, Adler et al. (2011) reported tha t increased drilling speed (100 RPM to 1,000 RPM) reduced mtDNA quantity. Throughout the research presented mechanical stresses drilling imparts on bone may have contr ibuted to more DNA loss at the diaphysis than the metaphyses and epiphyses. Furthermore, drilling the harder bovine bones might have increased nuclear DNA degradation, resulting in smaller amplicons generated from bovine DNA. MtDNA would likely be damage d as well; however, as discussed above, it is better protected and present in higher copy number than nuclear DNA, either of which could have resulted in less detected degradation. Drilling as a method to obtain bone powder for DNA isolation is fairly com mon (e.g., Bille et al., 2004; Rennick et al., 2005; Adler et al., 2011; Caputo et al., 2013; Mundorff et al., 2013; Hebda and Foran, 2015), therefore a systematic investigation of how drilling speed and pressure affect DNA quantity and quality from bone w ould be useful to forensic and archeological investigators. 127 Interesting comparisons can be made between this study and the research done by Mundorff and Davoren (2014) to assess inter - versus intra - elemental variation in DNA quantity and quality. Those au thors skeletonized three human males (47, 50, and 60 years old at the time of death who died within one year of each other), tested 55 elements from each cadaver, and ranked them from greatest to least based on the quantity and quality of nuclear DNA recov ered. The talus, calcaneus, the femur all produced full STR profiles, and ranked 5 th , 15 th , and 49 th in DNA quantification respectively. According to their associated National Institute of Justice Technical Report (Mundorff et al., 2013), the authors sam pled locations on each element so as to minimize interference for future research by avoiding common points where metric and visual rd above or below the midshaf t. Short bones (e.g., tarsals) were sampled at locations with that a single site was sufficient to collect the 200 mg of bone powder in most cases. This strate gy assumes homogeneity of DNA content throughout a bone, which the current research clearly demonstrates is inaccurate. In spite of this, the research presented here supports a couple lar among all three elements in both studies. Second, the talus and calcaneus had higher DNA quantity than the femoral diaphysis, based on the sampling location reported in Mundorff et al. (2013). However, the research presented here demonstrates that wh en other locations of the femur were assayed, it contained relatively more, similar, or less DNA than the tarsals. The fact that femoral DNA heterogeneity affected rank order among elements reinforces the importance of considering its influence on DNA rec overy in future investigations. 128 Another interesting result in the current research was that the demineralization method resulted in higher DNA yields from the femoral mid - diaphyses, while the non - demineralization method led to more DNA from the epiphyses. Full demineralization of bone has been shown to enhance DNA quantity and quality (Loreille et al., 2007; Seo et al., 2010; Amory et al., 2011); however, the sections of bone used in those studies generally originated from long bone diaphysis. Thirty - nine of forty samples used by Amory et al. (2011) were cut from the diaphysis of a femur, tibia, ulna, or radius, the exception being one mandible fragment (also dense cortical bone). Seo et al. (2010) utilized the diaphyses from eight femora and two tibiae. Loreille et al. (2007) assayed 10 samples, nine of which originated from unspecified long bones or femora, and one vertebra (Kimberly Andreaggi, personal communication). These authors postulated that full demineralization increased the recovery of analyz able DNA bound in crystal aggregates deep within bone matrix, which was based on Salamon et al. (2005) demonstrating that DNA was present in such aggregates of modern and fossilized bovine, porcine, and human bone. The aggregates made up to 50% of the min eral phase by weight, and protected DNA in the presence of harsh chemical treatment (e.g., sodium hypochlorite). Furthermore, they proposed that when these aggregates were dissolved by demineralization, quality DNA was released for analysis. The increase in DNA quantity and quality when demineralization was used in the present study, particularly at the mid - diaphysis, may have resulted from the liberation of DNA trapped within such mineral aggregates. Another possible influence on nuclear DNA yields was a n interaction between detergent and proteinase K in the tissue lysis buffer. Both SDS and SLS have a twelve carbon hydrophobic tail and a hydrophilic anionic head; however, they differ in polar head groups: SDS contains a sulfate while SLS has a carboxyla te attached by an amide linkage (reviewed by Priv é , 129 2007). SDS denatures most proteins through the disruption of noncovalent bonds within and between them by binding in a 1:2 ratio, adding a net negative charge to the protein (reviewed by Johnson, 2013). SLS acts in a similar manner, but tends to bind proteins more weakly and disassociates from them more easily than SDS; SLS is often used when permanent denaturation is undesired (Burgess and Deutscher, 2009). Hilz et al. (1975) demonstrated that SDS and proteinase K have an additive effect when degrading proteins. The authors postulated that SDS breaks native protein structure, increasing the number of accessible peptide bonds for proteinase K to cleave. However, proteinase K was also sensitive to SDS c oncentration: at 0.1% concentration enzyme activity dropped to 86%, and at a 1% concentration activity decreased to 5%. How SLS concentration affected proteinase K activity in the current research is unclear, but similarities between the detergents indica activity in demineralization buffer relative to 0.1% SDS in tissue lysis buffer. Proteinase K may have also been affected by EDTA concentration. Researchers have proposed that proteinase K has two cal occupied. Bajorath et al. (1988) used EDTA to decalcify proteinase K, which caused an 80% reduction in enzymatic activity. Conversely, M ü ller et al. (1994) suggested that the major role of calcium in a substantial reduction in enzymatic activity, but instead causes irreversible precipitation of the enzyme, reducing its effective concentration. The deminerali zation buffer used in the present study had 0.5 M EDTA, which could sequester calcium from proteinase K and reduce overall efficiency. Researchers that compared demineralization to non - demineralization protocols (Loreille et al., 2007; Seo et al., 2010; A mory et al., 2011) used about three times more proteinase K than what was applied in the current research. Increasing proteinase K 130 concentration for future investigations might compensate for any loss of enzymatic activity resulting from EDTA. Though sp eculative, the lower detergent and EDTA concentrations in tissue lysis buffer, compared to demineralization buffer, potentially increased DNA yields at the epiphyses. However, this assumes that epiphyseal cortical bone contains less nuclear DNA bound to h ydroxyapatite than the diaphysis. Demineralization under this context would result in decreased nuclear DNA recovery at the epiphyses relative to the midshaft diaphysis. Why mtDNA quantity was not similarly affected remains unclear. Investigating the us e of other detergents in demineralization buffer, and adjusting detergent and proteinase K concentrations to determine how they influence DNA recovery from diaphyseal versus epiphyseal bone, may help resolve why each digestion buffer worked better when app lied to different regions of the same bone. PCR inhibition was not detected during DNA amplification from macerated bone; however, it did occur during amplification of 17 of 168 DNAs extracted from surface exposed and buried bones. Sixteen of those DNA i solates came from the epiphyses or tarsals, fourteen of which were from week two or four time points. DNAs tested from the midshaft diaphysis were not inhibited throughout any portion of this research. The cause of this sporadic PCR inhibition is not cle ar; however, collagen in bone is a known inhibitor (Scholz et al., 1998), which is primarily eliminated postmortem by bacterial collagenases and gas - gangrene bacteria like Clostridium (Janaway, 1997; Dent et al., 2004). In the non - macerated bone, collagen might have naturally degraded into soluble peptides that co - extracted at the week two and four time points, but was not as abundant at subsequent time points due to environmental degradation. 131 There were several instances of PCR inhibition during quality assessment that were not detected during qPCR, wherein amplification did not occur until DNA extracts were diluted five - Rad, 2015), which are designed to alleviate PCR inhibition during quantitation. In contrast, the PCR cocktail used for the quality assay did not include adjuvants. Bovine serum albumin (BSA) can effectively alleviate PCR inhibition associat ed with humic substances present in soil (Kreader, 1996) and collagen from bone (Hebda, 2013); however, because DNAs were of bovine origin, BSA was not added to avoid potential DNA contamination. Chemical adjuvants like dimethyl sulfoxide were not tested, although they may alleviate inhibition in some instances. Dilution worked well to overcome inhibition during mtDNA amplification, but not as well for nuclear DNA when initial quantities were low. If the inclusion of BSA with the quality assay does not i ntroduce DNA contamination, then using it would likely improve the assay by reducing future occurrences of inhibition, increasing the likelihood of amplifying samples with low DNA quantity. Alternatively, if serum albumins from other species (e.g., sheep) are effective as adjuvants, then they could be used instead of BSA when bovine DNA is tested. Overall, this body of research demonstrated that recoverable nuclear and mitochondrial DNA varied depending on the femoral location assayed, and that DNA yields can differ as much or more within an element as among elements. DNA quantity along the femur was consistent in both model systems; however, an important question for forensic applications still needs to be addressed: does this apply to humans? The femur supports static body weight, facilitates locomotion, and has a diverse morphology, osseous tissue distribution, and an abundance of both articulating and non - 132 history of being preferent ially selected for DNA testing in research and casework made it the ideal element to establish that DNA is heterogeneously distributed in bone. The causes of DNA yield heterogeneity remain hypothetical at this juncture, but it likely results from a comple x combination of the factors discussed above, and others yet to be considered. Specific morphological features of bone may turn out to be better indicators of DNA yield than which element is assayed; however, it seems unlikely that a single element, or lo cation within one, will be ideal for best recovering DNA under all circumstances. Investigators determining optimal bones for DNA isolation should consider, when possible, a complex set of variables such as morphology, density, antemortem age, and the env ironment in which skeletal material resides. 133 CONCLUSION Previous researchers have demonstrated that DNA yields vary between osseous tissue types and among different bones. The research conducted here was used to characterize DNA heterogenei ty in femora, one of the most commonly utilized skeletal elements in research and forensic casework. Femoral DNA quantity has a predictable proximal/distal heterogeneity that varies quite substantially, demonstrating that location on a bone can be just as important as which element is analyzed, a critical consideration for future investigations that focus on optimizing DNA recovery from bone. Furthermore, nuclear and mitochondrial DNA follow similar distributions of heterogeneity; therefore, the same loca tion on an element can be utilized regardless of which DNA type is assayed. Demineralization and non - demineralization techniques can significantly enhance (or reduce) DNA recovery; however, they do so at different locations. Though more research is neede d to understand such phenomena, these results exemplify the importance of not relying on a single digestion technique for DNA isolation. Finally, the environment and duration of exposure can drastically alter DNA quantity, quality, and heterogeneity of sk eletal material, potentially influencing which bone (or location on it) should be assayed to optimize DNA recovery. Overall, the research presented here clearly demonstrates that DNA is heterogeneously distributed in bone, DNA quantity can vary more withi n an element than among them, and that environmental conditions (e.g., burial) affect DNA quantity and quality substantially over time. Knowledge about all these factors will ultimately improve practices for recovering DNA in both research and forensic la boratories. 134 APPENDICES 135 APPENDIX A : NORMALITY TEST AND PAIRWISE COMPARISON TABLES FOR PORCINE AND BOVINE QUANTIF I CATION DATA Table A 1: p - Region num ber is listed in the first column, and correspond to Table 3. Bolded values represent regions that did not follow a normal - Darling test, SW = Shapiro - Wilk test Region Mitochondrial DNA Nuclear DNA Tissue Lysis Dem ineralization Tissue Lysis Demineralization AD SW AD SW AD SW AD SW 1 0.481 0.435 0.468 0.468 0.008 0.006 0.651 0.625 2 0.477 0.470 0.234 0.269 0.423 0.378 0.687 0.808 3 0.352 0.348 0.373 0.450 0.004 0.003 0.534 0.640 4 0.935 0.911 0.896 0.934 0.005 0.003 0.567 0.719 5 0.532 0.499 0.452 0.534 0.090 0.078 0.613 0.637 6 0.138 0.109 0.489 0.238 0.333 0.302 0.012 0.008 7 0.279 0.205 0.263 0.269 0.073 0.060 0.315 0.377 8 0.912 0.913 0.590 0.607 0.410 0.353 0.913 0.956 9 0.464 0.479 0.982 0.995 0.161 0 .145 0.380 0.320 10 0.091 0.072 < 0.001 0.001 0.567 0.538 0.481 0.347 11 0.006 0.005 0.187 0.145 0.005 0.005 0.735 0.725 12 0.251 0.200 0.204 0.149 0.719 0.689 0.332 0.245 13 0.070 0.069 0.455 0.420 0.509 0.778 0.607 0.663 14 0.178 0.247 0.252 0.280 0 .263 0.305 0.651 0.635 Pooled Regions 1 6 0.002 0.001 0.0004 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 7 9 0.242 0.118 0.316 0.243 0.018 0.019 0.063 0.030 10 12 0.022 0.004 < 0.0001 < 0.0001 0.866 0.785 0.585 0.297 13 and 14 0.507 0.542 0.628 0.604 0.173 0.346 0.389 0.323 13 6 Table A 2 : p - - 01 and C - 02) Region number is listed in the first column, and correspond to Table 3. Bolded values represent regions that did not follow a normal distr - Darling test, SW = Shapiro - Wilk test Region Mitochondrial DNA Nuclear DNA Tissue Lysis Demineralization Tissue Lysis Demineralization AD SW AD SW AD SW AD SW 1 0.010 0.009 0.003 0.003 0.009 0.006 0.051 0.053 2 0.002 0.001 0.028 0.022 0.144 0.131 0.097 0.127 3 0.001 0.002 0.024 0.019 0.502 0.611 0.006 0.006 4 0.232 0.169 0.022 0.017 0.144 0.177 0.070 0.114 5 0.003 0.002 0.290 0.235 0.393 0.465 0.014 0.014 6 0.072 0.062 0.098 0.084 0.424 0.464 0.301 0.277 7 0.029 0.018 0.044 0.032 0.048 0.032 0.104 0.131 8 0.027 0.014 0.152 0.133 0.178 0.190 0.098 0.094 9 0.054 0.048 0.140 0.171 < 0.001 < 0.001 0.008 0.006 10 0.082 0.074 0.004 0.003 < 0.001 < 0.001 0.002 0.001 11 0.062 0.052 0.129 0.126 0.001 < 0.001 0.001 0.0 01 12 0.716 0.842 0.952 0.946 0.009 0.011 0.010 0.007 13 0.552 0.549 0.356 0.348 0.002 0.001 0.001 < 0.001 14 0.142 0.109 0.021 0.020 0.007 0.006 0.003 0.002 Pooled Regions 1 6 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.021 0.028 < 0.0001 < 0.000 1 7 9 < 0.0001 0.0003 0.001 0.002 < 0.0001 < 0.0001 0.0004 0.001 10 12 0.004 0.003 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 13 and 14 0.069 0.034 0.005 0.006 < 0.0001 < 0.0001 < 0.0001 < 0.0001 137 Table A 3 : p - Values from Normality Test - 01 and C - 02) Region number is listed in the first column, and correspond to Table 3. Bolded values represent regions that did not follow a normal Anderson - Darling test, SW = Shapiro - Wilk test Region Mitochondrial DNA Nuclear DNA Tissue Lysis Demineralization Tissue Lysis Demineralization AD SW AD SW AD SW AD SW 1 0.592 0.553 0.799 0.835 0.465 0.465 0.438 0.515 2 0.932 0.982 0.102 0.118 0.060 0.053 0.503 0.763 3 0.529 0.572 0.240 0.195 0.701 0.709 0.963 0.988 4 0.341 0.340 0.051 0.054 0.789 0.856 0.032 0.032 5 0.151 0.167 0.370 0.313 0.304 0.326 0.119 0.300 6 0.084 0.092 0.032 0.022 0.281 0.300 0.455 0.461 7 0.328 0.340 0.305 0.363 0.494 0 .408 0.889 0.983 8 0.673 0.636 0.753 0.730 0.992 0.995 0.595 0.668 9 0.058 0.065 0.776 0.857 0.636 0.746 0.095 0.129 10 0.691 0.671 0.873 0.937 0.191 0.202 0.496 0.493 11 0.094 0.086 0.725 0.764 0.262 0.239 0.765 0.749 12 0.780 0.872 0.744 0.780 0.561 0.533 0.103 0.101 13 0.573 0.559 0.122 0.117 0.946 0.969 0.721 0.686 14 0.584 0.525 0.674 0.610 0.075 0.080 0.450 0.448 Pooled Regions 1 6 0.036 0.038 < 0.0001 < 0.0001 0.612 0.594 0.690 0.533 7 9 0.060 0.019 0.287 0.172 0.895 0.814 0.51 4 0.499 10 12 0.334 0.314 0.687 0.898 0.214 0.118 0.021 0.023 13 and 14 0.397 0.264 0.071 0.053 0.170 0.129 0.453 0.553 138 Table A4 : p - Values from Pairwise Comparisons of Median mtDNA Yields Derived from Porcine Bones Digested in Tissue Lysis Buffer R egion identifiers are listed in the first column and row, and correspond to identifiers in Table 3. Pairwise comparisons wer e - in dicate a significant difference between regions compared. P - values in red indicate disagreement in significance between tissue lysis and demineralization buffer. Region 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 1 0.845 0.913 0.792 0.089 0.410 0.008 0.020 0.002 < 0.0001 < 0.0001 < 0.0001 0.004 < 0.0001 2 0.845 1 0.764 0.952 0.066 0.322 0.005 0.014 0.002 < 0.0001 < 0.0001 < 0.0001 0.003 < 0.0001 3 0.913 0.764 1 0.709 0.111 0.475 0.010 0.026 0.003 < 0.0001 < 0.0001 < 0.0001 0.006 < 0.0001 4 0.792 0.952 0.709 1 0 .049 0.277 0.003 0.009 0.001 < 0.0001 < 0.0001 < 0.0001 0.002 < 0.0001 5 0.089 0.066 0.111 0.049 1 0.380 0.332 0.529 0.174 0.001 0.003 0.001 0.244 0.057 6 0.410 0.322 0.475 0.277 0.380 1 0.065 0.132 0.025 < 0.0001 < 0.0001 < 0.0001 0.041 0.005 7 0.008 0 .005 0.010 0.003 0.332 0.065 1 0.733 0.698 0.012 0.044 0.015 0.846 0.351 8 0.020 0.014 0.026 0.009 0.529 0.132 0.733 1 0.465 0.004 0.019 0.006 0.592 0.203 9 0.002 0.002 0.003 0.001 0.174 0.025 0.698 0.465 1 0.035 0.105 0.041 0.846 0.587 10 < 0.0001 < 0. 0001 < 0.0001 < 0.0001 0.001 < 0.0001 0.012 0.004 0.035 1 0.625 0.944 0.021 0.117 11 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.003 < 0.0001 0.044 0.019 0.105 0.625 1 0.675 0.069 0.280 12 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.001 < 0.0001 0.015 0.006 0.041 0. 944 0.675 1 0.025 0.134 13 0.004 0.003 0.006 0.002 0.244 0.041 0.846 0.592 0.846 0.021 0.069 0.025 1 0.461 14 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.057 0.005 0.351 0.203 0.587 0.117 0.280 0.134 0.461 1 139 Table A5 : p - Values from Pairwise Comparisons of M edian mtDNA Yields Derived from Porcine Bones Digested in Demineralization Buffer Region identifiers are listed in the first column and row, and correspond to identifiers in Table 3. Pairwise comparisons we re procedure (two - indicate a significant difference between regions compared. P - values in red indicate disagreement in significance between tissue lysis and demineralization buffer. Region 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 1 0.847 0.770 0.945 0.144 0.847 0.009 0.027 0.001 < 0.0001 < 0.0001 < 0.0001 0.005 0.002 2 0.847 1 0.628 0.794 0.204 1 0.016 0.044 0.001 < 0.0001 < 0.0001 < 0.0001 0.008 0.004 3 0.770 0.628 1 0.823 0.079 0.628 0.004 0.012 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.002 0.001 4 0.945 0.794 0.823 1 0.126 0.794 0.008 0.023 0.001 < 0.0001 < 0.0001 < 0.0001 0.004 0.002 5 0.144 0.204 0.079 0.126 1 0.204 0.255 0.455 0.053 0.001 0.001 0.001 0.171 0.109 6 0.847 1 0.628 0.794 0.204 1 0 .016 0.044 0.001 < 0.0001 < 0.0001 < 0.0001 0.008 0.004 7 0.009 0.016 0.004 0.008 0.255 0.016 1 0.695 0.428 0.020 0.031 0.028 0.817 0.644 8 0.027 0.044 0.012 0.023 0.455 0.044 0.695 1 0.236 0.007 0.011 0.009 0.533 0.393 9 0.001 0.001 < 0.0001 0.001 0.05 3 0.001 0.428 0.236 1 0.127 0.173 0.159 0.574 0.741 10 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.001 < 0.0001 0.020 0.007 0.127 1 0.872 0.908 0.037 0.064 11 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.001 < 0.0001 0.031 0.011 0.173 0.872 1 0.963 0.054 0.090 12 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.001 < 0.0001 0.028 0.009 0.159 0.908 0.963 1 0.049 0.082 13 0.005 0.008 0.002 0.004 0.171 0.008 0.817 0.533 0.574 0.037 0.054 0.049 1 0.817 14 0.002 0.004 0.001 0.002 0.109 0.004 0.644 0.393 0.741 0.064 0.090 0.082 0.8 17 1 140 Table A6 : p - Values from Multiple and Individual Pairwise Comparisons of Pooled Median mtDNA and DNA Yields Derived from Fresh Porcine and Bovine Bones Digested in Tissue Lysis and Demineralization Buffer Pooled region identifiers are listed in the f irst column, and correspond to identifiers in Table 3. Multiple pairwise comparisons were - Bolded values indicate a significant diffe rence between regions compared. Pooled Regions Multiple Individual Porcine 1 6 7 -- 9 10 12 13 14 mtDNA TL DM TL DM TL DM TL DM 1 6 1 1 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 7 -- 9 < 0.0001 < 0.0001 1 1 0.001 0.002 0.041 0.42 0 10 -- 12 < 0.0001 < 0.0001 0.001 0.002 1 1 < 0.0001 < 0.0001 Nuclear DNA TL DM TL DM TL DM TL DM 1 6 1 1 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 7 9 < 0.0001 < 0.0001 1 1 0.001 0.001 0.035 0.002 10 12 < 0.0001 < 0.0001 0.001 0. 001 1 1 < 0.0001 < 0.0001 Pooled Regions Multiple Individual Bovine 1 6 7 -- 9 10 12 13 14 mtDNA TL DM TL DM TL DM TL DM 1 6 1 1 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0001 0.006 7 -- 9 < 0.0001 < 0.0001 1 1 0.020 0.0004 0.345 0.838 10 -- 12 < 0.0001 < 0.0001 0.020 0.0004 1 1 0.070 0.012 Nuclear DNA TL DM TL DM TL DM TL DM 1 6 1 1 < 0.0001 0.011 < 0.0001 < 0.0001 < 0.0001 0.008 7 9 < 0.0001 0.011 1 1 0.0158 0.008 0.079 0.576 10 12 < 0.0001 < 0.0001 0.0158 0.008 1 1 0.041 0.014 141 Table A7 : p - Values from Pairwise Comparisons of Median Nuclear DNA Yields Derived from Porcine Bones Digested in Tissue Lysis Buffer Region identifiers are listed in the first column and row, and correspond to identifiers in Table 3. Pairwise comparisons were - indicate a significant difference between regions compared. P - values in green indicate disagreement in significance between tissue lysis and demineralization buffer. Region 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 1 0.893 0.658 0.969 0.023 0.073 0.003 0.026 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.001 < 0.0001 2 0.893 1 0.770 0.922 0.040 0.110 0.006 0.044 0.001 < 0.0001 < 0.0001 < 0. 0001 0.002 < 0.0001 3 0.658 0.770 1 0.686 0.068 0.177 0.011 0.074 0.001 < 0.0001 < 0.0001 < 0.0001 0.004 0.001 4 0.969 0.922 0.686 1 0.026 0.079 0.003 0.029 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.001 < 0.0001 5 0.023 0.040 0.068 0.026 1 0.636 0.457 0.969 0.151 0.005 0.007 0.001 0.277 0.098 6 0.073 0.110 0.177 0.079 0.636 1 0.235 0.664 0.056 0.001 0.002 < 0.0001 0.118 0.033 7 0.003 0.006 0.011 0.003 0.475 0.235 1 0.451 0.470 0.035 0.049 0.011 0.709 0.347 8 0.026 0.044 0.074 0.029 0.969 0.664 0.451 1 0.1 40 0.004 0.007 0.001 0.260 0.090 9 < 0.0001 0.001 0.001 < 0.0001 0.969 0.056 0.470 0.140 1 0.167 0.214 0.068 0.727 0.828 10 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.005 0.001 0.035 0.004 0.167 1 0.889 0.658 0.083 0.244 11 < 0.0001 < 0.0001 < 0.0001 < 0.000 1 0.007 0.002 0.049 0.007 0.214 0.889 1 0.560 0.111 0.305 12 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.001 < 0.0001 0.001 0.001 0.068 0.658 0.560 1 0.030 0.108 13 0.001 0.002 0.004 0.001 0.277 0.118 0.709 0.260 0.727 0.083 0.111 0.030 1 0.571 14 < 0.0001 < 0.0001 0.001 < 0.0001 0.098 0.033 0.347 0.090 0.828 0.244 0.305 0.108 0.571 1 142 Table A8 : p - Values from Pairwise Comparisons of Median Nuclear DNA Yields Derived from Porcine Bones Digested in Demineralization Buffer Region identifiers are listed in the fi rst column and row, and correspond to identifiers in Table 3. Pairwise comparisons were - indicate a significant difference betwee n regions compared. P - values in green indicate disagreement in significance between tissue lysis and demineralization buffer. Region 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 1 0.994 0.776 0.872 0.201 0.678 0.016 0.027 0.014 < 0.0001 < 0.0001 < 0.0001 0.002 0.0 01 2 0.994 1 0.770 0.878 0.204 0.683 0.016 0.027 0.015 < 0.0001 < 0.0001 < 0.0001 0.002 0.001 3 0.776 0.770 1 0.655 0.118 0.484 0.007 0.012 0.006 < 0.0001 < 0.0001 < 0.0001 0.001 < 0.0001 4 0.872 0.878 0.655 1 0.264 0.799 0.025 0.040 0.022 < 0.0001 < 0. 0001 < 0.0001 0.003 0.001 5 0.201 0.204 0.118 0.264 1 0.389 0.258 0.348 0.242 0.001 < 0.0001 < 0.0001 0.070 0.035 6 0.678 0.683 0.484 0.799 0.389 1 0.046 0.072 0.042 < 0.0001 < 0.0001 < 0.0001 0.007 0.003 7 0.016 0.016 0.007 0.025 0.258 0.046 1 0.847 0. 969 0.025 0.018 0.011 0.496 0.326 8 0.027 0.027 0.012 0.040 0.348 0.072 0.847 1 0.817 0.015 0.010 0.006 0.382 0.240 9 0.014 0.015 0.006 0.022 0.242 0.042 0.969 0.817 1 0.027 0.020 0.012 0.520 0.346 10 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.001 < 0.0001 0 .025 0.015 0.027 1 0.902 0.764 0.117 0.205 11 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.018 0.010 0.020 0.902 1 0.859 0.091 0.165 12 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.011 0.006 0.012 0.764 0.859 1 0.062 0.117 13 0.002 0.002 0.001 0.003 0.070 0.007 0.496 0.382 0.520 0.117 0.091 0.062 1 0.764 14 0.001 0.001 < 0.0001 0.001 0.035 0.003 0.326 0.240 0.346 0.205 0.165 0.117 0.764 1 143 Table A9 : p - Values from Pairwise Comparisons of Median mtDNA Yields Derived from Bovine Bone s Digested in Tissue Lysis Buffer Region identifiers are listed in the first column and row, and correspond to identifiers in Table 3. Pairwise comparisons we re - tailed). Significance was determ indicate a significant difference between regions compared. P - values in blue indicate disagreement in significance between tissue lysis and demineralization buffer. Region 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 1 0.376 0.553 0.559 0.498 0.376 0.018 0.045 0.002 < 0.0001 < 0.0001 < 0.0001 0.007 0.004 2 0.723 1 0.811 0.817 0.746 0.595 0.045 0.099 0.005 < 0.0001 0.001 0.001 0.019 0.011 3 0.553 0.811 1 0.994 0.933 0.770 0.077 0.159 0.011 < 0.0001 0.003 0.001 0.036 0.021 4 0.559 0.817 0.811 1 0.926 0.764 0.075 0.157 0.011 < 0.0001 0.003 0.001 0.035 0.021 5 0.498 0.746 0.933 0.926 1 0.835 0.092 0.185 0.014 < 0.0001 0.003 0.002 0.044 0.027 6 0.376 0.595 0.770 0.764 0.835 1 0.139 0.264 0.024 0.001 0.007 0.004 0.070 0.045 7 0.018 0 .045 0.077 0.075 0.092 0.139 1 0.099 0.437 0.048 0.215 0.157 0.741 0.595 8 0.045 0.099 0.159 0.157 0.185 0.264 0.717 1 0.255 0.019 0.109 0.075 0.488 0.372 9 0.002 0.005 0.011 0.011 0.014 0.024 0.437 0.255 1 0.230 0.644 0.523 0.655 0.805 10 < 0.0001 < 0. 0001 < 0.0001 < 0.0001 < 0.0001 0.001 0.048 0.019 0.230 1 0.460 0.574 0.099 0.148 11 < 0.0001 0.001 0.003 0.003 0.003 0.007 0.215 0.109 0.644 0.460 1 0.859 0.364 0.479 12 < 0.0001 0.001 0.001 0.001 0.002 0.004 0.157 0.075 0.523 0.574 0.859 1 0.278 0.376 13 0.007 0.019 0.036 0.035 0.044 0.070 0.741 0.488 0.655 0.099 0.364 0.278 1 0.841 14 0.004 0.011 0.021 0.021 0.027 0.045 0.595 0.372 0.805 0.148 0.479 0.376 0.841 1 144 Table A10 : p - Values from Pairwise Comparisons of Median mtDNA Yields Derived from Bovi ne Bones Digested in Demineralization Buffer Region identifiers are listed in the first column and row, and correspond to identifiers in Table 3. Pairwise comparisons we re - tailed). Significance indicate a significant difference between regions compared. P - values in blue indicate disagreement in significance between tissue lysis and demineralization buffer. Region 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 1 0 .945 0.951 0.811 0.585 0.836 0.031 0.144 0.031 < 0.0001 0.001 0.005 0.085 0.111 2 0.945 1 0.986 0.866 0.538 0.782 0.051 0.126 0.026 < 0.0001 < 0.0001 0.004 0.073 0.096 3 0.951 0.896 1 0.764 0.628 0.884 0.069 0.161 0.036 0.001 0.001 0.006 0.096 0.126 4 0 .811 0.866 0.764 1 0.432 0.655 0.034 0.089 0.016 < 0.0001 < 0.0001 0.002 0.050 0.067 5 0.585 0.538 0.628 0.432 1 0.735 0.183 0.360 0.106 0.003 0.004 0.025 0.239 0.295 6 0.835 0.782 0.884 0.655 0.735 1 0.095 0.210 0.051 0.001 0.001 0.010 0.129 0.166 7 0. 060 0.051 0.069 0.034 0.183 0.095 1 0.678 0.776 0.098 0.124 0.360 0.878 0.776 8 0.144 0.126 0.161 0.089 0.360 0.210 0.678 1 0.484 0.038 0.051 0.183 0.794 0.896 9 0.031 0.026 0.036 0.016 0.106 0.051 0.776 0.484 1 0.171 0.210 0.528 0.661 0.569 10 < 0.0001 < 0.0001 0.001 < 0.0001 0.003 0.001 0.098 0.038 0.171 1 0.908 0.460 0.070 0.052 11 0.001 < 0.0001 0.001 < 0.0001 0.004 0.001 0.124 0.051 0.210 0.908 1 0.533 0.090 0.068 12 0.005 0.004 0.006 0.002 0.025 0.010 0.360 0.187 0.528 0.070 0.533 1 0.285 0.230 13 0.085 0.073 0.096 0.050 0.239 0.129 0.878 0.794 0.661 0.070 0.090 0.285 1 0.896 14 0.111 0.096 0.126 0.067 0.295 0.166 0.776 0.896 0.569 0.052 0.068 0.230 0.869 1 145 Table A11 : p - Values from Pairwise Comparisons of Median Nuclear DNA Yields Derived fro m Bovine Bones Digested in Tissue Lysis Buffer Region identifiers are listed in the first column and row, and correspond to identifiers in Table 3. Pairwise comparisons we re - tailed). Significan indicate a significant difference between regions compared. P - values in purple indicate disagreement in significance between tissue lysis and demineralization buffer. Region 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 1 0.378 0.758 0.441 0.393 0.378 0.002 0.006 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 2 0.378 1 0.566 0.911 0.979 0.709 0.029 0.065 0.009 < 0.0001 0.001 < 0.0001 0.003 0.002 3 0.758 0.566 1 0.644 0.585 0.344 0.006 0.016 0.001 < 0.0001 < 0.00 01 < 0.0001 < 0.0001 < 0.0001 4 0.441 0.911 0.644 1 0.933 0.628 0.022 0.051 0.007 < 0.0001 < 0.0001 < 0.0001 0.002 0.002 5 0.393 0.979 0.585 0.933 1 0.689 0.027 0.061 0.008 < 0.0001 0.001 < 0.0001 0.003 0.002 6 0.210 0.709 0.344 0.628 0.689 1 0.070 0.14 1 0.026 < 0.0001 0.003 < 0.0001 0.002 0.002 7 0.002 0.029 0.006 0.022 0.027 0.070 1 0.732 0.675 0.083 0.228 0.075 0.448 0.391 8 0.006 0.065 0.016 0.051 0.061 0.141 0.732 1 0.446 0.038 0.122 0.034 0.271 0.230 9 < 0.0001 0.009 0.001 0.007 0.008 0.026 0.67 5 0.446 1 0.188 0.432 0.173 0.735 0.661 10 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.083 0.038 0.188 1 0.595 0.963 0.328 0.380 11 < 0.0001 0.001 < 0.0001 < 0.0001 0.001 0.003 0.228 0.122 0.432 0.595 1 0.564 0.655 0.729 12 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.075 0.034 0.173 0.963 0.564 1 0.306 0.356 13 < 0.0001 0.003 < 0.0001 0.002 0.003 0.002 0.448 0.271 0.735 0.328 0.655 0.306 1 0.920 14 < 0.0001 0.002 < 0.0001 0.002 0.002 0.002 0.391 0.230 0.661 0.380 0.729 0.356 0.9 20 1 146 Table A12 : p - Values from Pairwise Comparisons of Median Nuclear DNA Yields Derived from Bovine Bones Digested in Demineralization Buffer Region identifiers are listed in the first column and row, and correspond to identifiers in Table 3. Pairwise co mparisons were - indicate a significant difference between regions compared. P - values in purple indicate disagreement in significa nce between tissue lysis and demineralization buffer. Region 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 1 0.805 0.859 0.805 0.853 0.513 0.079 0.210 0.133 < 0.0001 0.002 0.009 0.064 0.106 2 0.805 1 0.945 1 0.951 0.683 0.131 0.313 0.210 0.001 0.004 0.019 0.108 0.1 71 3 0.859 0.945 1 0.945 0.994 0.633 0.115 0.285 0.185 0.001 0.003 0.016 0.093 0.150 4 0.805 1 0.945 1 0.951 0.683 0.131 0.313 0.210 0.001 0.004 0.019 0.108 0.171 5 0.853 0.951 0.994 0.951 1 0.639 0.116 0.285 0.188 0.001 0.003 0.016 0.095 0.152 6 0.513 0.683 0.633 0.683 0.639 1 0.271 0.548 0.397 0.004 0.013 0.052 0.230 0.336 7 0.079 0.131 0.115 0.131 0.116 0.271 1 0.617 0.799 0.078 0.164 0.401 0.920 0.890 8 0.210 0.313 0.285 0.313 0.285 0.548 0.617 1 0.805 0.024 0.058 0.180 0.548 0.717 9 0.133 0.210 0.185 0.210 0.188 0.397 0.799 0.805 1 0.044 0.099 0.274 0.723 0.908 10 < 0.0001 0.001 0.001 0.001 0.001 0.004 0.078 0.024 0.044 1 0.712 0.356 0.096 0.057 11 0.002 0.004 0.003 0.004 0.003 0.013 0.164 0.058 0.099 0.712 1 0.579 0.196 0.126 12 0.009 0.019 0 .016 0.019 0.016 0.052 0.401 0.180 0.274 0.356 0.579 1 0.460 0.328 13 0.064 0.108 0.093 0.108 0.095 0.230 0.920 0.548 0.723 0.096 0.196 0.460 1 0.811 14 0.106 0.171 0.150 0.171 0.152 0.336 0.890 0.717 0.908 0.057 0.126 0.328 0.811 1 147 APPENDIX B: MITOCHO NDRIAL AND NUCLEAR DNA QUANTIFICATION DATA AND INDIVIDUAL PCR AMPLIFICATION RESULTS FROM FRESH PORCINE FEMORA AND TARSALS Table B 1: mtDNA Quantification of P - 01 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extrac t volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. P - 01 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL ) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 49 28.0 9.30 5.31 260.40 2 Midshaft Diaphysis (Distal to Region 1) 50 29.0 6.45 3.74 187.05 3 Diaphysis (Proximal to Region 1) 49 26.0 18.00 9.55 468.00 4 Diaphysis (Distal to Regio n 2) 50 26.0 8.56 4.45 222.56 5 Diaphysis (Proximal to Region 3) 51 30.0 31.20 18.35 936.00 6 Diaphysis (Distal to Region 4) 49 29.5 35.10 21.13 1035.45 7 Proximal Metaphysis 49 27.0 56.30 31.02 1520.10 8 Distal Metaphysis 50 28.0 55.60 31.14 1556.80 9 Articulating Surface 49 30.0 97.60 59.76 2928.00 10 Distal Epiphysis 51 35.0 472.00 323.92 16520.00 11 Femoral Head 51 36.0 248.00 175.06 8928.00 12 Trochanter 50 34.0 253.00 172.04 8602.00 13 Calcaneus 50 29.0 74.90 43.44 2172.10 14 Talus 50 35.0 118.00 82.60 4130.00 RB Reagent Blank - 29.5 0.02 - 0.59 148 Table B 2: mtDNA Quantification of P - 01 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in or der to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. P - 01 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midsha ft Diaphysis 51 25.5 26.00 13.00 663.00 2 Midshaft Diaphysis (Distal to Region 1) 51 29.5 18.20 10.53 536.90 3 Diaphysis (Proximal to Region 1) 20 29.0 24.60 14.27 713.40 4 Diaphysis (Distal to Region 2) 50 29.5 22.00 12.98 649.00 5 Diaphysis (Proximal to Region 3) 51 30.0 38.20 22.47 1146.00 6 Diaphysis (Distal to Region 4) 49 30.0 30.40 18.61 912.00 7 Proximal Metaphysis 51 28.0 66.50 36.51 1862.00 8 Distal Metaphysis 50 28.0 62.90 35.22 1761.20 9 Articulating Surface 50 30.0 123.00 73.80 3690.00 10 Distal Epiphysis 51 32.0 311.00 195.14 9952.00 11 Femoral Head 51 34.5 358.00 242.18 12351.00 12 Trochanter 51 35.0 316.00 216.86 11060.00 13 Calcaneus 49 35.0 70.90 50.64 2481.50 14 Talus 49 29.5 112.00 67.43 3304.00 RB Reagent Blank - 29.0 0.0 0 - 0.10 149 Table B3 : mtDNA Quantification of P - 02 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. P - 02 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 26.0 23.50 11.98 611.00 2 Midshaft D iaphysis (Distal to Region 1) 51 28.0 9.22 5.06 258.16 3 Diaphysis (Proximal to Region 1) 50 30.5 9.53 5.81 290.67 4 Diaphysis (Distal to Region 2) 49 30.0 9.15 5.60 274.50 5 Diaphysis (Proximal to Region 3) 50 29.0 16.60 9.63 481.40 6 Diaphysis (Dista l to Region 4) 49 27.5 12.30 6.90 338.25 7 Proximal Metaphysis 51 26.0 57.40 29.26 1492.40 8 Distal Metaphysis 50 28.0 45.60 25.54 1276.80 9 Articulating Surface 51 30.0 55.00 32.35 1650.00 10 Distal Epiphysis 51 40.0 187.00 146.67 7480.00 11 Femoral Head 50 40.0 107.00 85.60 4280.00 12 Trochanter 49 40.0 110.00 89.80 4400.00 13 Calcaneus 50 34.0 57.50 39.10 1955.00 14 Talus 50 40.0 64.40 51.52 2576.00 RB Reagent Blank - 29.0 0.00 - 0.09 150 Table B4 : mtDNA Quantification of P - 02 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. P - 02 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 28.0 29.20 16.35 817.60 2 Midshaft Diaphysis (Distal to Region 1) 49 27.0 27.20 14.99 734.40 3 Dia physis (Proximal to Region 1) 51 30.5 14.60 8.73 445.30 4 Diaphysis (Distal to Region 2) 49 29.5 15.90 9.57 469.05 5 Diaphysis (Proximal to Region 3) 51 29.0 21.60 12.28 626.40 6 Diaphysis (Distal to Region 4) 51 30.0 17.90 10.53 537.00 7 Proximal Meta physis 51 30.0 61.20 36.00 1836.00 8 Distal Metaphysis 51 28.5 48.80 27.27 1390.80 9 Articulating Surface 49 30.0 76.70 46.96 2301.00 10 Distal Epiphysis 51 38.0 131.00 97.61 4978.00 11 Femoral Head 50 39.0 169.00 131.82 6591.00 12 Trochanter 50 39.0 268.00 209.04 10452.00 13 Calcaneus 51 35.0 62.90 43.17 2201.50 14 Talus 50 38.5 68.90 53.05 2652.65 RB Reagent Blank - 28.0 0.00 - 0.00 151 Table B5 : mtDNA Quantification of P - 03 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. ND = No Data P - 03 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extrac t Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 31.0 27.90 16.96 864.90 2 Midshaft Diaphysis (Distal to Region 1) 51 0.0 ND ND ND 3 Diaphysis (Proximal to Region 1) 50 29.0 44.70 25.93 1296.30 4 Diaphysis (Distal to Region 2) 51 30.0 26.20 15.41 786.00 5 Diaphysis (Proximal to Region 3) 49 30.0 66.70 40.84 2001.00 6 Diaphysis (Distal to Region 4) 51 27.5 46.60 25.13 1281.50 7 Proximal Metaphysis 49 30.0 128.00 73.14 3584.00 8 Distal Metaph ysis 49 27.0 79.40 43.75 2143.80 9 Articulating Surface 49 30.0 118.00 72.24 3540.00 10 Distal Epiphysis 50 35.0 251.00 175.70 8785.00 11 Femoral Head 51 37.0 136.00 98.67 5032.00 12 Trochanter 49 32.0 275.00 179.59 8800.00 13 Calcaneus 51 31.5 90.30 55.77 2844.45 14 Talus 51 32.0 108.00 67.76 3456.00 RB Reagent Blank - 25.0 0.06 - 1.50 152 Table B6 : mtDNA Quantification of P - 03 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. P - 03 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 28.5 42.40 23.69 1208.40 2 Midshaft Diaphysis (Distal to Region 1) 51 28.0 41.40 22.73 1159.20 3 Diaphysis (Proximal to Region 1) 49 27.0 33.00 18.18 891.00 4 Diaphysis (Distal to Region 2) 49 26.0 39.0 0 20.69 1014.00 5 Diaphysis (Proximal to Region 3) 49 29.0 79.20 46.87 2296.80 6 Diaphysis (Distal to Region 4) 50 26.0 36.70 19.08 954.20 7 Proximal Metaphysis 51 29.5 138.00 79.82 4071.00 8 Distal Metaphysis 49 30.0 99.40 53.68 2683.80 9 Articulatin g Surface 51 27.0 93.10 49.29 2513.70 10 Distal Epiphysis 49 30.0 200.00 122.45 6000.00 11 Femoral Head 49 36.0 193.00 141.80 6948.00 12 Trochanter 50 29.5 195.00 115.05 5752.50 13 Calcaneus 51 29.5 99.30 57.44 2929.35 14 Talus 50 30.0 107.00 64.20 3210.00 RB Reagent Blank - 27.0 0.00 - 0.00 153 Table B7 : mtDNA Quantification of P - 04 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. P - 04 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 2 8.0 11.90 6.66 333.20 2 Midshaft Diaphysis (Distal to Region 1) 50 25.0 10.60 5.30 265.00 3 Diaphysis (Proximal to Region 1) 50 29.0 13.10 7.60 379.90 4 Diaphysis (Distal to Region 2) 52 28.0 19.80 10.66 554.40 5 Diaphysis (Proximal to Region 3) 51 30. 0 61.10 35.94 1833.00 6 Diaphysis (Distal to Region 4) 49 27.0 32.20 17.74 869.40 7 Proximal Metaphysis 51 31.5 83.90 51.82 2642.85 8 Distal Metaphysis 49 24.5 91.70 45.85 2246.65 9 Articulating Surface 50 27.0 71.60 38.66 1933.20 10 Distal Epiphysis 51 32.5 257.00 163.77 8352.50 11 Femoral Head 51 35.0 133.00 91.27 4655.00 12 Trochanter 51 32.5 141.00 89.85 4582.50 13 Calcaneus 51 30.0 66.10 38.88 1983.00 14 Talus 50 30.0 85.90 51.54 2577.00 RB Reagent Blank - 23.5 0.00 - 0.00 154 Table B8 : mtDN A Quantification of P - 04 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per m g of bone powder. P - 04 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 29.5 19.50 11.51 575.25 2 Midshaft Diaphysis (Distal to R egion 1) 51 23.5 29.80 13.73 700.30 3 Diaphysis (Proximal to Region 1) 50 28.5 22.40 12.77 638.40 4 Diaphysis (Distal to Region 2) 50 24.0 30.20 14.50 724.80 5 Diaphysis (Proximal to Region 3) 50 26.0 52.70 27.40 1370.20 6 Diaphysis (Distal to Region 4 ) 51 26.0 28.50 14.53 741.00 7 Proximal Metaphysis 49 29.0 56.70 33.56 1644.30 8 Distal Metaphysis 51 28.0 67.70 37.17 1895.60 9 Articulating Surface 51 27.0 70.80 37.48 1911.60 10 Distal Epiphysis 51 35.0 162.00 111.18 5670.00 11 Femoral Head 50 33.0 128.00 84.48 4224.00 12 Trochanter 49 38.5 76.50 60.11 2945.25 13 Calcaneus 51 28.5 81.70 45.66 2328.45 14 Talus 50 31.0 61.90 38.38 1918.90 RB Reagent Blank - 26.0 0.00 - 0.00 155 Table B9 : mtDNA Quantification of P - 05 Extracts Digested in Tissue Lys is Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. P - 05 Mitochondrial DNA ( ATPase ) Region Lo cation Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 28.0 13.20 7.39 369.60 2 Midshaft Diaphysis (Distal to Region 1) 49 27.0 37.30 20.55 1007.10 3 Diaphysis (Proximal t o Region 1) 50 26.5 19.10 10.12 506.15 4 Diaphysis (Distal to Region 2) 51 27.0 17.10 9.05 461.70 5 Diaphysis (Proximal to Region 3) 50 27.5 73.40 40.37 2018.50 6 Diaphysis (Distal to Region 4) 49 26.5 31.20 16.87 826.80 7 Proximal Metaphysis 50 27.5 9 5.80 52.69 2634.50 8 Distal Metaphysis 51 25.5 75.90 37.95 1935.45 9 Articulating Surface 51 27.0 68.40 36.21 1846.80 10 Distal Epiphysis 50 38.0 270.00 205.20 10260.00 11 Femoral Head 50 40.0 214.00 171.20 8560.00 12 Trochanter 50 32.0 317.00 202.88 10144.00 13 Calcaneus 49 28.0 129.00 73.71 3612.00 14 Talus 51 33.0 82.30 53.25 2715.90 RB Reagent Blank - 23.5 0.00 - 0.00 156 Table B10 : mtDNA Quantification of P - 05 Extracts Digested in Demineralization Buffer Region and location correspond with Ta ble 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. P - 05 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantif ication (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 30.0 31.20 18.35 936.00 2 Midshaft Diaphysis (Distal to Region 1) 50 27.5 48.40 26.62 1331.00 3 Diaphysis (Proximal to Region 1) 51 26.0 24.60 12.54 639.60 4 Diaphy sis (Distal to Region 2) 49 26.0 29.10 15.44 756.60 5 Diaphysis (Proximal to Region 3) 50 29.0 83.30 48.31 2415.70 6 Diaphysis (Distal to Region 4) 50 25.0 38.30 19.15 957.50 7 Proximal Metaphysis 49 29.0 84.80 50.19 2459.20 8 Distal Metaphysis 49 29.5 72.90 43.89 2150.55 9 Articulating Surface 50 31.0 87.50 54.25 2712.50 10 Distal Epiphysis 49 34.0 634.00 439.92 21556.00 11 Femoral Head 50 39.0 193.00 150.54 7527.00 12 Trochanter 50 32.0 622.00 398.08 19904.00 13 Calcaneus 49 28.0 122.00 69.71 34 16.00 14 Talus 49 31.5 93.00 59.79 2929.50 RB Reagent Blank - 26.0 0.00 - 0.00 157 Table B1 1: mtDNA Quantification of P - 06 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. P - 06 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Rec overed (ng) 1 Midshaft Diaphysis 49 32.0 25.80 16.85 825.60 2 Midshaft Diaphysis (Distal to Region 1) 51 25.0 26.70 13.09 667.50 3 Diaphysis (Proximal to Region 1) 50 25.5 29.30 14.94 747.15 4 Diaphysis (Distal to Region 2) 49 27.0 22.60 12.45 610.20 5 Diaphysis (Proximal to Region 3) 50 26.0 55.90 29.07 1453.40 6 Diaphysis (Distal to Region 4) 50 26.5 44.50 23.59 1179.25 7 Proximal Metaphysis 50 28.0 73.00 40.88 2044.00 8 Distal Metaphysis 51 28.0 79.60 43.70 2228.80 9 Articulating Surface 49 26.5 101.00 54.62 2676.50 10 Distal Epiphysis 49 34.0 226.00 156.82 7684.00 11 Femoral Head 51 32.0 138.00 86.59 4416.00 12 Trochanter 49 30.5 253.00 157.48 7716.50 13 Calcaneus 51 30.5 54.30 32.47 1656.15 14 Talus 50 32.0 90.00 57.60 2880.00 RB Reagen t Blank - 28.0 0.00 - 0.00 158 Table B1 2: mtDNA Quantification of P - 06 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields . Normalized val ues are reported as ng per mg of bone powder. P - 06 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 28.0 32.50 17 .84 910.00 2 Midshaft Diaphysis (Distal to Region 1) 51 28.0 40.30 22.13 1128.40 3 Diaphysis (Proximal to Region 1) 50 27.5 34.80 19.14 957.00 4 Diaphysis (Distal to Region 2) 50 28.0 41.50 23.24 1162.00 5 Diaphysis (Proximal to Region 3) 51 27.0 55.50 29.38 1498.50 6 Diaphysis (Distal to Region 4) 50 28.0 44.40 24.86 1243.20 7 Proximal Metaphysis 50 29.0 77.50 44.95 2247.50 8 Distal Metaphysis 50 33.0 65.50 35.96 1834.00 9 Articulating Surface 51 27.0 117.00 61.94 3159.00 10 Distal Epiphysis 50 33 .0 204.00 134.64 6732.00 11 Femoral Head 49 33.0 134.00 90.24 4422.00 12 Trochanter 49 31.0 111.00 70.22 3441.00 13 Calcaneus 51 24.5 73.40 35.26 1798.30 14 Talus 51 31.5 58.70 36.26 1849.05 RB Reagent Blank - 28.0 0.00 - 0.00 159 Table B13 : mtDNA Qua ntification of P - 07 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. P - 07 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 49 25.5 19.70 10.25 502.35 2 Midshaft Diaphysis (Distal to Region 1) 49 26.5 21.30 11.52 564.45 3 Diaphysis (Proximal to Region 1) 51 28.5 29.50 16.49 840.75 4 Diaphysis (Distal to Region 2) 50 27.0 14.90 8.05 402.30 5 Diaphysis (Proximal to Region 3) 50 29.0 71.30 41.35 2067.70 6 Diaphysis (Distal to Region 4) 50 26.5 37.00 19.61 980.50 7 Proximal Metaphysis 51 27.0 84.80 44.89 2289.60 8 Distal Metaphysis 50 26.0 75.70 39.36 1968.20 9 Articulating Surface 50 26.5 87.80 46.53 2326.70 10 Distal Epiphysis 49 30.5 177.00 110.39 5398.50 11 Femoral Head 49 33.0 155.00 10 4.39 5115.00 12 Trochanter 49 34.0 286.00 198.45 9724.00 13 Calcaneus 50 25.5 104.00 53.04 2652.00 14 Talus 51 30.0 118.00 69.41 3540.00 RB Reagent Blank - 27.0 0.00 - 0.00 160 Table B14 : mtDNA Quantification of P - 07 Extracts Digested in Demineralizat ion Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. P - 07 Mitochondrial DNA ( ATPase ) Region L ocation Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 49 27.5 30.50 17.12 838.75 2 Midshaft Diaphysis (Distal to Region 1) 50 26.0 25.50 13.26 663.00 3 Diaphysis (Proximal to Region 1) 50 25.5 37.10 18.92 946.05 4 Diaphysis (Distal to Region 2) 50 30.0 33.30 19.98 999.00 5 Diaphysis (Proximal to Region 3) 51 28.5 58.20 32.52 1658.70 6 Diaphysis (Distal to Region 4) 49 28.0 37.60 21.49 1052.80 7 Proximal Metaphysis 50 27. 0 98.90 53.41 2670.30 8 Distal Metaphysis 49 31.0 70.50 44.60 2185.50 9 Articulating Surface 49 29.5 94.80 57.07 2796.60 10 Distal Epiphysis 50 32.5 160.00 104.00 5200.00 11 Femoral Head 49 32.0 111.00 72.49 3552.00 12 Trochanter 50 38.5 233.00 179.41 8970.50 13 Calcaneus 49 29.0 76.50 45.28 2218.50 14 Talus 49 32.0 92.50 60.41 2960.00 RB Reagent Blank - 29.0 0.00 - 0.00 161 Table B15 : mtDNA Quantification of P - 08 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. P - 08 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantificatio n (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 26.0 35.80 18.62 930.80 2 Midshaft Diaphysis (Distal to Region 1) 49 23.0 34.20 16.05 786.60 3 Diaphysis (Proximal to Region 1) 51 28.0 28.80 15.81 806.40 4 Diaphysis (Di stal to Region 2) 51 26.0 27.60 14.07 717.60 5 Diaphysis (Proximal to Region 3) 49 26.0 53.50 28.39 1391.00 6 Diaphysis (Distal to Region 4) 49 29.0 39.10 23.14 1133.90 7 Proximal Metaphysis 49 28.5 48.60 28.27 1391.00 8 Distal Metaphysis 49 28.0 58.20 33.26 1629.60 9 Articulating Surface 49 27.0 99.00 54.55 2673.00 10 Distal Epiphysis 49 35.0 156.38 111.70 5473.30 11 Femoral Head 49 35.5 120.00 86.94 4260.00 12 Trochanter 49 34.0 219.00 151.96 7446.00 13 Calcaneus 49 30.5 58.90 36.66 1796.45 14 Talus 50 29.0 76.40 44.31 2215.60 RB Reagent Blank - 27.0 0.00 - 0.00 162 T able B16 : mtDNA Quantification of P - 08 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. P - 08 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Reco vered (ng) 1 Midshaft Diaphysis 49 25.5 45.90 23.89 1170.45 2 Midshaft Diaphysis (Distal to Region 1) 50 32.5 38.70 25.16 1257.75 3 Diaphysis (Proximal to Region 1) 49 30.5 33.30 20.73 1015.65 4 Diaphysis (Distal to Region 2) 50 27.0 34.50 18.63 931.50 5 Diaphysis (Proximal to Region 3) 50 27.5 42.90 23.60 1179.75 6 Diaphysis (Distal to Region 4) 51 27.0 36.10 19.11 974.70 7 Proximal Metaphysis 49 29.0 43.40 25.69 1258.60 8 Distal Metaphysis 49 28.0 47.40 27.09 1327.20 9 Articulating Surface 49 27. 0 124.00 68.33 3348.00 10 Distal Epiphysis 50 35.5 157.00 111.47 5573.50 11 Femoral Head 51 38.0 139.00 103.57 5282.00 12 Trochanter 50 36.0 126.00 90.72 4536.00 13 Calcaneus 49 30.0 57.80 35.39 1734.00 14 Talus 49 31.0 43.60 27.58 1351.60 RB Reage nt Blank - 29.0 0.00 - 0.00 163 Table B17 : Nuclear DNA Quantification of P - 01 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields . Normalized values are reported as ng of per mg of bone powder. P - 01 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 49 28.0 70.10 40.06 1 962.80 2 Midshaft Diaphysis (Distal to Region 1) 50 29.0 59.20 34.34 1716.80 3 Diaphysis (Proximal to Region 1) 49 26.0 144.00 76.41 3744.00 4 Diaphysis (Distal to Region 2) 50 26.0 92.20 47.94 2397.20 5 Diaphysis (Proximal to Region 3) 51 30.0 319.00 187.65 9570.00 6 Diaphysis (Distal to Region 4) 49 29.5 314.00 189.04 9263.00 7 Proximal Metaphysis 49 27.0 697.00 384.06 18819.00 8 Distal Metaphysis 50 28.0 423.00 236.88 11844.00 9 Articulating Surface 49 30.0 902.00 552.24 27060.00 10 Distal Epiph ysis 51 35.0 221.00 151.67 7735.00 11 Femoral Head 51 36.0 1600.00 1129.41 57600.00 12 Trochanter 50 34.0 1930.00 1312.40 65620.00 13 Calcaneus 50 29.0 686.00 397.88 19894.00 14 Talus 50 35.0 817.00 571.90 28595.00 RB Reagent Blank - 29.5 0.00 - 0.0 0 164 Table B18 : Nuclear DNA Quantification of P - 01 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder. P - 01 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 25.5 291.00 145.50 7420.50 2 Midshaft Diaphysis (Distal to Region 1) 51 29.5 246.00 142.29 7257.00 3 Diaphysis (Proximal to Region 1) 20 29.0 257.00 149.06 7453.00 4 Diaphysis (Distal to Region 2) 50 29.5 255.00 150.45 7522.50 5 Diaphysis (Proximal to Region 3) 51 30.0 384.00 225.88 11520.0 0 6 Diaphysis (Distal to Region 4) 49 30.0 223.00 136.53 6690.00 7 Proximal Metaphysis 51 28.0 512.00 281.10 14336.00 8 Distal Metaphysis 50 28.0 517.00 289.52 14476.00 9 Articulating Surface 50 30.0 810.00 486.00 24300.00 10 Distal Epiphysis 51 32.0 1640.00 1029.02 52480.00 11 Femoral Head 51 34.5 1710.00 1156.76 58995.00 12 Trochanter 51 35.0 1760.00 1207.84 61600.00 13 Calcaneus 49 35.0 640.00 457.14 22400.00 14 Talus 49 29.5 718.00 432.27 21181.00 RB Reagent Blank - 29.0 0.43 - 12.47 165 Table B19 : Nuclear DNA Quantification of P - 02 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported a s ng of per mg of bone powder. P - 02 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 26.0 21.00 10.71 546.00 2 Midshaft Diaphysis (Distal to Region 1) 51 28.0 12.60 6.92 352.80 3 Diaphysis (Proximal to Region 1) 50 30.5 32.10 19.58 979.05 4 Diaphysis (Distal to Region 2) 49 30.0 21.70 13.29 651.00 5 Diaphysis (Proximal to Region 3) 50 29.0 78.40 45.47 2273.60 6 Diaphysis (Distal to Regi on 4) 49 27.5 102.00 57.24 2805.00 7 Proximal Metaphysis 51 26.0 443.00 225.84 11518.00 8 Distal Metaphysis 50 28.0 288.00 161.28 8064.00 9 Articulating Surface 51 30.0 436.00 256.47 13080.00 10 Distal Epiphysis 51 40.0 1890.00 1482.35 75600.00 11 Fem oral Head 50 40.0 580.00 464.00 23200.00 12 Trochanter 49 40.0 1640.00 1338.78 65600.00 13 Calcaneus 50 34.0 508.00 345.44 17272.00 14 Talus 50 40.0 661.00 528.80 26440.00 RB Reagent Blank - 29.0 0 .25 - 7 .25 166 Table B20 : Nuclear DNA Quantification of P - 02 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder. P - 02 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 28.0 266.00 148.96 7448.00 2 Midshaft Diaphysis (Distal to Region 1) 49 27.0 173.00 95.33 4671.00 3 Diaphysis (Proximal to Region 1) 51 30.5 177.00 105.85 5398.50 4 Diaphysis (Distal to Region 2) 49 29.5 186.00 111.98 5487.00 5 Diaphysis (Proximal to Region 3) 51 29.0 201.00 114.29 5829.00 6 Diaphysis (Distal to Region 4) 51 30.0 187 .00 110.00 5610.00 7 Proximal Metaphysis 51 30.0 381.00 224.12 11430.00 8 Distal Metaphysis 51 28.5 469.00 262.09 13366.50 9 Articulating Surface 49 30.0 531.00 325.10 15930.00 10 Distal Epiphysis 51 38.0 1300.00 968.63 49400.00 11 Femoral Head 50 39. 0 1220.00 951.60 47580.00 12 Trochanter 50 39.0 1090.00 850.20 42510.00 13 Calcaneus 51 35.0 430.00 295.10 15050.00 14 Talus 50 38.5 667.00 513.59 25679.50 RB Reagent Blank - 28.0 0.35 - 9.80 167 Table B2 1: Nuclear DNA Quantification of P - 03 Extracts D igested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder. ND = No Data P - 03 N uclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 31.0 10.30 6.26 319.30 2 Midshaft Diaphysis (Distal to Region 1) 51 0.0 ND ND ND 3 Diaph ysis (Proximal to Region 1) 50 29.0 33.00 19.14 957.00 4 Diaphysis (Distal to Region 2) 51 30.0 15.10 8.88 453.00 5 Diaphysis (Proximal to Region 3) 49 30.0 494.00 302.45 14820.00 6 Diaphysis (Distal to Region 4) 51 27.5 230.00 124.02 6325.00 7 Proxima l Metaphysis 49 30.0 1260.00 720.00 35280.00 8 Distal Metaphysis 49 27.0 591.00 325.65 15957.00 9 Articulating Surface 49 30.0 906.00 554.69 27180.00 10 Distal Epiphysis 50 35.0 4560.00 3192.00 159600.00 11 Femoral Head 51 37.0 2330.00 1690.39 86210.00 12 Trochanter 49 32.0 4890.00 3193.47 156480.00 13 Calcaneus 51 31.5 631.00 389.74 19876.50 14 Talus 51 32.0 970.00 608.63 31040.00 RB Reagent Blank - 25.0 0.29 - 7.25 168 Table B2 2: Nuclear DNA Quantification of P - 03 Extracts Digested in Demineraliza tion Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder. P - 03 Nuclear DNA ( MC1R ) Region Locatio n Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 28.5 233.00 130.21 6640.50 2 Midshaft Diaphysis (Distal to Region 1) 51 28.0 294.00 161.41 8232.00 3 Diaphysis (Proximal to Region 1) 49 27.0 210.00 115.71 5670.00 4 Diaphysis (Distal to Region 2) 49 26.0 332.00 176.16 8632.00 5 Diaphysis (Proximal to Region 3) 49 29.0 539.00 319.00 15631.00 6 Diaphysis (Distal to Region 4) 50 26.0 330.00 171.60 8580.00 7 Proximal Metaph ysis 51 29.5 899.00 520.01 26520.50 8 Distal Metaphysis 49 30.0 454.00 245.16 12258.00 9 Articulating Surface 51 27.0 451.00 238.76 12177.00 10 Distal Epiphysis 49 30.0 1280.00 783.67 38400.00 11 Femoral Head 49 36.0 1160.00 852.24 41760.00 12 Trochan ter 50 29.5 2050.00 1209.50 60475.00 13 Calcaneus 51 29.5 835.00 482.99 24632.50 14 Talus 50 30.0 694.00 416.40 20820.00 RB Reagent Blank - 27.0 0.14 - 3.89 169 Table B23 : Nuclear DNA Quantification of P - 04 Extracts Digested in Tissue Lysis Buffer Regi on and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder. P - 04 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 28.0 4.32 2.44 122.08 2 Midshaft Diaphysis (Distal to Region 1) 50 25.0 3.20 1.60 80.00 3 Diaphysis (Proximal to Region 1) 50 29.0 3.46 2. 01 100.34 4 Diaphysis (Distal to Region 2) 52 28.0 5.68 3.06 159.04 5 Diaphysis (Proximal to Region 3) 51 30.0 125.00 73.53 3750.00 6 Diaphysis (Distal to Region 4) 49 27.0 126.00 69.43 3402.00 7 Proximal Metaphysis 51 31.5 384.00 237.18 12096.00 8 Di stal Metaphysis 49 24.5 143.00 71.50 3503.50 9 Articulating Surface 50 27.0 470.00 253.80 12690.00 10 Distal Epiphysis 51 32.5 3750.00 2389.71 121875.00 11 Femoral Head 51 35.0 4140.00 2841.18 144900.00 12 Trochanter 51 32.5 3460.00 2204.90 112450.00 13 Calcaneus 51 30.0 512.00 301.18 15360.00 14 Talus 50 30.0 703.00 421.80 21090.00 RB Reagent Blank - 23.5 0.26 - 6.11 170 Table B24 : Nuclear DNA Quantification of P - 04 Extracts Digested in Demineralization Buffer Region and location correspond with Ta ble 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder. P - 04 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 29.5 140.00 82.60 4130.00 2 Midshaft Diaphysis (Distal to Region 1) 51 23.5 271.00 124.87 6368.50 3 Diaphysis (Proximal to Region 1) 50 28.5 173.00 98.61 4930.50 4 Diaphys is (Distal to Region 2) 50 24.0 264.00 126.72 6336.00 5 Diaphysis (Proximal to Region 3) 50 26.0 302.00 157.04 7852.00 6 Diaphysis (Distal to Region 4) 51 26.0 255.00 130.00 6630.00 7 Proximal Metaphysis 49 29.0 441.00 261.00 12789.00 8 Distal Metaphys is 51 28.0 368.00 202.04 10304.00 9 Articulating Surface 51 27.0 486.00 257.29 13122.00 10 Distal Epiphysis 51 35.0 1530.00 1050.00 53550.00 11 Femoral Head 50 33.0 1170.00 772.20 38610.00 12 Trochanter 49 38.5 1460.00 1147.14 56210.00 13 Calcaneus 5 1 28.5 757.00 423.03 21574.50 14 Talus 50 31.0 569.00 352.78 17639.00 RB Reagent Blank - 26.0 0.08 - 2.03 171 Table B25 : Nuclear DNA Quantification of P - 05 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract vol ume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder. P - 05 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 28.0 11.20 6.27 313.60 2 Midshaft Diaphysis (Distal to Region 1) 49 27.0 37.60 20.72 1015.20 3 Diaphysis (Proximal to Region 1) 50 26.5 16.60 8.80 439.90 4 Diaphysis (Distal to Region 2) 51 27.0 23.90 12.65 645.30 5 Diaphysis (Proximal to Region 3) 50 27.5 335.00 184.25 9212.50 6 Diaphysis (Distal to Region 4) 49 26.5 96.10 51.97 2546.65 7 Proximal Metaphysis 50 27.5 241.00 132.55 6627.50 8 Distal Metaphysis 51 25.5 143.00 71.50 3646.50 9 Articulating Surface 51 27.0 498.00 263.65 13446.00 10 Distal Epiphysis 50 38.0 1360.00 1033.60 51680.00 11 Femoral Head 50 40.0 1090.00 872.00 43600.00 12 Trochanter 50 32.0 2990.00 1913.60 95680.00 13 Calcaneus 49 28.0 966.00 552.00 27048.00 14 T alus 51 33.0 565.00 365.59 18645.00 RB Reagent Blank - 23.5 0.21 - 4.94 172 Table B26 : Nuclear DNA Quantification of P - 05 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powd er was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder. P - 05 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovere d (ng) 1 Midshaft Diaphysis 51 30.0 193.00 113.53 5790.00 2 Midshaft Diaphysis (Distal to Region 1) 50 27.5 239.00 131.45 6572.50 3 Diaphysis (Proximal to Region 1) 51 26.0 244.00 124.39 6344.00 4 Diaphysis (Distal to Region 2) 49 26.0 219.00 116.20 56 94.00 5 Diaphysis (Proximal to Region 3) 50 29.0 438.00 254.04 12702.00 6 Diaphysis (Distal to Region 4) 50 25.0 241.00 120.50 6025.00 7 Proximal Metaphysis 49 29.0 427.00 252.71 12383.00 8 Distal Metaphysis 49 29.5 461.00 277.54 13599.50 9 Articulati ng Surface 50 31.0 334.00 207.08 10354.00 10 Distal Epiphysis 49 34.0 691.00 479.47 23494.00 11 Femoral Head 50 39.0 914.00 712.92 35646.00 12 Trochanter 50 32.0 1350.00 864.00 43200.00 13 Calcaneus 49 28.0 566.00 323.43 15848.00 14 Talus 49 31.5 76 4.00 491.14 24066.00 RB Reagent Blank - 26.0 0.14 - 3.61 173 Table B27 : Nuclear DNA Quantification of P - 06 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder. P - 06 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft D iaphysis 49 32.0 10.50 6.86 336.00 2 Midshaft Diaphysis (Distal to Region 1) 51 25.0 6.57 3.22 164.25 3 Diaphysis (Proximal to Region 1) 50 25.5 21.90 11.17 558.45 4 Diaphysis (Distal to Region 2) 49 27.0 5.66 3.12 152.82 5 Diaphysis (Proximal to Regio n 3) 50 26.0 175.00 91.00 4550.00 6 Diaphysis (Distal to Region 4) 50 26.5 187.00 99.11 4955.50 7 Proximal Metaphysis 50 28.0 197.00 110.32 5516.00 8 Distal Metaphysis 51 28.0 176.00 96.63 4928.00 9 Articulating Surface 49 26.5 694.00 375.33 18391.00 10 Distal Epiphysis 49 34.0 2680.00 1859.59 91120.00 11 Femoral Head 51 32.0 1560.00 978.82 49920.00 12 Trochanter 49 30.5 3170.00 1973.16 96685.00 13 Calcaneus 51 30.5 270.00 161.47 8235.00 14 Talus 50 32.0 600.00 384.00 19200.00 RB Reagent Blank - 28.0 0.18 - 5.04 174 Table B28 : Nuclear DNA Quantification of P - 06 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Nor malized values are reported as ng of per mg of bone powder. P - 06 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 28.0 157.00 86.20 4396.0 0 2 Midshaft Diaphysis (Distal to Region 1) 51 28.0 162.00 88.94 4536.00 3 Diaphysis (Proximal to Region 1) 50 27.5 154.00 84.70 4235.00 4 Diaphysis (Distal to Region 2) 50 28.0 164.00 91.84 4592.00 5 Diaphysis (Proximal to Region 3) 51 27.0 201.00 106 .41 5427.00 6 Diaphysis (Distal to Region 4) 50 28.0 198.00 110.88 5544.00 7 Proximal Metaphysis 50 29.0 318.00 184.44 9222.00 8 Distal Metaphysis 50 33.0 279.00 153.18 7812.00 9 Articulating Surface 51 27.0 316.00 167.29 8532.00 10 Distal Epiphysis 5 0 33.0 771.00 508.86 25443.00 11 Femoral Head 49 33.0 857.00 577.16 28281.00 12 Trochanter 49 31.0 887.00 561.16 27497.00 13 Calcaneus 51 24.5 454.00 218.10 11123.00 14 Talus 51 31.5 355.00 219.26 11182.50 RB Reagent Blank - 28.0 0.04 - 0.99 175 Table B29 : Nuclear DNA Quantification of P - 07 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported a s ng of per mg of bone powder. P - 07 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 49 25.5 15.10 7.86 385.05 2 Midshaft Diaphysis (Distal to Region 1) 49 26.5 25.00 13.52 662.50 3 Diaphysis (Proximal to Region 1) 51 28.5 35.80 20.01 1020.30 4 Diaphysis (Distal to Region 2) 50 27.0 1270.00 672.35 221.67 5 Diaphysis (Proximal to Region 3) 50 29.0 835.00 484.30 24215.00 6 Diaphysis (Distal to Region 4) 50 26.5 457.00 242.21 12110.50 7 Proximal Metaphysis 51 27.0 1270.00 672.35 34290.00 8 Distal Metaphysis 50 26.0 671.00 348.92 17446.00 9 Articulating Surface 50 26.5 1310.00 694.30 34715.00 10 Distal Epiphysis 49 30.5 3540.00 2203.47 1079 70.00 11 Femoral Head 49 33.0 1240.00 835.10 40920.00 12 Trochanter 49 34.0 2480.00 1720.82 84320.00 13 Calcaneus 50 25.5 718.00 366.18 18309.00 14 Talus 51 30.0 718.00 366.18 21240.00 RB Reagent Blank - 27.0 0.04 - 1.08 176 Table B30 : Nuclear DNA Qua ntification of P - 07 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder. P - 07 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 49 27.5 294.00 165.00 8085.00 2 Midshaft Diaphysis (Distal to Region 1) 50 26.0 229.00 119.08 5954.00 3 Diaphysis (Proximal to Region 1) 50 25.5 321.00 163.71 8185.50 4 Diaphysis (Distal to Region 2) 50 30.0 269.00 161.40 8070.00 5 Diaphysis (Proximal to Region 3) 51 28.5 404.00 225.76 11514.00 6 Diaphysis (Distal to Regio n 4) 49 28.0 438.00 250.29 12264.00 7 Proximal Metaphysis 50 27.0 670.00 361.80 18090.00 8 Distal Metaphysis 49 31.0 543.00 343.53 16833.00 9 Articulating Surface 49 29.5 579.00 348.58 17080.50 10 Distal Epiphysis 50 32.5 1080.00 702.00 35100.00 11 Fe moral Head 49 32.0 957.00 624.98 30624.00 12 Trochanter 50 38.5 1210.00 931.70 46585.00 13 Calcaneus 49 29.0 567.00 335.57 16443.00 14 Talus 49 32.0 700.00 457.14 22400.00 RB Reagent Blank - 29.0 0.05 - 1.45 177 Table B 31: Nuclear DNA Quantification of P - 08 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder. P - 08 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 26.0 26.20 13.62 681.20 2 Midshaft Diaphysis (Distal to Region 1) 49 23.0 23.40 10.98 5 38.20 3 Diaphysis (Proximal to Region 1) 51 28.0 23.80 13.07 666.40 4 Diaphysis (Distal to Region 2) 51 26.0 16.70 8.51 434.20 5 Diaphysis (Proximal to Region 3) 49 26.0 404.00 214.37 10504.00 6 Diaphysis (Distal to Region 4) 49 29.0 273.00 161.57 7917 .00 7 Proximal Metaphysis 49 28.5 281.00 163.44 8008.50 8 Distal Metaphysis 49 28.0 395.00 225.71 11060.00 9 Articulating Surface 49 27.0 1060.00 584.08 28620.00 10 Distal Epiphysis 49 35.0 3600.00 2571.43 126000.00 11 Femoral Head 49 35.5 1420.00 102 8.78 50410.00 12 Trochanter 49 34.0 3910.00 2713.06 132940.00 13 Calcaneus 49 30.5 652.00 405.84 19886.00 14 Talus 50 29.0 759.00 440.22 22011.00 RB Reagent Blank - 27.0 0.06 - 1.62 178 Table B 32: Nuclear DNA Quantification of P - 08 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng of per mg of bone powder. P - 08 Nuclear DNA ( MC1R ) R egion Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 49 25.5 243.00 126.46 6196.50 2 Midshaft Diaphysis (Distal to Region 1) 50 32.5 198.00 128.70 6435.00 3 Diaphys is (Proximal to Region 1) 49 30.5 174.00 108.31 5307.00 4 Diaphysis (Distal to Region 2) 50 27.0 220.00 118.80 5940.00 5 Diaphysis (Proximal to Region 3) 50 27.5 293.00 161.15 8057.50 6 Diaphysis (Distal to Region 4) 51 27.0 248.00 131.29 6696.00 7 Pro ximal Metaphysis 49 29.0 222.00 131.39 6438.00 8 Distal Metaphysis 49 28.0 320.00 182.86 8960.00 9 Articulating Surface 49 27.0 331.00 182.39 8937.00 10 Distal Epiphysis 50 35.5 886.00 629.06 31453.00 11 Femoral Head 51 38.0 1060.00 789.80 40280.00 12 Trochanter 50 36.0 722.00 519.84 25992.00 13 Calcaneus 49 30.0 449.00 274.90 13470.00 14 Talus 49 31.0 498.00 315.06 15438.00 RB Reagent Blank - 29.0 0.03 - 0.74 179 Table B 33: Porcine Mitochondrial DNA PCR Amplification Chart for Quality Assay (Tissue Lysis Buffer) Region and location correspond to Table 3. Values (1 4) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 181 bp amplicon, 2 = 414 bp amplicon, 3 = 604 bp amplicon, 4 = 1017 bp amplicon. N T = Not Tested NA = No Amplification Region Location Mitochondrial DNA ( ATPase ) P - 01 P - 02 P - 03 P - 04 P - 05 P - 06 P - 07 P - 08 1 Midshaft Diaphysis 4 4 4 4 4 4 4 4 2 Midshaft Diaphysis (Distal to Region 1) 4 4 NT 4 4 4 4 4 3 Diaphysis (Proximal to R egion 1) 4 4 4 4 4 4 4 4 4 Diaphysis (Distal to Region 2) 4 4 4 4 4 4 4 4 5 Diaphysis (Proximal to Region 3) 4 4 4 4 4 4 4 4 6 Diaphysis (Distal to Region 4) 4 4 4 4 4 4 4 4 7 Proximal Metaphysis 4 4 4 4 4 4 4 4 8 Distal Metaphysis 4 4 4 4 4 4 4 4 9 Articulating Surface 4 4 4 4 4 4 4 4 10 Distal Epiphysis 4 4 4 4 4 4 4 4 11 Femoral Head 4 4 4 4 4 4 4 4 12 Trochanter 4 4 4 4 4 4 4 4 13 Calcaneus 4 4 4 4 4 4 4 4 14 Talus 4 4 4 4 4 4 4 4 RB Reagent Blank NA NA NA NA NA NA NA NA 180 Tab le B 34: Porcine Mitochondrial DNA PCR Amplification Chart for Quality Assay (Demineralization Buffer) Region and location correspond to Table 3. Values (1 4) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 181 bp amplicon, 2 = 414 bp amplicon, 3 = 604 bp amplicon, 4 = 1017 bp amplicon. NA = No Amplification Region Location Mitochondrial DNA ( ATPase ) P - 01 P - 02 P - 03 P - 04 P - 05 P - 06 P - 07 P - 08 1 Midshaft Diaphysis 4 4 4 4 4 4 4 4 2 Midshaft Diaphysis (D istal to Region 1) 4 4 4 4 4 4 4 4 3 Diaphysis (Proximal to Region 1) 4 4 4 4 4 4 4 4 4 Diaphysis (Distal to Region 2) 4 4 4 4 4 4 4 4 5 Diaphysis (Proximal to Region 3) 4 4 4 4 4 4 4 4 6 Diaphysis (Distal to Region 4) 4 4 4 4 4 4 4 4 7 Proximal Meta physis 4 4 4 4 4 4 4 4 8 Distal Metaphysis 4 4 4 4 4 4 4 4 9 Articulating Surface 4 4 4 4 4 4 4 4 10 Distal Epiphysis 4 4 4 4 4 4 4 4 11 Femoral Head 4 4 4 4 4 4 4 4 12 Trochanter 4 4 4 4 4 4 4 4 13 Calcaneus 4 4 4 4 4 4 4 4 14 Talus 4 4 4 4 4 4 4 4 RB Reagent Blank NA NA NA NA NA NA NA NA 181 Table B 35: Porcine Nuclear DNA PCR Amplification Chart for Quality Assay (Tissue Lysis Buffer) Region and location correspond to Table 3. Values (1 3) correspond to the length of PCR amplicon s uccessfully generated for each region per replicate: 1 = 257 bp amplicon, 2 = 457 b p amplicon, 3 = 642 bp amplicon. NT = Not Tested NA = No Amplification Region Location Nuclear DNA ( IGF - 1 ) P - 01 P - 02 P - 03 P - 04 P - 05 P - 06 P - 07 P - 08 1 Midshaft Di aphysis 3 3 3 3 3 3 3 3 2 Midshaft Diaphysis (Distal to Region 1) 3 3 NT 3 3 3 3 3 3 Diaphysis (Proximal to Region 1) 3 3 3 3 3 3 3 3 4 Diaphysis (Distal to Region 2) 3 3 3 3 3 3 3 3 5 Diaphysis (Proximal to Region 3) 2 3 3 3 3 3 3 3 6 Diaphysis (Dis tal to Region 4) 2 3 3 3 3 3 3 3 7 Proximal Metaphysis 3 3 2 3 3 3 3 3 8 Distal Metaphysis 3 3 3 3 3 3 3 3 9 Articulating Surface 3 3 3 3 3 3 3 3 10 Distal Epiphysis 3 3 3 3 3 3 3 3 11 Femoral Head 3 3 3 3 3 3 3 3 12 Trochanter 2 3 3 3 3 3 3 3 13 Ca lcaneus 3 3 3 3 3 3 3 3 14 Talus 3 3 3 3 3 3 3 3 RB Reagent Blank NA NA NA NA NA NA NA NA 182 Table B 36: Porcine Nuclear DNA PCR Amplification Chart for Quality Assay (Demineralization Buffer) Region and location correspond to Table 3. Values (1 3) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 257 bp amplicon, 2 = 457 b p amplicon, 3 = 642 bp amplicon. NA = No Amplification. Region Location Nuclear DNA ( IGF - 1 ) P - 01 P - 02 P - 03 P - 04 P - 05 P - 06 P - 07 P - 08 1 Midshaft Diaphysis 3 3 3 3 3 3 3 3 2 Midshaft Diaphysis (Distal to Region 1) 2 3 3 3 3 3 3 3 3 Diaphysis (Proximal to Region 1) 3 3 3 3 3 3 3 2 4 Diaphysis (Distal to Region 2) 3 3 3 3 3 3 3 3 5 Diaphysis (Proximal to Region 3) 3 3 3 3 3 3 3 3 6 Diaphysis (Distal to Region 4) 2 3 3 3 3 3 3 3 7 Proximal Metaphysis 2 3 3 3 3 3 3 3 8 Distal Metaphysis 3 3 3 3 3 3 3 3 9 Articulating Surface 3 3 3 3 3 3 3 3 10 Distal Epiphysis 3 3 3 3 3 3 3 3 11 Femoral Head 3 3 3 3 3 3 3 3 12 Trochanter 2 3 3 3 3 3 3 3 13 Calcaneus 3 3 3 3 3 3 3 3 14 Talus 3 3 3 3 3 3 3 3 RB Reagent Blank NA NA NA NA NA NA NA NA 183 APPENDIX C: MITOCHONDRIAL AND NUCLEAR DNA QUANTIFICATION DATA AND INDIVIDUAL PCR AMPLIFICATION RESULTS FROM FRESH BOVINE FEMORA AN D TARSALS Table C1: mtDNA Quantification of C - 01 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. C - 01 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 30.0 12.90 7.59 387.00 2 Midshaft Di aphysis (Distal to Region 1) 50 25.0 10.60 5.30 265.00 3 Diaphysis (Proximal to Region 1) 51 29.0 14.60 8.30 423.30 4 Diaphysis (Distal to Region 2) 49 29.0 12.90 7.63 373.87 5 Diaphysis (Proximal to Region 3) 50 30.0 9.86 5.92 296.00 6 Diaphysis (Dist al to Region 4) 51 30.0 9.83 5.78 294.78 7 Proximal Metaphysis 49 26.0 25.30 13.42 657.58 8 Distal Metaphysis 50 30.0 22.50 14.56 742.56 9 Articulating Surface 49 30.0 46.60 28.53 1397.97 10 Distal Epiphysis 50 30.0 104.00 62.40 3120.00 11 Femoral Hea d 51 29.0 89.50 50.89 2595.39 12 Trochanter 51 29.0 50.60 28.77 1467.27 13 Calcaneus 50 29.0 30.50 17.69 884.50 14 Talus 51 27.0 109.00 57.71 2943.21 RB Reagent Blank - 26.0 0.06 - 1.56 184 Table C2: mtDNA Quantification of C - 01 Extracts Digested in Dem ineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. C - 01 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 49 28.0 32.60 18.63 912.87 2 Midshaft Diaphysis (Distal to Region 1) 49 25.0 21.90 11.17 547.33 3 Diaphysis (Proximal to Region 1) 50 30.0 21.80 13.08 654.00 4 Diaphysis (Distal to Region 2) 51 30.0 18.40 10.82 551.82 5 Diaphysis (Proximal to Region 3) 49 30.0 20.10 12.31 603.19 6 Diaphysis (Distal to Region 4) 49 30.0 20.00 12.24 599.76 7 Proximal Metaphysi s 51 29.0 34.40 19.56 997.56 8 Distal Metaphysis 51 29.0 55.50 31.56 1609.56 9 Articulating Surface 49 29.0 65.70 38.88 1905.12 10 Distal Epiphysis 51 30.0 133.00 78.24 3990.24 11 Femoral Head 50 30.0 140.00 84.00 4200.00 12 Trochanter 51 30.0 68.60 4 0.35 2057.85 13 Calcaneus 49 30.0 43.10 26.39 1293.11 14 Talus 49 31.0 87.30 55.23 2706.27 RB Reagent Blank - 30.0 0.06 - 1.80 185 Table C3 : mtDNA Quantification of C - 02 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Tabl e 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. C - 02 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantific ation (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 49 34.0 16.40 11.38 557.60 2 Midshaft Diaphysis (Distal to Region 1) 51 30.0 27.80 16.35 834.00 3 Diaphysis (Proximal to Region 1) 49 30.0 37.30 22.84 1119.00 4 Diaphysi s (Distal to Region 2) 49 30.0 19.10 11.69 573.00 5 Diaphysis (Proximal to Region 3) 51 29.0 28.20 16.04 817.80 6 Diaphysis (Distal to Region 4) 49 28.0 20.20 11.54 565.60 7 Proximal Metaphysis 50 32.0 67.50 43.20 2160.00 8 Distal Metaphysis 50 32.0 16 4.00 102.90 1913.60 9 Articulating Surface 50 30.0 87.60 52.56 2628.00 10 Distal Epiphysis 51 32.0 164.00 102.90 5248.00 11 Femoral Head 51 32.0 137.00 85.96 4384.00 12 Trochanter 51 31.0 92.50 56.23 2867.50 13 Calcaneus 49 30.0 64.30 39.37 1929.00 14 Talus 50 31.0 96.40 59.77 2988.40 RB Reagent Blank - 30.0 0.04 - 1.20 186 Table C4 : mtDNA Quantification of C - 02 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder w as considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. C - 02 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recov ered (ng) 1 Midshaft Diaphysis 50 30.0 30.90 18.54 927.00 2 Midshaft Diaphysis (Distal to Region 1) 51 30.0 32.10 18.88 963.00 3 Diaphysis (Proximal to Region 1) 51 29.0 35.10 19.96 1017.90 4 Diaphysis (Distal to Region 2) 50 29.0 27.40 15.89 764.60 5 Diaphysis (Proximal to Region 3) 50 29.0 39.20 22.74 1136.80 6 Diaphysis (Distal to Region 4) 49 25.0 31.90 16.28 797.50 7 Proximal Metaphysis 50 26.0 95.00 49.40 2470.00 8 Distal Metaphysis 50 29.0 79.40 46.05 2302.60 9 Articulating Surface 49 29.0 7 9.60 47.11 2308.40 10 Distal Epiphysis 50 32.0 244.00 156.16 7808.00 11 Femoral Head 49 30.0 124.00 75.92 3720.00 12 Trochanter 50 30.0 76.00 45.60 2280.00 13 Calcaneus 51 30.0 70.20 41.29 2106.00 14 Talus 49 34.0 76.20 52.87 2590.80 RB Reagent Bla nk - 29.0 0.04 - 1.16 187 Table C5 : mtDNA Quantification of C - 03 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normal ized val ues are reported as ng per mg of bone powder. C - 03 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 29.0 3.10 1.80 89.90 2 Midshaft Diaphysis (Distal to Region 1) 49 26.0 4.46 2.37 115.96 3 Diaphysis (Proximal to Region 1) 50 30.0 3.94 2.36 118.20 4 Diaphysis (Distal to Region 2) 50 27.0 5.06 2.73 136.62 5 Diaphysis (Proximal to Region 3) 50 29.0 3.26 1.89 94.54 6 Diap hysis (Distal to Region 4) 49 30.0 4.25 2.60 127.50 7 Proximal Metaphysis 50 32.0 7.52 4.81 240.64 8 Distal Metaphysis 50 30.0 5.18 3.11 155.40 9 Articulating Surface 51 31.0 9.75 5.93 302.25 10 Distal Epiphysis 51 31.0 21.20 12.89 657.20 11 Femoral Head 49 30.0 18.80 11.51 564.00 12 Trochanter 50 25.0 22.80 11.40 570.00 13 Calcaneus 50 30.0 4.81 2.89 144.30 14 Talus 50 30.0 6.22 3.73 186.60 RB Reagent Blank - 26.0 0.00 - 0.00 188 Table C6 : mtDNA Quantification of C - 03 Extracts Digested in Demin eralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. C - 03 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 30.0 5.62 3.31 168.60 2 Midshaft Diaphysis (Distal to Region 1) 51 30.0 3.78 2.22 113.40 3 Diaphysis (Proxi mal to Region 1) 51 27.0 4.83 2.51 133.38 4 Diaphysis (Distal to Region 2) 49 32.0 4.36 2.85 139.52 5 Diaphysis (Proximal to Region 3) 50 26.0 4.83 2.51 125.58 6 Diaphysis (Distal to Region 4) 50 29.0 5.05 2.93 146.45 7 Proximal Metaphysis 50 28.0 7.33 4.10 205.24 8 Distal Metaphysis 51 29.0 7.18 4.08 208.22 9 Articulating Surface 51 26.0 9.01 4.59 234.26 10 Distal Epiphysis 49 31.0 20.80 13.16 644.80 11 Femoral Head 49 31.0 18.80 11.89 582.80 12 Trochanter 49 26.0 20.80 11.04 540.80 13 Calcaneus 50 32.0 4.98 3.19 159.36 14 Talus 51 30.0 5.63 3.31 168.90 RB Reagent Blank - 28.0 0.01 - 0.28 189 Table C7 : mtDNA Quantification of C - 04 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligr ams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. C - 04 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng /mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 32.0 5.79 3.63 185.28 2 Midshaft Diaphysis (Distal to Region 1) 50 27.0 2.67 1.44 72.09 3 Diaphysis (Proximal to Region 1) 50 30.0 4.30 2.58 129.00 4 Diaphysis (Distal to Region 2) 50 27.0 3.99 2.15 107.73 5 Diaphysis (Proximal to Region 3) 51 29.0 4.22 2.40 122.38 6 Diaphysis (Distal to Region 4) 50 25.0 6.07 3.04 151.75 7 Proximal Metaphysis 50 27.0 6.09 3.29 164.43 8 Distal Metaphysis 51 30.0 4.61 2.71 138.30 9 Articulating Surface 51 29.0 13 .50 7.68 391.50 10 Distal Epiphysis 50 29.0 35.10 20.36 1017.90 11 Femoral Head 50 30.0 15.80 9.48 474.00 12 Trochanter 50 30.0 8.71 5.23 261.30 13 Calcaneus 49 30.0 4.88 2.99 146.40 14 Talus 49 30.0 3.74 2.29 112.20 RB Reagent Blank - 28.0 0.03 - 0.84 190 Table C8 : mtDNA Quantification of C - 04 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues ar e reported as ng per mg of bone powder. C - 04 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 49 28.0 3.51 2.01 98.28 2 Midshaft Dia physis (Distal to Region 1) 50 30.0 4.97 2.98 149.10 3 Diaphysis (Proximal to Region 1) 49 31.0 5.00 3.16 155.00 4 Diaphysis (Distal to Region 2) 51 28.0 5.74 3.15 160.72 5 Diaphysis (Proximal to Region 3) 51 28.0 6.16 3.38 172.48 6 Diaphysis (Distal t o Region 4) 51 29.0 5.24 2.98 151.96 7 Proximal Metaphysis 50 29.0 7.41 4.30 214.89 8 Distal Metaphysis 51 31.0 4.76 2.89 147.56 9 Articulating Surface 51 27.0 19.90 10.54 537.30 10 Distal Epiphysis 50 30.0 31.30 18.78 939.00 11 Femoral Head 51 31.0 2 3.80 14.47 737.80 12 Trochanter 51 28.0 9.44 5.18 264.32 13 Calcaneus 50 31.0 4.99 3.09 154.69 14 Talus 51 26.0 4.59 2.34 119.34 RB Reagent Blank - 29.0 0.00 - 0.00 191 Table C9 : mtDNA Quantification of C - 05 Extracts Digested in Tissue Lysis Buffe r Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. C - 05 Mitochondrial DNA ( ATPase ) Region Location B one Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 30.0 4.84 2.90 145.20 2 Midshaft Diaphysis (Distal to Region 1) 50 25.5 7.56 3.86 192.78 3 Diaphysis (Proximal to Region 1) 50 27.0 7.43 4.01 200.61 4 Diaphysis (Distal to Region 2) 50 37.0 7.95 5.88 294.15 5 Diaphysis (Proximal to Region 3) 51 23.0 10.70 4.83 246.10 6 Diaphysis (Distal to Region 4) 50 37.0 8.94 5.72 286.08 7 Proximal Metaphysis 50 30.0 13.90 8.34 417.00 8 Distal Metaphysis 49 30.0 14.40 8.82 432.00 9 Articulating Surface 50 32.0 43.40 27.78 1388.80 10 Distal Epiphysis 50 28.0 41.10 23.02 1150.80 11 Femoral Head 51 30.0 45.20 26.59 1356.00 12 Trochanter 51 26.0 77.90 39.71 2025.40 13 Calcaneus 51 30.5 51.30 30.68 1564.65 14 Talus 50 27.5 49.70 27.34 1366.75 RB Reagent Blank - 28.0 0.02 - 0.56 192 Table C10 : mtDNA Quantification of C - 05 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and m illigrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. C - 05 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized D NA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 29.0 9.83 5.70 285.07 2 Midshaft Diaphysis (Distal to Region 1) 51 28.0 13.90 7.63 389.20 3 Diaphysis (Proximal to Region 1) 49 28.0 10.90 6.23 305.20 4 Diaphysis (Distal to Region 2) 49 31.5 7.52 4.83 236.88 5 Diaphysis (Proximal to Region 3) 50 32.0 17.40 11.14 556.80 6 Diaphysis (Distal to Region 4) 50 30.0 10.70 6.42 321.00 7 Proximal Metaphysis 51 29.0 27.10 15.41 785.90 8 Distal Metaphysis 50 28.0 22.60 12.66 632.80 9 Articulating Su rface 51 28.0 38.00 20.86 1064.00 10 Distal Epiphysis 50 29.5 43.00 25.37 1268.50 11 Femoral Head 51 31.0 51.10 31.06 1584.10 12 Trochanter 50 27.0 45.10 24.35 1217.70 13 Calcaneus 51 28.0 32.90 18.06 921.20 14 Talus 51 30.0 26.30 15.47 789.00 RB R eagent Blank - 26.0 0.05 - 1.30 193 Table C1 1: mtDNA Quantification of C - 06 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA y ields. Normalized val ues are reported as ng per mg of bone powder. C - 06 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 27.0 3.4 3 1.82 92.61 2 Midshaft Diaphysis (Distal to Region 1) 50 31.0 20.40 3.48 174.22 3 Diaphysis (Proximal to Region 1) 49 28.0 3.36 1.92 94.08 4 Diaphysis (Distal to Region 2) 49 28.5 7.44 4.33 212.04 5 Diaphysis (Proximal to Region 3) 50 30.0 7.12 4.27 2 13.60 6 Diaphysis (Distal to Region 4) 49 30.0 9.06 5.55 271.80 7 Proximal Metaphysis 50 27.5 12.80 7.04 352.00 8 Distal Metaphysis 49 28.5 23.50 13.67 669.75 9 Articulating Surface 50 30.0 19.90 18.47 597.00 10 Distal Epiphysis 50 28.0 66.80 37.41 18 70.40 11 Femoral Head 50 28.5 46.00 26.22 1311.00 12 Trochanter 50 29.0 41.10 23.84 1191.90 13 Calcaneus 50 31.0 31.80 19.72 985.80 14 Talus 49 27.5 42.00 23.57 1155.00 RB Reagent Blank - 29.0 0.06 - 1.74 194 Table C1 2: mtDNA Quantification of C - 06 E xtracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. C - 06 Mit ochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 29.5 7.51 4.34 221.55 2 Midshaft Diaphysis (Distal to Region 1) 50 29.5 7.35 4.34 2 16.83 3 Diaphysis (Proximal to Region 1) 51 31.0 4.34 2.64 134.54 4 Diaphysis (Distal to Region 2) 49 27.5 11.20 6.29 308.00 5 Diaphysis (Proximal to Region 3) 50 30.5 17.60 10.74 536.80 6 Diaphysis (Distal to Region 4) 50 28.0 24.00 13.44 672.00 7 Pr oximal Metaphysis 50 21.5 30.60 13.16 657.90 8 Distal Metaphysis 50 30.0 31.50 18.90 945.00 9 Articulating Surface 50 28.0 32.80 18.37 918.40 10 Distal Epiphysis 51 29.5 52.90 30.60 1560.55 11 Femoral Head 51 28.0 77.20 42.38 2161.60 12 Trochanter 50 29.0 53.20 30.86 1542.80 13 Calcaneus 50 29.0 28.70 16.65 832.30 14 Talus 61 29.0 31.60 17.97 916.40 RB Reagent Blank - 27.5 0.01 - 0.28 195 Table C13 : mtDNA Quantification of C - 07 Extracts Digested in Tissue Lysis Buffer Region and location corre spond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. C - 07 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 29.0 4.62 2.68 133.98 2 Midshaft Diaphysis (Distal to Region 1) 50 30.0 7.71 4.63 231.30 3 Diaphysis (Proximal to Region 1) 50 29.0 9.65 5.60 279.85 4 Diaphysis (Distal to Region 2) 49 25.0 8.87 4.53 221.75 5 Diaphysis (Proximal to Region 3) 51 26.5 8.84 4.59 234.26 6 Diaphysis (Distal to Region 4) 51 30.0 10.70 6.29 321.00 7 Proximal Metaphysis 51 29.0 35.50 20.19 1029.50 8 Distal Metaphysis 51 28.0 10.30 5.65 288.40 9 Articulating Surface 51 28.0 20.70 11.36 579.60 10 Distal Epiphysis 50 31.0 46.20 28.64 1432.20 11 Femoral Head 51 30.0 11.60 6.82 348.00 12 Trochanter 50 28.0 54.20 30.35 1517.60 13 Calcaneus 50 26.5 33.80 17.91 895.70 14 Talus 49 31.0 23.70 14.99 734.70 RB Reagent Blank - 28.0 0.02 - 0.56 196 Table C14 : mtDNA Quantification of C - 07 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was co nsidered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. C - 07 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 49 30.5 6.78 4.22 206.79 2 Midshaft Diaphysis (Distal to Region 1) 49 32.0 5.59 2.65 178.88 3 Diaphysis (Proximal to Region 1) 50 26.0 11.20 5.82 291.20 4 Diaphysis (Distal to Region 2) 51 30.0 5.26 3.09 157.80 5 Diaphysis (P roximal to Region 3) 49 28.5 12.70 7.39 361.95 6 Diaphysis (Distal to Region 4) 51 28.5 5.29 2.96 150.77 7 Proximal Metaphysis 50 30.5 34.10 20.80 1040.05 8 Distal Metaphysis 51 28.5 12.60 7.04 359.10 9 Articulating Surface 50 29.0 24.60 14.27 713.40 10 Distal Epiphysis 50 26.5 49.10 26.02 1301.15 11 Femoral Head 51 30.5 39.40 23.56 1201.70 12 Trochanter 51 28.5 46.40 25.93 1322.40 13 Calcaneus 49 29.0 22.30 13.20 646.70 14 Talus 50 29.5 15.10 8.91 445.45 RB Reagent Blank - 28.0 0.01 - 0.28 197 Table C15 : mtDNA Quantification of C - 08 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are report ed as ng per mg of bone powder. C - 08 Mitochondrial DNA ( ATPase ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 30.0 3.73 2.24 111.90 2 Midshaft Diaphysis (Distal to Region 1) 50 28.5 4.67 2.66 133.10 3 Diaphysis (Proximal to Region 1) 50 26.0 7.00 3.64 182.00 4 Diaphysis (Distal to Region 2) 49 28.5 3.79 2.20 108.02 5 Diaphysis (Proximal to Region 3) 51 29.0 7.10 4.04 205.90 6 Diaphysis (Distal to Regio n 4) 50 25.0 5.87 2.94 146.75 7 Proximal Metaphysis 50 27.0 21.70 11.72 585.90 8 Distal Metaphysis 50 28.5 18.00 10.26 513.00 9 Articulating Surface 49 26.0 24.20 12.84 629.20 10 Distal Epiphysis 50 27.0 71.20 38.45 1922.40 11 Femoral Head 50 29.0 42. 00 24.36 1218.00 12 Trochanter 51 27.0 47.10 24.94 1271.70 13 Calcaneus 51 29.0 19.80 11.26 574.20 14 Talus 50 27.5 17.30 9.52 475.75 RB Reagent Blank - 28.0 0.01 - 0.28 198 Table C16 : mtDNA Quantification of C - 08 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powder. C - 08 Mitochondrial DNA ( ATPase ) Region Loca tion Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 28.5 4.36 2.44 124.26 2 Midshaft Diaphysis (Distal to Region 1) 49 28.0 5.75 3.29 161.00 3 Diaphysis (Proximal to Regi on 1) 50 26.5 8.97 4.75 237.71 4 Diaphysis (Distal to Region 2) 51 28.5 4.87 2.72 138.80 5 Diaphysis (Proximal to Region 3) 49 28.5 7.88 4.58 224.58 6 Diaphysis (Distal to Region 4) 50 26.0 9.92 5.16 257.92 7 Proximal Metaphysis 51 31.5 22.60 13.96 711 .90 8 Distal Metaphysis 50 28.0 19.10 10.70 534.80 9 Articulating Surface 49 29.0 17.80 10.53 516.20 10 Distal Epiphysis 50 27.5 64.20 35.31 1765.50 11 Femoral Head 51 28.0 54.70 30.03 1531.60 12 Trochanter 51 29.0 32.20 18.31 933.80 13 Calcaneus 51 26.0 26.70 13.61 694.20 14 Talus 50 27.0 22.20 11.99 599.40 RB Reagent Blank - 26.5 0.00 - 0.00 199 Table C17 : Nuclear DNA Quantification of C - 01 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder. C - 01 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg ) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 30.0 306.00 180.00 9180.00 2 Midshaft Diaphysis (Distal to Region 1) 50 25.0 386.00 193.00 9650.00 3 Diaphysis (Proximal to Region 1) 51 29.0 282.00 160.35 8178.00 4 Diaphysis (Distal to Region 2) 49 2 9.0 288.00 170.45 8352.00 5 Diaphysis (Proximal to Region 3) 50 30.0 274.00 164.40 8220.00 6 Diaphysis (Distal to Region 4) 51 30.0 157.00 92.35 4710.00 7 Proximal Metaphysis 49 26.0 660.00 350.20 17160.00 8 Distal Metaphysis 50 30.0 448.00 289.88 1478 4.00 9 Articulating Surface 49 30.0 410.00 251.02 12300.00 10 Distal Epiphysis 50 30.0 1200.00 720.00 36000.00 11 Femoral Head 51 29.0 813.00 462.29 23577.00 12 Trochanter 51 29.0 1460.00 830.20 42340.00 13 Calcaneus 50 29.0 482.00 279.56 13978.00 1 4 Talus 51 27.0 1240.00 656.47 33480.00 RB Reagent Blank - 26.0 0.29 - 7.54 200 Table C18 : Nuclear DNA Quantification of C - 01 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder. C - 01 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recove red (ng) 1 Midshaft Diaphysis 49 28.0 392.00 224.00 10976.00 2 Midshaft Diaphysis (Distal to Region 1) 49 25.0 433.00 220.92 10825.00 3 Diaphysis (Proximal to Region 1) 50 30.0 350.00 210.00 10500.00 4 Diaphysis (Distal to Region 2) 51 30.0 394.00 231. 76 11820.00 5 Diaphysis (Proximal to Region 3) 49 30.0 300.00 183.67 9000.00 6 Diaphysis (Distal to Region 4) 49 30.0 354.00 216.73 10620.00 7 Proximal Metaphysis 51 29.0 492.00 279.76 14268.00 8 Distal Metaphysis 51 29.0 482.00 274.08 13978.00 9 Arti culating Surface 49 29.0 586.00 346.82 16994.00 10 Distal Epiphysis 51 30.0 920.00 541.18 27600.00 11 Femoral Head 50 30.0 1360.00 816.00 40800.00 12 Trochanter 51 30.0 672.00 395.29 20160.00 13 Calcaneus 49 30.0 357.00 218.57 10710.00 14 Talus 49 3 1.0 878.00 555.47 27218.00 RB Reagent Blank - 30.0 0.31 - 9.30 201 Table C19 : Nuclear DNA Quantification of C - 02 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was conside red in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder. C - 02 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaf t Diaphysis 49 34.0 48.20 33.44 1638.80 2 Midshaft Diaphysis (Distal to Region 1) 51 30.0 250.00 147.06 7500.00 3 Diaphysis (Proximal to Region 1) 49 30.0 143.00 87.55 4290.00 4 Diaphysis (Distal to Region 2) 49 30.0 148.00 90.61 4440.00 5 Diaphysis (P roximal to Region 3) 51 29.0 272.00 154.67 7888.00 6 Diaphysis (Distal to Region 4) 49 28.0 182.00 104.00 5096.00 7 Proximal Metaphysis 50 32.0 1060.00 678.40 33920.00 8 Distal Metaphysis 50 32.0 461.00 295.04 14752.00 9 Articulating Surface 50 30.0 14 20.00 852.00 42600.00 10 Distal Epiphysis 51 32.0 3050.00 1913.73 97600.00 11 Femoral Head 51 32.0 1860.00 1167.06 59520.00 12 Trochanter 51 31.0 1730.00 1051.57 53630.00 13 Calcaneus 49 30.0 1110.00 679.59 33300.00 14 Talus 50 31.0 1790.00 1109.80 55490.00 RB Reagent Blank - 30.0 0.00 - 0.00 202 Table C20 : Nuclear DNA Quantification of C - 02 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order t o normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder. C - 02 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 30.0 496.00 297.60 14880.00 2 Midshaft Diaphysis (Distal to Region 1) 51 30.0 416.00 244.71 12480.00 3 Diaphysis (Proximal to Region 1) 51 29.0 521.00 296.25 15109.00 4 Diaphysis (Distal to Region 2) 50 29.0 341.00 197.78 9889.00 5 Diaphysis (Proximal to Region 3) 50 29.0 566.00 328.28 16414.00 6 Diaphysis (Distal to Region 4) 49 25.0 518.00 264.29 12950.00 7 Proximal Metaphysis 50 26.0 632.00 328.64 16432.00 8 Distal Metaphysis 50 29.0 620.00 359.60 17980.00 9 Articulating Surface 49 29.0 203.00 1 20.14 5887.00 10 Distal Epiphysis 50 32.0 1430.00 915.20 45760.00 11 Femoral Head 49 30.0 1730.00 1059.18 51900.00 12 Trochanter 50 30.0 1470.00 882.00 44100.00 13 Calcaneus 51 30.0 1280.00 752.94 38400.00 14 Talus 49 34.0 1510.00 1047.76 51340.00 RB Reagent Blank - 29.0 1.51 - 43.79 203 Table C2 1: Nuclear DNA Quantification of C - 03 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize D NA yields. Normaliz ed values are reported as ng per mg of bone powder. C - 03 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 29.0 68.70 3 9.85 1992.30 2 Midshaft Diaphysis (Distal to Region 1) 49 26.0 173.00 91.80 4498.00 3 Diaphysis (Proximal to Region 1) 50 30.0 129.00 77.40 3870.00 4 Diaphysis (Distal to Region 2) 50 27.0 186.00 100.44 5022.00 5 Diaphysis (Proximal to Region 3) 50 2 9.0 165.00 95.70 4785.00 6 Diaphysis (Distal to Region 4) 49 30.0 186.00 113.88 5580.00 7 Proximal Metaphysis 50 32.0 366.00 234.24 11712.00 8 Distal Metaphysis 50 30.0 324.00 194.40 9720.00 9 Articulating Surface 51 31.0 348.00 211.53 10788.00 10 Di stal Epiphysis 51 31.0 618.00 375.65 19158.00 11 Femoral Head 49 30.0 530.00 324.49 15900.00 12 Trochanter 50 25.0 758.00 379.00 18950.00 13 Calcaneus 50 30.0 358.00 214.80 10740.00 14 Talus 50 30.0 286.00 171.60 8580.00 RB Reagent Blank - 26.0 0.0 2 - 0.52 204 Table C 2 2: Nuclear DNA Quantification of C - 03 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder. C - 03 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 30.0 138.00 81.18 4140.00 2 Mid shaft Diaphysis (Distal to Region 1) 51 30.0 152.00 89.41 4560.00 3 Diaphysis (Proximal to Region 1) 51 27.0 165.00 87.35 4455.00 4 Diaphysis (Distal to Region 2) 49 32.0 175.00 114.29 5600.00 5 Diaphysis (Proximal to Region 3) 50 26.0 181.00 94.12 4706 .00 6 Diaphysis (Distal to Region 4) 50 29.0 200.00 116.00 5800.00 7 Proximal Metaphysis 50 28.0 274.00 153.44 7672.00 8 Distal Metaphysis 51 29.0 215.00 122.25 6235.00 9 Articulating Surface 51 26.0 310.00 158.04 8060.00 10 Distal Epiphysis 49 31.0 3 61.00 228.39 11191.00 11 Femoral Head 49 31.0 394.00 249.27 12214.00 12 Trochanter 49 26.0 455.00 241.43 11830.00 13 Calcaneus 50 32.0 213.00 136.32 6816.00 14 Talus 51 30.0 198.00 116.47 5940.00 RB Reagent Blank - 28.0 2.09 - 58.52 205 Table C23 : Nuclear DNA Quantification of C - 04 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder. C - 04 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 32.0 96.50 60.55 3088.00 2 Midshaft Diaphysis (Distal to Reg ion 1) 50 27.0 94.40 50.98 2548.80 3 Diaphysis (Proximal to Region 1) 50 30.0 88.00 52.80 2640.00 4 Diaphysis (Distal to Region 2) 50 27.0 147.00 79.38 3969.00 5 Diaphysis (Proximal to Region 3) 51 29.0 126.00 71.65 3654.00 6 Diaphysis (Distal to Regio n 4) 50 25.0 211.00 105.50 5275.00 7 Proximal Metaphysis 50 27.0 288.00 155.52 7776.00 8 Distal Metaphysis 51 30.0 210.00 123.53 6300.00 9 Articulating Surface 51 29.0 349.00 198.45 10121.00 10 Distal Epiphysis 50 29.0 607.00 352.06 17603.00 11 Femora l Head 50 30.0 346.00 207.60 10380.00 12 Trochanter 50 30.0 367.00 220.20 11010.00 13 Calcaneus 49 30.0 255.00 156.12 7650.00 14 Talus 49 30.0 213.00 130.41 6390.00 RB Reagent Blank - 28.0 3.16 - 88.48 206 Table C24 : Nuclear DNA Quantification of C - 04 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder. C - 04 Nucl ear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 49 28.0 91.90 52.51 2573.20 2 Midshaft Diaphysis (Distal to Region 1) 50 30.0 159.00 95.40 4770 .00 3 Diaphysis (Proximal to Region 1) 49 31.0 157.00 99.33 4876.00 4 Diaphysis (Distal to Region 2) 51 28.0 201.00 110.35 5628.00 5 Diaphysis (Proximal to Region 3) 51 28.0 178.00 97.73 4984.00 6 Diaphysis (Distal to Region 4) 51 29.0 216.00 122.82 62 64.00 7 Proximal Metaphysis 50 29.0 221.00 128.18 6409.00 8 Distal Metaphysis 51 31.0 166.00 100.90 5146.00 9 Articulating Surface 51 27.0 312.00 165.18 8424.00 10 Distal Epiphysis 50 30.0 444.00 266.40 13320.00 11 Femoral Head 51 31.0 372.00 226.12 1 1532.00 12 Trochanter 51 28.0 207.00 113.65 5796.00 13 Calcaneus 50 31.0 157.00 97.34 4867.00 14 Talus 51 26.0 154.00 78.51 4004.00 RB Reagent Blank - 29.0 0.23 - 6.67 207 Table C25 : Nuclear DNA Quantification of C - 05 Extracts Digested in Tissue Ly sis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder. C - 05 Nuclear DNA ( MC1R ) Region Location Bo ne Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 30.0 46.30 27.78 1389.00 2 Midshaft Diaphysis (Distal to Region 1) 50 25.5 99.00 50.49 2524.50 3 Diaphysis (Proximal to Regio n 1) 50 27.0 67.60 30.49 1441.80 4 Diaphysis (Distal to Region 2) 50 37.0 54.40 40.26 2012.80 5 Diaphysis (Proximal to Region 3) 51 23.0 67.60 30.49 1554.80 6 Diaphysis (Distal to Region 4) 50 37.0 115.00 73.60 3680.00 7 Proximal Metaphysis 50 30.0 156 .00 93.60 4680.00 8 Distal Metaphysis 49 30.0 147.00 90.00 4410.00 9 Articulating Surface 50 32.0 335.00 214.40 10720.00 10 Distal Epiphysis 50 28.0 441.00 246.96 12348.00 11 Femoral Head 51 30.0 468.00 275.29 14040.00 12 Trochanter 51 26.0 604.00 307 .92 15704.00 13 Calcaneus 51 30.5 427.00 255.36 13023.00 14 Talus 50 27.5 501.00 275.55 13777.50 RB Reagent Blank - 28.0 0.88 - 24.64 208 Table C26 : Nuclear DNA Quantification of C - 05 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder. C - 05 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL ) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 29.0 161.00 93.38 4669.00 2 Midshaft Diaphysis (Distal to Region 1) 51 28.0 261.00 143.29 7308.00 3 Diaphysis (Proximal to Region 1) 49 28.0 186.00 106.29 5 208.00 4 Diaphysis (Distal to Region 2) 49 31.5 168.00 108.00 5292.00 5 Diaphysis (Proximal to Region 3) 50 32.0 205.00 131.20 6560.00 6 Diaphysis (Distal to Region 4) 50 30.0 178.00 106.80 5340.00 7 Proximal Metaphysis 51 29.0 280.00 159.22 8120.00 8 Distal Metaphysis 50 28.0 261.00 146.16 7308.00 9 Articulating Surface 51 28.0 333.00 182.82 9324.00 10 Distal Epiphysis 50 29.5 378.00 223.02 11151.00 11 Femoral Head 51 31.0 402.00 244.35 12462.00 12 Trochanter 50 27.0 446.00 240.35 12042.00 13 Cal caneus 51 28.0 421.00 231.14 11788.00 14 Talus 51 30.0 322.00 189.41 9660.00 RB Reagent Blank - 26.0 0.73 - 18.98 209 Table C27 : Nuclear DNA Quantification of C - 06 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder. C - 06 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 27.0 31.10 16.46 839.70 2 Midshaft Diaphysis (Distal to Region 1) 50 31.0 74.70 46.31 2315.70 3 Diaphysis (Proximal to Region 1) 49 28.0 13.80 7.89 386.40 4 Diaphysis (Distal to Re gion 2) 49 28.5 98.60 57.35 2810.10 5 Diaphysis (Proximal to Region 3) 50 30.0 84.00 50.40 2520.00 6 Diaphysis (Distal to Region 4) 49 30.0 190.00 116.33 5700.00 7 Proximal Metaphysis 50 27.5 151.00 83.05 4152.50 8 Distal Metaphysis 49 28.5 248.00 144. 24 7068.00 9 Articulating Surface 50 30.0 433.00 259.80 12990.00 10 Distal Epiphysis 50 28.0 481.00 269.36 13468.00 11 Femoral Head 50 28.5 420.00 239.40 11970.00 12 Trochanter 50 29.0 492.00 285.36 14268.00 13 Calcaneus 50 31.0 444.00 275.28 13764.0 0 14 Talus 49 27.5 490.00 275.00 13475.00 RB Reagent Blank - 29.0 0.27 - 7.69 210 Table C28 : Nuclear DNA Quantification of C - 06 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bo ne powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder. C - 06 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Reco vered (ng) 1 Midshaft Diaphysis 51 29.5 238.00 137.67 7021.00 2 Midshaft Diaphysis (Distal to Region 1) 50 29.5 189.00 111.51 5575.50 3 Diaphysis (Proximal to Region 1) 51 31.0 186.00 113.06 5766.00 4 Diaphysis (Distal to Region 2) 49 27.5 202.00 113.3 7 5555.00 5 Diaphysis (Proximal to Region 3) 50 30.5 165.00 100.65 5032.50 6 Diaphysis (Distal to Region 4) 50 28.0 300.00 168.00 8400.00 7 Proximal Metaphysis 50 21.5 244.00 104.92 5246.00 8 Distal Metaphysis 50 30.0 313.00 187.80 9390.00 9 Articulat ing Surface 50 28.0 301.00 168.56 8428.00 10 Distal Epiphysis 51 29.5 374.00 216.33 11033.00 11 Femoral Head 51 28.0 377.00 206.98 10556.00 12 Trochanter 50 29.0 354.00 205.32 10266.00 13 Calcaneus 50 29.0 363.00 210.54 10527.00 14 Talus 61 29.0 337 .00 191.63 9773.00 RB Reagent Blank - 27.5 0.04 - 1.02 211 Table C29 : Nuclear DNA Quantification of C - 07 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in o rder to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder. C - 07 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphy sis 50 29.0 113.00 65.54 3277.00 2 Midshaft Diaphysis (Distal to Region 1) 50 30.0 156.00 93.60 4680.00 3 Diaphysis (Proximal to Region 1) 50 29.0 151.00 87.58 4379.00 4 Diaphysis (Distal to Region 2) 49 25.0 175.00 89.29 4375.00 5 Diaphysis (Proximal to Region 3) 51 26.5 170.00 88.33 4505.00 6 Diaphysis (Distal to Region 4) 51 30.0 142.00 83.53 4260.00 7 Proximal Metaphysis 51 29.0 393.00 223.47 11397.00 8 Distal Metaphysis 51 28.0 250.00 137.25 7000.00 9 Articulating Surface 51 28.0 299.00 164.16 8372.00 10 Distal Epiphysis 50 31.0 460.00 285.20 14260.00 11 Femoral Head 51 30.0 349.00 205.29 10470.00 12 Trochanter 50 28.0 673.00 376.88 18844.00 13 Calcaneus 50 26.5 440.00 233.20 11660.00 14 Talus 49 31.0 424.00 268.24 13144.00 RB Reagent Bl ank - 28.0 0.70 - 19.60 212 Table C30 : Nuclear DNA Quantification of C - 07 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields . Normaliz ed values are reported as ng per mg of bone powder. C - 07 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 49 30.5 137.00 85.28 417 8.50 2 Midshaft Diaphysis (Distal to Region 1) 49 32.0 141.00 92.08 4512.00 3 Diaphysis (Proximal to Region 1) 50 26.0 178.00 92.56 4628.00 4 Diaphysis (Distal to Region 2) 51 30.0 126.00 74.12 3780.00 5 Diaphysis (Proximal to Region 3) 49 28.5 162.00 94.22 4617.00 6 Diaphysis (Distal to Region 4) 51 28.5 132.00 73.76 3762.00 7 Proximal Metaphysis 50 30.5 313.00 190.93 9546.50 8 Distal Metaphysis 51 28.5 154.00 86.06 4389.00 9 Articulating Surface 50 29.0 267.00 154.86 7743.00 10 Distal Epiphysis 5 0 26.5 544.00 288.32 14416.00 11 Femoral Head 51 30.5 335.00 200.34 10217.50 12 Trochanter 51 28.5 464.00 259.29 13224.00 13 Calcaneus 49 29.0 295.00 174.59 8555.00 14 Talus 50 29.5 233.00 137.47 6873.50 RB Reagent Blank - 28.0 0.63 - 17.64 213 Ta ble C31: Nuclear DNA Quantification of C - 08 Extracts Digested in Tissue Lysis Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reporte d as ng per mg of bone powder. C - 08 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 50 30.0 96.00 57.60 2880.00 2 Midshaft Diaphysis (Dista l to Region 1) 50 28.5 148.00 84.36 4218.00 3 Diaphysis (Proximal to Region 1) 50 26.0 131.00 68.12 3406.00 4 Diaphysis (Distal to Region 2) 49 28.5 131.00 76.19 3733.50 5 Diaphysis (Proximal to Region 3) 51 29.0 156.00 88.71 4524.00 6 Diaphysis (Dista l to Region 4) 50 25.0 208.00 104.00 5200.00 7 Proximal Metaphysis 50 27.0 345.00 186.30 9315.00 8 Distal Metaphysis 50 28.5 291.00 165.87 8293.50 9 Articulating Surface 49 26.0 288.00 152.82 7488.00 10 Distal Epiphysis 50 27.0 476.00 257.04 12852.00 11 Femoral Head 50 29.0 380.00 220.40 11020.00 12 Trochanter 51 27.0 484.00 256.24 13068.00 13 Calcaneus 51 29.0 335.00 190.49 9715.00 14 Talus 50 27.5 311.00 171.05 8552.50 RB Reagent Blank - 28.0 0.96 - 26.88 214 Table C32: Nuclear DNA Quantification of C - 08 Extracts Digested in Demineralization Buffer Region and location correspond with Table 3. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normaliz ed values are reported as ng per mg of bone powder. C - 08 Nuclear DNA ( MC1R ) Region Location Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 1 Midshaft Diaphysis 51 28.5 113.00 63.15 3220.50 2 Midshaft Diaphysis (Distal to Region 1) 49 28.0 101.00 57.71 2828.00 3 Diaphysis (Proximal to Region 1) 50 26.5 146.00 77.38 3869.00 4 Diaphysis (Distal to Region 2) 51 28.5 108.00 60.35 3078.00 5 Diaphysis (Proximal to Region 3) 49 28.5 94.60 55.02 2696.10 6 Diaphysis (Distal to Region 4) 50 26.0 143.00 7 4.36 3718.00 7 Proximal Metaphysis 51 31.5 234.00 144.53 7371.00 8 Distal Metaphysis 50 28.0 204.00 114.24 5712.00 9 Articulating Surface 49 29.0 180.00 106.53 5220.00 10 Distal Epiphysis 50 27.5 446.00 245.30 12265.00 11 Femoral Head 51 28.0 317.00 1 74.04 8876.00 12 Trochanter 51 29.0 179.00 101.78 5191.00 13 Calcaneus 51 26.0 227.00 115.73 5902.00 14 Talus 50 27.0 199.00 107.46 5373.00 RB Reagent Blank - 26.5 0.00 - 0.00 215 Table C33: Bovine Mitochondrial DNA PCR Amplification Chart for Quality Assay (Tissue Lysis Buffer) Region and location correspond to Table 3. Values (1 4) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 201 bp amplicon, 2 = 390 bp amplicon, 3 = 607 b p amplicon, 4 = 994 bp amplicon. NA = No Amplification Region Location Mitochondrial DNA ( ATPase ) C - 01 C - 02 C - 03 C - 04 C - 05 C - 06 C - 07 C - 08 1 Midshaft Diaphysis 4 4 3 3 4 4 4 4 2 Midshaft Diaphysis (Distal to Region 1) 4 4 3 3 3 3 3 4 3 Diaphysis (Proximal to Region 1) 4 4 2 4 4 4 4 4 4 Diaphysis (Distal to Region 2) 4 4 2 4 4 3 4 4 5 Diaphysis (Proximal to Region 3) 4 4 3 4 4 4 4 4 6 Diaphysis (Distal to Region 4) 4 4 2 4 3 4 4 4 7 Proximal Metaphysis 4 4 3 2 4 4 4 4 8 Distal Metaphysis 4 4 2 4 4 4 4 4 9 Articulating Surface 4 4 3 3 4 3 4 4 10 Distal Epiphysis 4 4 3 2 4 4 4 4 11 Femoral Head 4 4 4 4 4 4 4 4 12 Trochanter 4 4 4 3 4 4 4 4 13 Calcaneus 4 4 3 4 4 4 4 4 14 Talus 4 4 3 4 4 4 4 4 RB Reagent Blank 4 4 NA NA NA NA NA NA 216 Table C34: Bovine Mitochondrial DNA PCR Amplification Chart for Quality Assay (Demineralization Buffer) Region and location correspond to Table 3. Values (1 4) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 201 bp ampli con, 2 = 390 bp amplicon, 3 = 607 b p amplicon, 4 = 994 bp amplicon. NA = No Amplification Region Location Mitochondrial DNA ( ATPase ) C - 01 C - 02 C - 03 C - 04 C - 05 C - 06 C - 07 C - 08 1 Midshaft Diaphysis 4 4 4 4 3 3 4 4 2 Midshaft Diaphysis (Distal to Region 1) 4 4 3 3 4 3 4 4 3 Diaphysis (Proximal to Region 1) 4 4 4 3 4 4 4 4 4 Diaphysis (Distal to Region 2) 4 4 3 4 4 4 4 4 5 Diaphysis (Proximal to Region 3) 4 4 4 3 4 4 4 4 6 Diaphysis (Distal to Region 4) 4 4 3 3 3 4 4 4 7 Proximal Metaphysis 4 4 4 4 4 4 4 4 8 Distal Metaphysis 4 4 4 4 4 4 4 4 9 Articulating Surface 4 4 4 3 4 4 4 3 10 Distal Epiphysis 4 4 3 4 4 4 4 4 11 Femoral Head 4 4 4 4 4 4 4 4 12 Trochanter 4 4 3 4 4 4 4 4 13 Calcaneus 4 4 3 4 4 4 4 4 14 Talus 4 4 4 4 4 4 4 4 RB Reagent Bl ank 4 4 NA NA NA NA NA NA 217 Table C35: Bovine Nuclear DNA PCR Amplification Chart for Quality Assay (Tissue Lysis Buffer) Region and location correspond to Table 3. Values (1 4) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 200 bp amplicon, 2 = 410 bp amplicon, 3 = 599 bp amplicon, 4 = 989 bp amplicon. NA = No Amplification Region Location Nuclear DNA ( MC1R ) C - 01 C - 02 C - 03 C - 04 C - 05 C - 06 C - 07 C - 08 1 Midshaft Diaphysis 2 2 2 1 2 2 2 2 2 Mid shaft Diaphysis (Distal to Region 1) 2 2 1 2 1 1 2 2 3 Diaphysis (Proximal to Region 1) 2 2 2 1 2 1 2 3 4 Diaphysis (Distal to Region 2) 2 2 1 2 1 1 2 2 5 Diaphysis (Proximal to Region 3) 2 2 2 1 1 1 2 2 6 Diaphysis (Distal to Region 4) 2 2 1 2 2 2 2 2 7 Proximal Metaphysis 2 2 1 1 2 2 2 2 8 Distal Metaphysis 2 2 1 1 2 2 2 2 9 Articulating Surface 2 2 2 2 2 2 2 2 10 Distal Epiphysis 2 2 1 2 2 2 2 2 11 Femoral Head 2 2 2 2 2 2 2 2 12 Trochanter 2 2 2 2 2 2 2 2 13 Calcaneus 2 2 1 1 2 2 2 2 14 T alus 2 1 2 2 2 2 1 2 RB Reagent Blank NA 2 NA NA 2 NA NA NA 218 Table C36: Bovine Nuclear DNA PCR Amplification Chart for Quality Assay (Demineralization Buffer) Region and location correspond to Table 3. Values (1 4) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 200 bp amplicon, 2 = 410 bp amplicon, 3 = 599 b p amplicon, 4 = 989 bp amplicon. NA = No Amplification Region Location Nuclear DNA ( MC1R ) C - 01 C - 02 C - 03 C - 04 C - 05 C - 06 C - 07 C - 08 1 M idshaft Diaphysis 2 2 2 2 2 2 2 2 2 Midshaft Diaphysis (Distal to Region 1) 2 2 2 2 2 2 2 2 3 Diaphysis (Proximal to Region 1) 3 2 1 2 2 1 2 2 4 Diaphysis (Distal to Region 2) 2 2 2 2 2 2 2 2 5 Diaphysis (Proximal to Region 3) 2 2 2 2 2 2 2 2 6 Diaph ysis (Distal to Region 4) 2 2 1 2 2 2 2 2 7 Proximal Metaphysis 2 2 2 2 2 2 2 2 8 Distal Metaphysis 2 2 2 2 2 2 2 2 9 Articulating Surface 2 2 2 1 2 2 2 2 10 Distal Epiphysis 2 2 2 2 2 2 2 2 11 Femoral Head 2 2 2 2 2 2 2 2 12 Trochanter 2 2 2 2 2 2 2 2 13 Calcaneus 2 2 2 1 2 2 1 2 14 Talus 2 1 2 2 2 2 2 2 RB Reagent Blank NA NA NA NA NA NA NA NA 219 APPENDIX D: MITOCHONDRIAL AND NUCLEAR DNA QUANTIFICATION DATA AND INDIVIDUAL PCR AMPLIFICATION RESULTS FROM SURFACE EXPOSED AND BURIED BO VINE FEMORA AND TARSALS Table D1: mtDNA Quantification of Day 0 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Nor malized va lues are reported as ng per mg of bone Day 0 Mitochondrial DNA ( ATPase ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (n g) Diaphysis 1 51 29.5 37.00 21.40 631.36 2 49 30.0 56.30 34.47 1034.08 Proximal Metaphysis 1 51 31.0 27.80 16.90 523.84 2 50 30.0 20.40 12.24 367.20 Distal Metaphysis 1 51 31.5 12.20 7.54 237.36 2 51 32.0 32.40 20.33 650.54 Femoral Head 1 51 31. 0 101.00 61.39 1903.00 2 51 32.0 118.00 74.04 2369.25 Distal Epiphysis 1 50 31.5 110.00 69.30 2182.95 2 50 33.0 128.00 84.48 2787.84 Calcaneus 1 51 27.5 76.40 41.20 1132.89 2 51 30.0 50.90 29.94 898.24 Talus 1 51 34.0 66.40 44.27 1505.07 2 50 29 .0 68.50 39.73 1152.17 Reagent Blank - 28.0 0.003 - 0.084 220 Table D2: mtDNA Quantification of Week 1 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized va lues are reported as ng per mg of bone Week 1 Mitochondrial DNA ( ATPase ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Nor malized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 49 27.0 4.66 2.57 69.33 2 51 27.0 1.55 0.82 22.16 Proximal Metaphysis 1 51 28.0 10.20 5.60 156.80 2 50 27.0 11.90 6.43 173.50 Distal Metaphysis 1 51 26.0 5.37 2.74 71.18 2 49 25.5 27.60 14.3 6 366.26 Femoral Head 1 51 30.0 35.20 20.71 621.18 2 50 29.0 111.00 64.38 1867.02 Distal Epiphysis 1 51 30.5 54.00 32.29 984.97 2 51 30.0 80.60 47.41 1422.35 Calcaneus 1 49 27.0 12.30 6.78 182.99 2 50 31.0 54.90 34.04 1055.18 Talus 1 50 30.0 34.9 0 20.94 628.20 2 51 30.0 46.50 27.35 820.59 Reagent Blank - 28.0 0.004 - 0.112 221 Table D3: mtDNA Quantification of Week 2 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized va lues are reported as ng per mg of bone Week 2 Mitochondrial DNA ( ATPase ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 50 29.0 13.00 7.54 218.66 2 50 26.5 21.90 11.61 307.59 Proximal Metaphysis 1 51 29.0 19.00 10.80 313.31 2 49 30.5 22.30 13.88 423.26 Distal Metaphysis 1 51 28.5 10.50 5.87 167.23 2 50 31.0 23.20 14.38 445.90 Femoral Head 1 51 30.5 51.00 30.50 930.25 2 50 32.0 72.90 46.66 1492.99 Distal Epiphysis 1 50 30.0 42.50 25.50 765.00 2 50 32.5 48.10 31.27 1016.11 Calcaneus 1 50 32.0 33.50 21.44 686.08 2 49 32.5 31.80 21.09 685.48 Talus 1 49 33.5 59.00 40.34 1351.28 2 51 30.5 61.70 36.90 1125.42 Reagent Blank - 27.0 0.00 - 0.00 222 Table D4: mtDNA Quantification of Week 4 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized va lues are reported as ng per mg of bone quantificati on obtained using a 1:10 dilution of extract. Week 4 Mitochondrial DNA ( ATPase ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 51 27.5 18.00 9.71 266.91 2 50 30.5 22.50 13.7 3 418.61 Proximal Metaphysis 1 49 32.0 14.10 9.21 294.66 2 50 31.0 32.20 19.96 618.88 Distal Metaphysis 1 50 30.0 15.40 9.24 277.20 2 49 29.5 36.70 22.09 651.80 Femoral Head 1 51 31.5 65.80 40.64 1280.20 2 50 31.5 5.85** 3.69 116.09 Distal Epiphy sis 1 51 28.0 39.50 21.69 607.22 2 51 27.5 4.72** 2.55 69.99 Calcaneus 1 51 29.5 32.30 18.68 551.16 2 50 31.0 31.20 19.34 599.66 Talus 1 49 28.0 27.30 15.60 436.80 2 51 27.5 11.90 6.42 176.46 Reagent Blank - 29.5 0.00 - 0.00 223 Table D5: mtD NA Quantification of Month 3 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized va lues are reported as ng pe r mg of bone Month 3 Mitochondrial DNA ( ATPase ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 51 29.5 9.26 5.36 158.01 2 51 26.5 8.59 4.46 118.28 Proximal Metaphysis 1 51 29.5 4.60 2.66 78.49 2 50 29.0 14.70 8.53 247.25 Distal Metaphysis 1 50 26.5 7.80 4.13 109.55 2 50 31.0 9.17 5.69 176.25 Femoral Head 1 51 27.5 48.50 26.15 719.18 2 50 29.0 68.00 39.44 1 143.76 Distal Epiphysis 1 49 29.0 17.10 10.12 293.49 2 50 25.5 44.00 22.44 572.22 Calcaneus 1 51 28.0 28.30 15.54 435.04 2 50 29.0 20.10 11.66 338.08 Talus 1 50 31.0 17.90 11.10 344.04 2 50 30.0 14.10 8.46 253.80 Reagent Blank - 29.0 0.00 - 0.00 224 Table D6: mtDNA Quantification of Month 6 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ue s are reported as ng per mg of bone quantification obtained using a 1:10 dilution of extract. Month 6 Mitochondrial DNA ( ATPase ) Region Bone Mass (mg) Extract Vo lume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 50 25.0 7.18 3.59 89.75 2 49 26.5 4.57 2.47 65.50 Proximal Metaphysis 1 49 27.0 6.10 3.36 90.75 2 50 28.0 8.21 4.60 128.73 Distal Metaphysis 1 50 31.0 6.98 4.33 134.16 2 50 29.0 8.32 4.83 139.94 Femoral Head 1 51 31.5 41.30 25.51 803.53 2 51 28.5 14.10 7.88 224.56 Distal Epiphysis 1 51 26.5 34.50 17.93 475.05 2 51 30.0 24.10 14.18 425.29 Calcaneus 1 49 30.5 11.10 6.91 210.73 2 50 27.5 27.70 15.24 4 18.96 Talus 1 49 30.0 32.10 19.65 589.59 2 49 29.0 0.37** 0.22 6.26 Reagent Blank - 29.0 0.00 - 0.00 225 Table D7 : mtDNA Normalized Quantification of Surface Exposed Bovine Skeletal Material Region bone powder was collected from in first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng calcanei, and tali exposed during the course of this exp eriment. Cells highlighted in yellow represent extracts that had PCR inhibition. ied by dilution factor. Surface Mitochondrial D NA (ng/mg) ( ATPase ) Region Day 0 Week 1 Week 2 Week 4 Month 3 Month 6 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 Diaphysis 21.40 34.47 2.57 0.82 7.54 11.61 9.71 13.73 5.36 4.46 3.59 2.47 Proximal Metaphysis 16.90 12.24 5.60 6.43 10.80 13.88 9.21 19.96 2.66 8 .53 3.36 4.60 Distal Metaphysis 7.54 20.33 2.74 14.36 5.87 14.38 9.24 22.09 4.13 5.69 4.33 4.83 Femoral Head 61.39 74.04 20.71 64.38 30.50 46.66 40.64 36.90** 26.15 39.44 25.51 7.88 Distal Epiphysis 69.30 84.48 32.29 47.41 25.50 31.27 21.69 47.20** 10.1 2 22.44 17.93 14.18 Calcaneus 41.20 29.94 6.78 34.04 21.44 21.09 18.68 19.34 15.54 11.66 6.91 15.24 Talus 44.27 39.73 20.94 27.35 40.34 36.90 15.60 6.42 11.10 8.46 19.65 2.20** 226 Table D8 : Bovine Mitochondrial DNA PCR Amplification Chart for Quality Assa y (Surface Exposed Skeletal Material) 4) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 201 bp amplicon, 2 = 390 bp ampl icon, 3 = 607 b p amplicon, 4 = 994 bp amplicon. NA = No Amplification Surface Mitochondrial DNA ( ATPase ) Amplicon Length Region Day 0 Week 1 Week 2 Week 4 Month 3 Month 6 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 Diaphysis 4 4 4 4 4 4 4 4 4 4 4 4 Proximal Metaphysis 4 4 4 4 4 4 4 4 4 4 4 4 Distal Metaphysis 4 4 4 4 4 4 4 4 4 4 4 4 Femoral Head 4 4 4 4 4 4 4 4 4 4 4 4 Distal Epiphysis 4 4 4 4 4 4 4 4 4 4 4 4 Calcaneus 4 4 4 4 4 4 4 4 4 4 4 4 Talus 4 4 4 4 4 4 4 4 4 4 4 4 Reagent Blank NA 4 NA NA NA NA 227 Table D9: Nuclear DNA Normalized Quantification of Surface Exposed Bovine Skeletal Material Region bone powder was collected from in first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yie lds. Normalized v alues are reported as ng calcanei, and tali exposed during the course of this experiment. Cells highlighted in yellow represent extracts that had PCR inhibition. quantification multiplied by dilution factor. Surface Nuclear DNA (ng/mg) ( MC1R ) Region Day 0 Week 1 Week 2 Week 4 Month 3 Month 6 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 Diaphysis 192.04 208.16 63.37 49.02 142.68 170.02 152.60 178.12 202.45 129.90 139.50 122.22 Proximal Metaphysis 147.71 193.20 76.31 170.10 170.02 262.05 148.24 293.88 175.26 335.82 138.86 171.36 Distal Metaphysis 218.65 158.12 4 8.38 401.76 126.85 236.22 184.80 345.57 279.31 319.30 258.54 175.74 Femoral Head 160.47 375.84 278.24 348.00 413.25 812.80 273.62 639.45* 352.65 446.02 282.88 488.41 Distal Epiphysis 158.76 413.82 257.75 243.53 211.20 338.65 322.27 413.60** 257.45 484.50 222.39 116.47 Calcaneus 216.23 206.47 150.98 294.50 248.32 519.34 211.13 511.27** 282.20 489.52 126.98 278.85 Talus 364.67 287.10 259.80 200.00 296.71 476.04 181.71 509.60** 358.36 200.40 287.76 174.60** 228 Table D10 : Nuclear DNA Quantification of Day 0 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized v alues are reported as ng per mg of bone powder. Day 0 Nuclear DNA ( MC1R ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 51 29.5 332.00 192.04 5665.16 2 49 30.0 340.00 208.16 624 4.90 Proximal Metaphysis 1 51 31.0 243.00 147.71 4578.88 2 50 30.0 322.00 193.20 5796.00 Distal Metaphysis 1 51 31.5 354.00 218.65 6887.38 2 51 32.0 252.00 158.12 5059.76 Femoral Head 1 51 31.0 264.00 160.47 4974.59 2 51 32.0 599.00 375.84 12026.9 8 Distal Epiphysis 1 50 31.5 252.00 158.76 5000.94 2 50 33.0 627.00 413.82 13656.06 Calcaneus 1 51 27.5 401.00 216.23 5946.20 2 51 30.0 351.00 206.47 6194.12 Talus 1 51 34.0 547.00 364.67 12398.67 2 50 29.0 495.00 287.10 8325.90 Reagent Blank - 2 8.0 0.02 - 0.56 229 Table D11 : Nuclear DNA Quantification of Week 1 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize DNA yiel ds. Normalized values are reported as ng per mg of bone powder. Week 1 Nuclear DNA ( MC1R ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered ( ng) Diaphysis 1 49 27.0 115.00 63.37 1710.92 2 51 27.0 92.60 49.02 1323.64 Proximal Metaphysis 1 51 28.0 139.00 76.31 2136.78 2 50 27.0 315.00 170.10 4592.70 Distal Metaphysis 1 51 26.0 94.90 48.38 1257.89 2 49 25.5 772.00 401.76 10244.76 Femoral Head 1 51 30.0 473.00 278.24 8347.06 2 50 29.0 600.00 348.00 10092.00 Distal Epiphysis 1 51 30.5 431.00 257.75 7861.52 2 51 30.0 414.00 243.53 7305.88 Calcaneus 1 49 27.0 274.00 150.98 4076.45 2 50 31.0 475.00 294.50 9129.50 Talus 1 50 30.0 433.0 0 259.80 7794.00 2 51 30.0 340.00 200.00 6000.00 Reagent Blank - 28.0 0.08 - 2.24 230 Table D12 : Nuclear DNA Quantification of Week 2 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume an d milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng per mg of bone powder. Week 2 Nuclear DNA ( MC1R ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 50 29.0 246.00 142.68 4137.72 2 50 26.5 343.00 181.79 4817.44 Proximal Metaphysis 1 51 29.0 299.00 170.02 4930.57 2 49 30.5 421.00 262.05 7992.56 Distal Metaphy sis 1 51 28.5 227.00 126.85 3615.31 2 50 31.0 381.00 236.22 7322.82 Femoral Head 1 51 30.5 691.00 413.25 12603.98 2 50 32.0 1270.00 812.80 26009.60 Distal Epiphysis 1 50 30.0 352.00 211.20 6336.00 2 50 32.5 521.00 338.65 11006.13 Calcaneus 1 50 32 .0 388.00 248.32 7946.24 2 49 32.5 783.00 519.34 16878.44 Talus 1 49 33.5 434.00 296.71 9939.93 2 51 30.5 796.00 476.04 14519.20 Reagent Blank - 27.0 1.13 - 30.51 231 Table D13 : Nuclear DNA Quantification of Week 4 Extracts from Surface E xposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized v alues are reported as ng per mg of bone powder. quantification obtained using a 1:5 or 1:10 dilution of extract respectively. Week 4 Nuclear DNA ( MC1R ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Tota l DNA Recovered (ng) Diaphysis 1 51 27.5 283.00 152.60 4196.45 2 50 30.5 292.00 178.12 5432.66 Proximal Metaphysis 1 49 32.0 227.00 148.24 4743.84 2 50 31.0 474.00 293.88 9110.28 Distal Metaphysis 1 50 30.0 308.00 184.80 5544.00 2 49 29.5 574.00 3 45.57 10194.36 Femoral Head 1 51 31.5 443.00 273.62 8618.96 2 50 31.5 203.00* 127.89 4028.54 Distal Epiphysis 1 51 28.0 587.00 322.27 9023.69 2 51 27.5 76.70** 41.36 1137.34 Calcaneus 1 51 29.5 365.00 211.13 6228.26 2 50 31.0 82.70** 51.27 1589.49 Talus 1 49 28.0 318.00 181.71 5088.00 2 51 27.5 94.50** 50.96 1401.29 Reagent Blank - 29.5 1.43 - 42.19 232 Table D14 : Nuclear DNA Quantification of Month 3 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first c olumn. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normali zed values are reported as ng per mg of bone powder. Month 3 Nuclear DNA ( MC1R ) Region Bon e Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 51 29.5 350.00 202.45 5972.30 2 51 26.5 250.00 129.90 3442.40 Proximal Metaphysis 1 51 29.5 303.00 175.26 5170.31 2 50 29.0 579.00 335. 82 9738.78 Distal Metaphysis 1 50 26.5 527.00 279.31 7401.72 2 50 31.0 515.00 319.30 9898.30 Femoral Head 1 51 27.5 654.00 352.65 9697.79 2 50 29.0 769.00 446.02 12934.58 Distal Epiphysis 1 49 29.0 435.00 257.45 7466.02 2 50 25.5 950.00 484.50 123 54.75 Calcaneus 1 51 28.0 514.00 282.20 7901.49 2 50 29.0 844.00 489.52 14196.08 Talus 1 50 31.0 578.00 358.36 11109.16 2 50 30.0 334.00 200.40 6012.00 Reagent Blank - 29.0 0.00 - 0.00 233 Table D15 : Nuclear DNA Quantification of Month 6 Extracts from Surface Exposed Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized v alues are reported as ng per mg of bone powder. obtained using a 1:10 dilution of extract. Month 6 Nuclear DNA ( MC1R ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 50 25.0 279.00 139.50 3487.50 2 49 26.5 226.00 122.22 3238.95 Proximal Metaphysis 1 49 27.0 252.00 138.86 3749.14 2 50 28.0 306.00 171.36 4798.08 Distal Metaphysis 1 50 31.0 417.00 258.54 8014.74 2 50 29.0 303.00 17 5.74 5096.46 Femoral Head 1 51 31.5 458.00 282.88 8910.79 2 51 28.5 874.00 488.41 13919.74 Distal Epiphysis 1 51 26.5 428.00 222.39 5893.39 2 51 30.0 198.00 116.47 3494.12 Calcaneus 1 49 30.5 204.00 126.98 3872.88 2 50 27.5 507.00 278.85 7668.38 Talus 1 49 30.0 470.00 287.76 8632.65 2 49 29.0 29.50** 17.46 506.32 Reagent Blank - 29.0 0.00 - 0.00 234 Table D16: Bovine Nuclear DNA PCR Amplification Chart for Quality Assay (Surface Exposed Skeletal Material) Region listed in first 4) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 200 bp amplicon, 2 = 410 bp amplicon, 3 = 599 bp amplicon, 4 = 989 bp amplicon, NA = No Ampl ification. Surface Nuclear DNA ( MC1R ) Amplicon Length Region Day 0 Week 1 Week 2 Week 4 Month 3 Month 6 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 Diaphysis 2 2 2 2 2 2 2 2 2 2 2 2 Proximal Metaphysis 2 2 2 2 2 2 2 2 2 2 2 2 Distal Metaphysis 2 2 2 2 2 2 2 2 2 2 2 2 Femoral Head 2 2 2 2 2 2 2 2 2 2 2 2 Distal Epiphysis 2 2 2 2 2 2 2 2 2 2 2 2 Calcaneus 2 2 2 2 2 2 2 2 2 2 2 2 Talus 2 2 2 2 2 2 2 2 2 2 2 2 Reagent Blank NA NA NA NA NA NA 235 Table D17: mtDNA Quantification of Day 0 Ex tracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone o region denotes replicate number. Day 0 Mitochondrial DNA ( ATPase ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 49 32.0 12.30 8.03 257.04 2 50 30.0 55.40 33.24 997.20 Pr oximal Metaphysis 1 50 31.0 21.30 13.21 409.39 2 50 31.0 40.00 24.80 768.80 Distal Metaphysis 1 51 32.0 46.80 29.36 939.67 2 50 30.5 74.90 45.69 1393.51 Femoral Head 1 50 27.0 29.90 16.15 435.94 2 50 33.0 112.00 73.92 2439.36 Distal Epiphysis 1 51 31.0 41.10 24.98 774.45 2 50 32.0 121.00 77.44 2478.08 Calcaneus 1 49 31.0 41.70 26.38 817.83 2 50 31.0 42.10 26.10 809.16 Talus 1 50 34.0 56.60 38.49 1308.59 2 49 33.0 65.00 43.78 1444.59 Reagent Blank - 27.0 0.004 - 0.108 236 Table D18: m tDNA Quantification of Week 1 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone Week 1 Mitochondrial DNA ( ATPase ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 50 29.5 1.30 0.77 22.63 2 51 29.5 1.03 0.60 17.58 Proximal Metaphysis 1 51 32.0 1.71 1.07 34.33 2 51 30.0 1.09 0.64 19.24 Distal Metaphysis 1 51 27.0 0.80 0.43 11.49 2 50 31.5 1.27 0.80 25.20 Femoral Head 1 51 29.0 6.29 3.58 103.72 2 51 31.0 5.20 3.16 97.98 Distal Epiphy sis 1 51 30.5 3.37 2.02 61.47 2 51 26.0 2.12 1.08 28.10 Calcaneus 1 49 28.0 4.81 2.75 76.96 2 51 33.5 3.20 2.10 70.42 Talus 1 50 29.5 2.99 1.76 52.04 2 51 31.0 2.79 1.70 52.57 Reagent Blank - 24.0 0.00 - 0.00 237 Table D19: mtDNA Quan tification of Week 2 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone powd quantification obtained using a 1:10 dilution of extract. Week 2 Mitochondrial DNA ( ATPase ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normaliz ed DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 50 28.0 0.72 0.40 11.31 2 50 30.5 0.88 0.54 16.39 Proximal Metaphysis 1 51 30.5 0.63 0.38 11.47 2 51 31.0 1.33 0.81 25.06 Distal Metaphysis 1 49 27.5 0.27 0.15 4.18 2 50 30.0 1.32 0.79 23.76 Fem oral Head 1 51 34.0 1.90 1.27 43.07 2 49 30.5 7.50 4.67 142.39 Distal Epiphysis 1 51 30.0 1.60 0.94 28.24 2 50 30.0 0.12** 0.07 2.11 Calcaneus 1 49 28.5 2.19 1.27 36.30 2 51 30.0 0.11** 0.07 1.96 Talus 1 51 29.0 3.24 1.84 53.43 2 50 30.0 2.82 1. 69 50.76 Reagent Blank - 30.0 0.001 - 0.03 238 Table D20: mtDNA Quantification of Week 4 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtD NA yields. Normalized val ues are reported as ng per mg of bone represents quantification obtained using a 1:5 or 1:10 dilution of extract respectively. Week 4 Mitochon drial DNA ( ATPase ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 51 29.5 0.69 0.40 11.77 2 49 32.0 0.62 0.40 15.12 Proximal Metaphysis 1 49 28.0 0.95 0.54 15.15 2 51 32.0 0.75 0.47 15.12 Distal Metaphysis 1 49 30.0 0.002** 0.001 0.036 2 51 31.5 2.79 1.72 54.28 Femoral Head 1 50 30.5 2.66 1.62 49.49 2 50 27.5 3.77 2.07 57.02 Distal Epiphysis 1 50 27.0 0.07* 0.04 0.96 2 50 29.0 2.31 1.34 38.85 Calcaneus 1 49 32.0 0 .19* 0.12 3.93 2 51 30.5 1.38 0.83 25.17 Talus 1 49 32.5 0.20* 0.13 4.27 2 51 30.5 2.04 1.22 37.21 Reagent Blank - 27.5 0.00 - 0.00 239 Table D21: mtDNA Quantification of Month 3 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng per mg of bone Month 3 Mitochondrial DNA ( ATPase ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 51 30.0 1.11 0.65 19.59 2 49 31.0 0.81 0.51 15.85 Proximal Metaphysis 1 51 28.0 0.50 0.28 7.70 2 50 31.0 0.64 0.40 12.26 D istal Metaphysis 1 51 31.0 0.19 0.11 3.52 2 50 29.0 0.35 0.20 5.94 Femoral Head 1 51 31.0 1.15 0.70 21.67 2 51 29.0 0.92 0.52 15.11 Distal Epiphysis 1 50 31.0 1.06 0.66 20.37 2 51 30.0 0.58 0.34 10.20 Calcaneus 1 49 26.0 0.92 0.49 12.69 2 51 28. 5 0.77 0.43 12.33 Talus 1 50 29.5 2.46 1.45 42.82 2 51 29.5 1.76 1.02 30.03 Reagent Blank - 30.0 0.00 - 0.00 240 Table D22: mtDNA Quantification of Month 6 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized va lues are reported as ng per mg of bone Month 6 Mitochondrial DNA ( ATPase ) Region Bone Mass (mg) E xtract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 50 32.0 0.95 0.61 19.42 2 50 27.0 0.58 0.31 8.49 Proximal Metaphysis 1 49 30.0 0.52 0.32 9.51 2 51 28.5 2.49 1.39 39.66 Distal Metaphysis 1 50 27.0 0.45 0.24 6.59 2 49 27.0 0.21 0.12 3.14 Femoral Head 1 51 31.0 1.29 0.78 24.31 2 50 28.0 1.92 1.08 30.11 Distal Epiphysis 1 51 30.0 1.06 0.62 18.71 2 49 30.0 1.06 0.65 19.47 Calcaneus 1 50 27.5 1.52 0.84 22.99 2 49 29.0 0.94 0.56 16.10 Talus 1 50 27.5 3.80 2.09 57.48 2 51 29.5 1.64 0.95 27.98 Reagent Blank - 29.0 0.00 - 0.00 241 Table D23: mtDNA Normalized Quantification of Buried Bovine Skeletal Material Region bone powder was collected from in first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized val ues are reported as ng calcanei, and tali buried during the course of this experiment. Cells highlig hted in yellow represent extracts that had PCR inhibition. quantification multiplied by dilution factor. Burial Mitochondrial DNA (ng/mg) ( ATPase ) Region Day 0 Week 1 Week 2 Week 4 Month 3 Month 6 B1 B2 B1 B2 B1 B2 B1 B2 B1 B2 B1 B2 Diaphysis 8.03 33.24 0.77 0.60 0.40 0.54 0.40 0.40 0.65 0.51 0.61 0.31 Proximal Metaphysis 13.21 24.80 1.07 0.64 0.38 0.81 0.54 0.47 0.28 0.40 0.32 1.39 Distal Metaphysis 29.36 45.69 0.43 0.80 0.15 0.79 0.01** 1.72 0.11 0.20 0.24 0.12 Femoral Head 16.15 73.92 3.58 3.16 1.27 4.67 1.62 2.07 0.70 0.52 1.08 0.78 Distal Epiphysis 24.98 77.44 2.02 1.08 0.94 0.70** 0.20* 1.34 0.66 0.34 0.62 0.65 Calcane us 26.38 26.10 2.75 2.10 1.27 0.70** 0.60* 0.83 0.49 0.43 0.84 0.56 Talus 38.49 43.78 1.76 1.70 1.84 1.69 0.65* 1.22 1.45 1.02 2.09 0.95 242 Table D24 : Bovine Mitochondrial DNA PCR Amplification Chart for Quality Assay (Buried Skeletal Material) Region list 4) correspond to the length of PCR amplicon successfully generated for each region per replicate: 1 = 201 bp amplicon, 2 = 390 bp amplicon, 3 = 607 bp amplicon, 4 = 994 bp amplicon, N A = No Amplification. Surface Mitochondrial DNA ( ATPase ) Amplicon Length Region Day 0 Week 1 Week 2 Week 4 Month 3 Month 6 B1 B2 B1 B2 B1 B2 B1 B2 B1 B2 B1 B2 Diaphysis 4 4 4 4 4 4 4 4 4 4 4 4 Proximal Metaphysis 4 4 4 4 4 4 4 4 4 4 4 4 Distal Meta physis 4 4 4 4 4 4 4 4 4 4 4 4 Femoral Head 4 4 4 4 4 4 4 4 4 4 4 4 Distal Epiphysis 4 4 4 4 4 4 4 4 4 4 4 4 Calcaneus 4 4 4 4 4 4 4 4 4 4 4 4 Talus 4 4 4 4 4 4 4 4 4 4 4 4 Reagent Blank 4 NA NA NA NA NA 243 Table D25: Nuclear DNA Quantifica tion of Day 0 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized v alues are reported as ng per mg of bone powder. Day 0 Nuclear DNA ( MC1R ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 49 32.0 240.00 156.73 5015.51 2 50 30.0 435.00 261.0 0 7830.00 Proximal Metaphysis 1 50 31.0 312.00 193.44 5996.64 2 50 31.0 454.00 281.48 8725.88 Distal Metaphysis 1 51 32.0 494.00 309.96 9918.75 2 50 30.5 486.00 296.46 9042.03 Femoral Head 1 50 27.0 288.00 155.52 4199.04 2 50 33.0 658.00 434.28 14 331.24 Distal Epiphysis 1 51 31.0 403.00 244.96 7593.78 2 50 32.0 635.00 406.40 13004.80 Calcaneus 1 49 31.0 359.00 227.12 7040.80 2 50 31.0 426.00 264.12 8187.72 Talus 1 50 34.0 280.00 190.40 6473.60 2 49 33.0 456.00 307.10 10134.37 Reagent Blan k - 27.0 0.15 - 4.05 244 Table D26: Nuclear DNA Quantification of Week 1 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized v alues are reported as ng per mg of bone powder. Week 1 Nuclear DNA ( MC1R ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 50 29.5 9.00 5.31 156.65 2 51 29.5 4.22 2.44 72.01 Proximal Metaphysis 1 51 32.0 2.55 1.60 51.20 2 51 30.0 0.26 0.15 4.50 Distal Metaphysis 1 51 27.0 1.67 0.88 23.87 2 50 31.5 0.11 0.07 2.26 Femoral Head 1 51 29.0 46.60 26.50 768.44 2 51 31.0 72.20 43.89 1360.47 Distal Epiphysis 1 51 30.5 18.30 10.94 333.80 2 51 26.0 7.07 3.60 93.71 Calcaneus 1 49 28.0 70.90 40.51 1134.40 2 51 33.5 43.00 28.25 946.21 Talus 1 50 29.5 27.90 16.46 485.60 2 51 31.0 57.60 35.01 1085.36 Reagent B lank - 24.0 0.01 - 0.24 245 Table D27: Nuclear DNA Quantification of Week 2 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng per mg of bone powder. obtained using a 1:10 dilution of extract. Week 2 Nuclear DNA ( MC1R ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 50 28.0 1.37 0.77 21.48 2 50 30.5 3.60 2.20 66.98 Proximal Metaphysis 1 51 30.5 0.90 0.54 16.34 2 51 31.0 1.49 0.91 28.08 Distal Metaphysis 1 49 27.5 1.30 0.73 20.06 2 50 30.0 0.43 0.26 7.70 Femoral Head 1 51 34.0 12.30 8.20 278.80 2 49 30.5 43.90 27.33 833.43 Distal Epiphysis 1 51 30.0 13.30 7.82 234.71 2 50 30.0 3.61** 2.17 64.98 Calcaneus 1 49 28.5 14.90 8.67 246.99 2 51 30.0 0.8 5** 0.50 15.05 Talus 1 51 29.0 21.60 12.28 356.19 2 50 30.0 19.30 11.58 347.40 Reagent Blank - 30.0 0.09 - 2.70 246 Table D28: Nuclear DNA Quantification of Week 4 Extracts from Buried Bovine Skeletal Material Region listed in the first colu mn. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng per mg of bone powder. Week 4 Nuclear DNA ( MC1R ) Region Bone Ma ss (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 51 29.5 4.09 2.37 69.79 2 49 32.0 2.67 1.74 55.80 Proximal Metaphysis 1 49 28.0 1.55 0.89 24.80 2 51 32.0 1.15 0.72 23.09 Distal Metaphysi s 1 49 30.0 2.01 1.23 36.92 2 51 31.5 0.31 0.19 5.99 Femoral Head 1 50 30.5 7.63 4.65 141.96 2 50 27.5 11.30 6.22 170.91 Distal Epiphysis 1 50 27.0 0.00** 0.00 0.00 2 50 29.0 14.60 8.47 245.57 Calcaneus 1 49 32.0 0.00** 0.00 0.00 2 51 30.5 2.34 1.40 42.68 Talus 1 49 32.5 0.16** 0.10 3.36 2 51 30.5 5.62 3.36 102.51 Reagent Blank - 27.5 0.04 - 1.10 247 Table D29: Nuclear DNA Quantification of Month 3 Extracts from Buried Bovine Skeletal Material Region listed in the first column. E xtract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized v alues are reported as ng per mg of bone powder. Month 3 Nuclear DNA ( MC1R ) Region Bone Mass (m g) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 51 30.0 2.40 1.41 42.35 2 49 31.0 1.90 1.20 37.26 Proximal Metaphysis 1 51 28.0 1.21 0.66 18.60 2 50 31.0 0.31 0.19 5.94 Distal Metaphysis 1 51 31.0 0.62 0.38 11.68 2 50 29.0 0.08 0.05 1.34 Femoral Head 1 51 31.0 9.75 5.93 183.72 2 51 29.0 18.40 10.46 303.42 Distal Epiphysis 1 50 31.0 5.71 3.54 109.75 2 51 30.0 20.30 11.94 358.24 Calcaneus 1 49 26.0 5.45 2.89 75.19 2 51 28.5 4.50 2.51 71.67 Talus 1 50 29.5 17.40 10.27 302.85 2 51 29.5 10.30 5.96 175.76 Reagent Blank - 30.0 0.00 - 0.00 248 Table D30: Nuclear DNA Quantification of Month 6 Extracts from Buried Bovine Skeletal Material Region listed in the first column. Ex tract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized v alues are reported as ng per mg of bone powder. obta ined using a 1:5 dilution of extract. Month 6 Nuclear DNA ( MC1R ) Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis 1 50 32.0 2.00 1.28 40.96 2 50 27.0 3.95 2.13 57.59 Proximal M etaphysis 1 49 30.0 0.43 0.26 7.81 2 51 28.5 2.87 1.60 45.71 Distal Metaphysis 1 50 27.0 1.56 0.84 22.74 2 49 27.0 0.89 0.49 13.26 Femoral Head 1 51 31.0 19.60 11.91 369.33 2 50 28.0 7.51* 4.21 117.91 Distal Epiphysis 1 51 30.0 6.84 4.02 120.71 2 49 30.0 18.20 11.14 334.29 Calcaneus 1 50 27.5 23.10 12.71 349.39 2 49 29.0 4.42 2.62 75.86 Talus 1 50 27.5 39.20 21.56 592.90 2 51 29.5 18.70 10.82 319.09 Reagent Blank - 29.0 0.00 - 0.00 249 Table D31: Nuclear DNA Normalized Quantif ication of Buried Bovine Skeletal Material Region bone powder was collected from in first column. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized va lues are reported as ng per mg of bone powder. calcanei, and tali buried during the course of this experiment. Cells highlighted in yellow represent extracts that had PCR inhibition. te inhibition, listed value represents DNA quantification multiplied by dilution factor. Burial Nuclear DNA (ng/mg) ( MC1R ) Region Day 0 Week 1 Week 2 Week 4 Month 3 Month 6 B1 B2 B1 B2 B1 B2 B1 B2 B1 B2 B1 B2 Diaphysis 156.73 261.00 5.31 2.44 0.77 2.20 2.37 1.74 1.41 1.20 1.28 2.13 Proximal Metaphysis 193.44 281.48 1.60 0.15 0.54 0.91 0.89 0.72 0.66 0.19 0.26 1.60 Distal Metaphysis 309.96 296.46 0.88 0.07 0.73 0.26 1.23 0.19 0.38 0.05 0.84 0.49 Femoral Head 155.52 434.28 26.50 43.89 8.20 27.33 4.65 6 .22 5.93 10.46 11.91 21.05** Distal Epiphysis 244.96 406.40 10.94 3.60 7.82 21.70** 0.00** 8.47 3.54 11.94 4.02 11.14 Calcaneus 227.12 264.12 40.51 28.25 8.67 5.00** 0.00** 1.40 2.89 2.51 12.71 2.62 Talus 287.10 307.10 16.46 35.01 12.28 11.58 1.00* 3.36 10.27 5.96 21.56 10.82 250 Table D32 : Bovine Nuclear DNA PCR Amplification Chart for Quality Assay (Buried Skeletal Material) 4) correspond to the length of PCR amplicon su ccessfully generated for each region per replicate: 1 = 200 bp amplicon, 2 = 410 bp amplicon, 3 = 599 bp amplicon, 4 = 989 bp amplicon, NA = No Amplification. Surface Nuclear DNA ( MC1R ) Amplicon Length Region Day 0 Week 1 Week 2 Week 4 Month 3 Month 6 B 1 B2 B1 B2 B1 B2 B1 B2 B1 B2 B1 B2 Diaphysis 2 2 1 2 3 2 1 2 3 3 3 2 Proximal Metaphysis 2 2 2 2 3 2 1 2 3 3 3 2 Distal Metaphysis 2 2 3 2 2 1 NA 2 2 3 2 2 Femoral Head 2 2 3 2 3 1 1 2 3 2 2 NA Distal Epiphysis 2 2 3 2 3 NA NA 3 3 3 2 2 Calcaneus 2 3 1 2 3 NA NA 2 3 3 3 2 Talus 3 2 2 2 3 2 NA 3 2 2 3 2 Reagent Blank NA NA NA NA NA NA 251 APPENDIX E: ANCILLARY EXPERIMENT DATA Changes in the Recoverable Total DNA of Buried Bovine Bone Segments over a One Month Time Period Table E1: mtDN A Quantification of Buried Femoral Diaphysis (Segment Type A) replicate s that were retrieved, tested , and stored at - 20 ° C. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized values are reported as ng per mg of bone powd er. Mitochondrial DNA ( ATPase ) Segment Identifier Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 2D - 1 57 32.6 2.22 1.27 72.39 2D - 2 52 30.6 3.81 2.24 116.48 2D - 3 50 32.0 3.72 2.38 119.00 2D - 4 49 26.4 5.08 2.74 134.26 4D - 1 50 31.8 2.12 1.35 67.50 4D - 2 54 30.0 1.96 1.09 58.86 4D - 3 51 30.2 1.63 0.97 49.47 4D - 4 52 31.0 1.89 1.09 56.68 1W - 1 51 33.0 0.30 0.19 9.69 1W - 2 51 28.4 0.06 0.03 1.53 1W - 3 50 28.0 1.77 0.99 49.50 1W - 4 52 27.0 0.15 0.0 8 4.16 11D - 1 50 30.0 0.10 0.06 3.00 11D - 2 49 28.2 0.03 0.02 0.98 11D - 3 49 28.2 0.06 0.04 1.96 11D - 4 50 29.0 0.03 0.02 1.00 2W - 1 50 15.0 0.04 0.01 0.50 2W - 2 49 30.0 0.15 0.09 4.41 2W - 3 51 23.2 0.12 0.05 2.55 2W - 4 49 26.0 0.05 0.03 1.47 3W - 1 50 30.8 0.02 0.02 1.00 3W - 2 50 28.6 0.04 0.02 1.00 3W - 3 50 29.2 0.09 0.05 2.50 3W - 4 51 24.2 0.03 0.01 0.51 4W - 1 51 24.8 0.03 0.02 1.02 4W - 2 50 26.2 0.02 0.01 0.50 4W - 3 51 29.4 0.03 0.02 1.02 4W - 4 50 30.4 0.04 0.02 1.00 252 Table E2: mtDNA Quantification of Bu ried Femoral Diaphysis (Segment Type B) replicate of femoral diaphysis. cyclically retrieved, tested , and reburied. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized values are reported as ng per mg of bone powder. failed extract due to Amicon ® column damage. Mitochondrial DNA ( ATPase ) Segment Identifier Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 0D - 1 51 30.2 0.26 0.15 7.65 0D - 2 50 31.8 1.29 0.82 41.0 0 0D - 3 50 6.0* 1.44 0.17 8.50 0D - 4 50 31.0 0.59 0.36 18.00 1W - 1 53 25.0 0.61 0.29 15.37 1W - 2 50 26.4 0.58 0.31 15.50 1W - 3 55 25.2 0.50 0.23 12.65 1W - 4 51 26.0 0.94 0.48 24.48 2W - 1 49 27.4 0.02 0.01 0.49 2W - 2 51 26.0 0.04 0.02 1.02 2W - 3 49 26.2 0.0 5 0.03 1.47 2W - 4 50 24.6 0.04 0.02 1.00 3W - 1 50 27.6 0.01 0.01 0.50 3W - 2 49 28.6 0.03 0.01 0.49 3W - 3 51 29.0 0.04 0.02 1.02 3W - 4 51 26.2 0.02 0.01 0.51 4W - 1 49 27.6 0.02 0.01 0.49 4W - 2 50 24.8 0.04 0.02 1.00 4W - 3 50 27.4 0.07 0.04 2.00 4W - 4 51 28. 8 0.04 0.02 1.02 253 Table E3: Nuclear DNA Quantification of Buried Femoral Diaphysis (Segment Type A) s that were retrieved, tested , and stored at - 20 ° C. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized val ues are reported as ng per mg of bone powder. Nuclear DNA ( MC1R ) Segment Identifier Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 2D - 1 57 32.6 69.60 39.81 2269.17 2D - 2 52 30.6 61.20 36.01 187 2.52 2D - 3 50 32.0 97.70 62.53 3126.50 2D - 4 49 26.4 435.00 234.37 11484.13 4D - 1 50 31.8 3.94 2.51 125.50 4D - 2 54 30.0 140.00 77.78 4200.12 4D - 3 51 30.2 168.00 99.48 5073.48 4D - 4 52 31.0 127.00 75.71 3936.92 1W - 1 51 33.0 0.37 0.24 12.24 1W - 2 51 28.4 34.70 19.32 985.32 1W - 3 50 28.0 23.60 13.22 661.00 1W - 4 52 27.0 2.14 1.11 57.72 11D - 1 50 30.0 0.23 0.14 7.00 11D - 2 49 28.2 0.04 0.02 0.98 11D - 3 49 28.2 1.32 0.76 37.24 11D - 4 50 29.0 0.38 0.22 11.00 2W - 1 50 15.0 1.06 0.32 16.00 2W - 2 49 30.0 1.81 1.1 1 54.39 2W - 3 51 23.2 0.89 0.40 20.40 2W - 4 49 26.0 0.33 0.17 8.33 3W - 1 50 30.8 0.00 0.00 0.00 3W - 2 50 28.6 0.005 0.002 0.10 3W - 3 50 29.2 1.61 0.94 47.00 3W - 4 51 24.2 0.94 0.04 2.04 4W - 1 51 24.8 1.24 0.60 30.60 4W - 2 50 26.2 0.07 0.04 2.00 4W - 3 51 29 .4 0.36 0.20 10.20 4W - 4 50 30.4 0.03 0.02 1.00 254 Table E4: Nuclear DNA Quantification of Buried Femoral Diaphysis (Segment Type B) cyclically retrieved, tested , and reburied. Extract volume and milligrams of bone powder was considered in order to norma lize DNA yields. Normalized values are reported as ng per mg of bone powder. Nuclear DNA ( MC1R ) Segment Identifier Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 0D - 1 51 30.2 0.24 0.14 12.24 0D - 2 50 31.8 114.00 72.50 5700.00 0D - 3 50 6.0* 0.00* 0.00 0.00 0D - 4 50 31.0 7.51 4.66 375.50 1W - 1 53 25.0 15.30 7.22 810.90 1W - 2 50 26.4 20.80 10.98 1040.00 1W - 3 55 25.2 26.50 13.09 1457.50 1W - 4 51 26.0 158.00 80.03 8058.00 2W - 1 49 27.4 0.01 0.006 0 .49 2W - 2 51 26.0 0.94 0.48 47.94 2W - 3 49 26.2 2.59 1.38 126.91 2W - 4 50 24.6 1.00 0.49 50.00 3W - 1 50 27.6 2.53 1.40 126.50 3W - 2 49 28.6 0.82 0.48 40.18 3W - 3 51 29.0 1.03 0.59 52.53 3W - 4 51 26.2 0.08 0.04 4.08 4W - 1 49 27.6 0.002 0.001 0.10 4W - 2 50 2 4.8 0.18 0.09 9.00 4W - 3 50 27.4 3.74 2.16 187.00 4W - 4 51 28.8 0.85 0.52 43.35 255 Changes in the Recoverable Total DNA of Non - Buried Bovine Bone Segments over a One Month Time Period Table E5: mtDNA Quantification of Non - Buried Bovine Femoral Diap hysis replicate of femoral diaphysis. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized values are reported as ng per mg of bone powder. Mitochondrial DNA ( ATPase ) Segment Identifier Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) To tal DNA Recovered (ng) 0D - 1 50 24.0 6.67 3.20 160.00 0D - 2 50 28.0 3.12 1.75 87.50 0D - 3 50 30.8 1.58 0.97 48.50 0D - 4 51 28.0 3.38 1.85 94.35 2D - 1 50 30.0 9.40 5.64 282.00 2D - 2 49 29.2 5.61 3.34 163.66 2D - 3 51 33.8 5.11 3.39 172.89 2D - 4 50 31.6 6.83 4.32 216.00 4D - 1 50 26.0 10.7 5.56 278.00 4D - 2 50 29.0 6.09 3.43 171.50 4D - 3 50 25.0 9.33 4.67 233.50 4D - 4 51 25.0 7.74 3.79 193.29 1W - 1 49 27.0 6.10 3.36 164.64 1W - 2 49 26.2 4.07 2.18 106.82 1W - 3 50 26.8 6.91 3.70 185.00 1W - 4 51 28.4 5.80 3.23 164 .73 10D - 1 51 28.4 3.02 1.68 85.68 10D - 2 49 24.2 1.85 0.91 44.59 10D - 3 50 24.6 2.56 1.26 63.00 10D - 4 51 29.4 2.48 1.43 72.93 2W - 1 50 23.2 3.30 1.53 76.50 2W - 2 49 27.4 2.96 1.66 81.34 2W - 3 51 30.2 3.07 1.82 92.82 2W - 4 51 23.0 2.64 1.19 60.69 3W - 1 50 30.2 4.83 2.92 146.00 3W - 2 50 25.0 1.27 0.64 32.00 3W - 3 51 26.2 2.82 1.45 73.95 3W - 4 51 27.2 2.73 1.45 73.95 4W - 1 51 29.4 4.68 2.70 137.70 4W - 2 49 28.0 2.16 1.23 60.27 4W - 3 49 32.6 2.90 1.92 94.08 4W - 4 49 28.4 3.01 1.74 85.26 RB - 27.0 0.005 - 0.1 35 256 Table E6: Nuclear DNA Quantification of Non - Buried Bovine Femoral Diaphysis ological replicate of femoral diaphysis. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng per mg of bone powder. Nuclear DNA ( MC1R ) Segment Identifier Bone Mass (mg) Extrac t Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) 0D - 1 50 24.0 224.00 107.52 5376.00 0D - 2 50 28.0 139.00 77.84 3892.00 0D - 3 50 30.8 112.00 68.99 3449.50 0D - 4 51 28.0 162.00 88.94 4535.94 2D - 1 50 30.0 481.00 288.60 14 430.00 2D - 2 49 29.2 186.00 110.84 5431.16 2D - 3 51 33.8 291.00 192.29 9806.79 2D - 4 50 31.6 551.00 348.23 17411.50 4D - 1 50 26.0 407.00 211.64 10582.00 4D - 2 50 29.0 196.00 113.68 5684.00 4D - 3 50 25.0 276.00 138.00 6900.00 4D - 4 51 25.0 250.00 122.55 625 0.05 1W - 1 49 27.0 413.00 227.57 11150.93 1W - 2 49 26.2 265.00 141.69 6942.81 1W - 3 50 26.8 238.00 127.57 6378.50 1W - 4 51 28.4 207.00 115.27 5878.77 10D - 1 51 28.4 134.00 74.62 3805.62 10D - 2 49 24.2 145.00 71.61 3508.89 10D - 3 50 24.6 186.00 91.51 4575.5 0 10D - 4 51 29.4 159.00 91.66 4674.66 2W - 1 50 23.2 117.00 54.29 2714.50 2W - 2 49 27.4 191.00 106.80 5233.20 2W - 3 51 30.2 187.00 110.73 5647.23 2W - 4 51 23.0 184.00 82.98 4231.98 3W - 1 50 30.2 319.00 192.68 9634.00 3W - 2 50 25.0 103.00 51.50 2575.00 3W - 3 51 26.2 179.00 91.96 4689.96 3W - 4 51 27.2 186.00 99.20 5059.20 4W - 1 51 29.4 401.00 231.16 11789.16 4W - 2 49 28.0 156.00 89.14 4367.86 4W - 3 49 32.6 243.00 161.67 7921.83 4W - 4 49 28.4 219.00 126.93 6219.57 RB - 27.0 0.27 - 7.29 257 Organic versus SoilMa ster : Total DNA Yield Comparisons Over One Week Table E7: Total Quantification of Non - Buried Bovine Femoral Diaphysis Extracted Utilizing Organic and SoilMaster . th of kit Extraction. Extract volume and milligrams of bone powder was considered in order to normalize DNA yields. Normalized values are reported as ng per mg of bone powder. Mitochondria l DNA ( ATPase ) Segment Identifier Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) O - 0D 100 100.0 0.19 0.19 19.00 O - 2D 101 101.0 0.46 0.46 46.46 O - 5D 99 99.0 0.22 0.22 21.78 O - 7D 99 99.0 0.12 0. 12 11.88 S - 0D 101 25.0 9.62 2.38 240.38 S - 2D 100 25.0 7.19 1.80 180.00 S - 5D 101 25.0 5.05 1.25 126.25 S - 7D 99 25.0 1.64 0.42 41.58 Nuclear DNA ( MC1R ) Segment Identifier Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) O - 0D 100 100.0 0.63 0.63 63.00 O - 2D 101 101.0 12.70 12.70 1282.70 O - 5D 99 99.0 11.00 11.00 1089.00 O - 7D 99 99.0 4.64 4.64 459.36 S - 0D 101 25.0 91.00 22.52 2274.52 S - 2D 100 25.0 122.00 30.50 3050.00 S - 5D 101 25.0 85.80 21.16 2137.16 S - 7D 99 25.0 52.60 13.28 1314.72 258 Mass Difference between Wet and Dry Bone Table E8: Mass of Drying Bone over Time in Four Containers plus Whole Bone Drying time in hours listed in first column. Mass of bone powder in milligrams are listed in NT = Not Tested Drying Time (Hours) Mass of Bone Powder (mg) per Treatment Open Microcentrifuge Tube Closed Microcentrifuge Tube Weigh Boat (Bone Powder Pile) W eigh Boat (Bone Powder Spread Out) Whole Bone 1 2 1 2 1 2 1 2 1 0 51 50 50 50 51 51 50 51 51,286 0.5 50 49 51 50 49 52 50 49 51,120 1 50 49 50 50 50 52 50 49 51,027 2 50 49 51 50 50 52 50 48 50.880 3 50 49 51 50 50 52 50 49 50,784 4 50 49 50 50 50 52 50 49 50,702 5 50 49 50 50 50 52 50 49 50,632 6 50 49 51 50 50 52 50 49 50,569 23.5 49 49 51 50 50 51 49 49 49,974 48.5 49 49 51 50 50 51 49 50 49,519 75 49 48 51 50 49 51 49 49 49,302 100 49 48 50 50 49 51 49 49 49,154 175 NT NT NT NT NT NT NT N T 48,953 250 NT NT NT NT NT NT NT NT 48,706 325 NT NT NT NT NT NT NT NT 48,617 259 Effect of Proteinase K Concentration on Total DNA Yields Table E9: mtDNA Quantification of Bovine Bones Digested with Varied Concentrations of Proteinase K Bone ide ntifier listed in the first column. TL = tissue lysis buffer, DM = demineraliza tion buffer, RB = reagent blank. proteinase K added . technical replicate s p er treatment. Extract volume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized values are reported as ng per mg of bone powder. Mitochondrial DNA ( ATPase ) Bone Identifier Bone Mass (mg) Extract Volume (µL) Quant ification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) TL 0% (1) 49 29.0 8.74 5.17 253.46 TL 0% (2) 49 29.0 7.60 4.50 220.50 TL 0.5% (1) 49 31.0 14.90 9.43 462.07 TL 0.5% (2) 49 31.0 13.90 8.79 430.71 TL 1% (1) 50 26.0 15.20 7.90 395.00 TL 1% (2) 50 26.0 19.00 9.88 494.00 TL 2% (1) 49 30.0 16.20 9.92 486.08 TL 2% (2) 49 30.0 15.40 9.43 462.07 RB TL - 30.0 0.001 - 0.03 DM 0% (1) 50 30.0 8.89 5.33 266.50 DM 0% (2) 50 30.0 9.26 5.56 278.00 DM 0.5% (1) 49 30.0 18.30 11.20 548.80 DM 0.5% (2) 49 30.0 17.90 10.96 537.04 DM 1% (1) 50 30.0 17.40 10.40 520.00 DM 1% (2) 50 30.0 20.70 12.42 621.00 DM 2% (1) 50 30.0 20.30 12.18 596.82 DM 2% (2) 50 30.0 17.10 10.26 513.00 RB DM - 30.0 0.005 - 0.15 260 Table E10: Nuclear DNA Quantification of Bovine Bones Digested with Varied Concentrations of Proteinase K Bone identifier listed in the first column. TL = tissue lysis buffer, DM = demineraliza tion buffer, RB = reagent blank. 20 mg/mL proteinase K added . technical replicate s per treatment. Extract volume and milligrams of bone powder was con sidered in order to normalize nuclear DNA yields. Normalized values are reported as ng per mg of bone powder. Nu clear DNA ( MC1R ) Bone Identifier Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) TL 0% (1) 49 29.0 481.00 284.67 13948.83 TL 0% (2) 49 29.0 438.00 259.22 12701.78 TL 0.5% (1) 49 31.0 844.00 533. 96 26164.04 TL 0.5% (2) 49 31.0 1130.00 668.78 32770.22 TL 1% (1) 50 26.0 967.00 503.84 25192.00 TL 1% (2) 50 26.0 1480.00 769.60 38480.00 TL 2% (1) 49 30.0 1110.00 679.59 33299.91 TL 2% (2) 49 30.0 1060.00 648.98 31800.02 RB TL - 30.0 0.00 - 0.00 D M 0% (1) 50 30.0 337.00 202.20 10110.00 DM 0% (2) 50 30.0 423.00 253.80 12690.00 DM 0.5% (1) 49 30.0 482.00 295.10 14459.90 DM 0.5% (2) 49 30.0 550.00 336.73 16499.77 DM 1% (1) 50 30.0 436.00 261.60 13080.00 DM 1% (2) 50 30.0 694.00 416.40 20820.00 D M 2% (1) 49 30.0 555.00 333.00 16317.00 DM 2% (2) 49 30.0 382.00 229.20 11230.80 RB DM - 30.0 0.001 - 0.03 261 Comparison of Total DNA Yields from Bovine Bones Macerated by MSU Forensic Anthropologist versus MSU Forensic Biologist Table E11: Mitoch ondrial DNA Quantification of Bovine Bones Macerated by MSU Forensic Biology/Anthropology Laboratories Region tested is in the first column. FA = element macerated by MSU Forensic Anthropology. FB = element macerated by MSU Forensic Biology. Extract vol ume and milligrams of bone powder was considered in order to normalize mtDNA yields. Normalized values are reported as ng per mg of bone powder. mtDNA Tissue Lysis Buffer Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng /mg) Total DNA Recovered (ng) Diaphysis FA 50 25.5 4.51 2.30 115.01 FB 49 28.0 11.20 6.40 313.60 Femoral Head FA 50 29.0 46.10 26.74 1336.90 FB 49 31.0 80.70 51.06 2501.70 Distal Epiphysis FA 50 29.0 54.70 31.73 1586.30 FB 50 26.0 106.00 55.12 275 6.00 Calcaneus FA 51 27.0 8.00 4.23 216.00 FB 51 26.0 81.00 41.29 2106.00 Talus FA 51 26.0 12.70 6.47 330.20 FB 50 25.0 32.00 16.00 800.00 Reagent Blank - 24.0 0.00 - 0.00 mtDNA Demineralization Buffer Region Bone Mass (mg) Extract Volume (µL) Qua ntification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis FA 49 22.5 11.60 5.33 261.00 FB 51 28.0 28.90 10.71 564.00 Femoral Head FA 49 27.0 37.30 20.55 1007.10 FB 51 30.0 62.50 36.76 1875.00 Distal Epiphysis FA 51 24.5 40.90 19. 65 1002.05 FB 50 26.0 86.70 45.08 2254.20 Calcaneus FA 51 27.5 22.50 12.13 618.75 FB 50 22.5 53.80 24.21 1210.50 Talus FA 51 27.5 15.40 8.30 423.50 FB 49 28.0 24.50 14.00 392.00 Reagent Blank - 24.0 0.00 - 0.00 262 Table E12: Nuclear DNA Quantificat ion of Bovine Bones Macerated by MSU Forensic Biology/Anthropology Laboratories Region tested is in the first column. FA = element macerated by MSU Forensic Anthropology. FB = element macerated by MSU Forensic Biology. Extract volume and milligrams of b one powder was considered in order to normalize nuclear DNA yields. Normalized values are reported as ng per mg of bone powder. Nuclear DNA Tissue Lysis Buffer Region Bone Mass (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis FA 50 25.5 157.00 80.07 4003.50 FB 49 28.0 210.00 120.00 5880.00 Femoral Head FA 50 29.0 984.00 570.72 28536.00 FB 49 31.0 966.00 611.14 29946.00 Distal Epiphysis FA 50 29.0 709.00 411.22 20561.00 FB 50 26.0 953.00 495 .56 24778.00 Calcaneus FA 51 27.0 242.00 128.12 6534.00 FB 51 26.0 1270.00 647.45 33020.00 Talus FA 51 26.0 378.00 192.71 9828.00 FB 50 25.0 780.00 390.00 19500.00 Reagent Blank - 24.0 0.00 - 0.00 Nuclear DNA Demineralization Buffer Region Bone Ma ss (mg) Extract Volume (µL) Quantification (ng/µL) Normalized DNA (ng/mg) Total DNA Recovered (ng) Diaphysis FA 49 22.5 402.00 184.59 9045.00 FB 51 28.0 430.00 236.08 12040.00 Femoral Head FA 49 27.0 543.00 299.20 14661.00 FB 51 30.0 578.00 340.00 17 340.00 Distal Epiphysis FA 51 24.5 509.00 244.52 12470.50 FB 50 26.0 612.00 318.24 15912.00 Calcaneus FA 51 27.5 409.00 220.54 11247.50 FB 50 22.5 572.00 257.40 12870.00 Talus FA 51 27.5 485.00 261.52 13337.50 FB 49 28.0 522.00 298.29 8352.00 Rea gent Blank - 24.0 0.00 - 0.00 263 REFERENCES 264 REFERENCES Adler CJ, Haak W, Donlon D, Cooper A, The Genographic Consortium. 2011. Survival and recovery of DNA from ancient teeth and bones. J Archaeol Sci. 38:956 64. Aerssens J, Bo onen S, Lowet G, Dequeker J. 1998. Interspecies differences in bone composition, density, and quality: Potential implications for in vivo bone research. Endocrinology . 139(2):663 70. demineralization DNA extraction procedure from degraded skeletal remains. Forensic Sci Int Genet . 6(3):398 406. Anderson HC. 2003. Matrix vesicles and calcification. Curr Rheumatol Rep 5:222 6. Atkinson PJ, Weatherell JA, Weidmann SM. 1962. Changes in d ensity of the human femoral cortex with age. J Bone Joint Surg Br . 44(3):496 502. Bajorath J, Hinrichs W, Saenger W. 1988. The enzymatic activity of proteinase K is controlled by calcium. Eur J Biochem . 176:441 7. B är W, Kratzer A, Mächler M, Schmid W. 1 988. Postmortem stability of DNA. Forensic Sci Int. 89:59 70. Barta JL, Monroe C, Crockford SJ, Kemp BM. 2014. Mitochondrial DNA preservation across 3000 - year - old northern fur seal ribs is not related to bone density: Implications for forensic investigati ons. Forensic Sci Int. 239:11 8. Bass WM. 1995. Human osteology: A laboratory and field manual . 4 th edition, Missouri Archaeological Society. Bell LS, Skinner MF, Jones SJ. 1996. The speed of post mortem change to the human skeleton and its taphonomic si gnificance. Forensic Sci Int . 82:129 40. Bigi A, Cojazzi G, Panzavolta S, Ripamonti A, Roveri N, Romanello M, et al. 1997. Chemical and structural characterization of the mineral phase from cortical and trabecular bone. J Inorg Biochem . 68:45 51. Bickle y J, Short JK, McDowell DG, Parkes HC. 1996. Polymerase chain reaction (PCR) detection of Listeria monocytogenes in diluted milk and reversal of PCR inhibition caused by calcium ions. Lett Appl Microbiol . 22(2):153 8. Bille T, Wingrove M, Holland M, Holla nd C, Cave C, Schumm J. 2004. Novel method of DNA extraction from bones assisted DNA identification of World Trade Center victims. Int Congr Ser. 1261:553 5. 265 Bio - Rad. 2015. iQ Supermix protocol. http://www.bio - rad.com/webroot/web/pdf/lsr/literature/Bu lletin_4016204.pdf Bisgard JD, Bisgard ME. 1935. Longitudinal growth of long bones. Arch Surg . 31(4):568 78. Black TK III. 1978. A new method for assessing the sex of fragmentary skeletal remains: Femoral shaft circumference. Am J Phys Anthropol . 48:227 31. Brundin M, Figdor D, Sundqvist G, Sjögren U. 2013. DNA binding to hydroxyapatite: A potential mechanism for preservation of microbial DNA. J Endodont . 39(2):211 6. Burger EH, Klein - Nuland J, Smit TH. 2003. Strain - derived canalicular fluid flow regul ates osteoclast activity in a remodeling osteon: A proposal. J Biomech 36:1452 9. Burger J, Hummel S, Herrmann B, Henke W. 1999. DNA preservation: A microsatellite - DNA study on ancient skeletal remains. Electrophoresis . 20:733 41. Burgess RR, Deutscher M P. 2009. Refolding soluble inclusion body proteins . Methods in Enzymology: Guide to Protein Purification, Chapter 1 7, 266 7. Burgess RR, Deutscher MP, Eds. Ac ademic Press. Burr DB. 2002. Targeted and nontargeted remodeling. Bone . 30(1):2 4. Butler JM. 20 12. DNA extraction methods. Advanced topics in forensic DNA typing: methodology . San Diego: Elsevier Academic Press, 31 2. Campos PF, Craig OE, Turner - Walker G, Peacock E, Willerslev E. 2012. DNA in ancient bone Where is it located and how should we extra ct it? An Anat . 194:7 16. Canik JJ (AFDIL). 2013. DNA in the identification process. Briefing for the defense prisoner of war missing personnel office family update. [PowerPoint presentation] September. Caputo M, Irisarri M, Alechine E, Corach D. 2013. A DNA extraction method of small quantities of bone for high - quality genotyping. Forensic Sci Int Gen. 7:488 93. Carter DR . 1984. Mechanical loading histories and cortical bone remodeling. Calcif Tissue Int . 36:S19 S24. Carter YC, Thomas DL, Clement JG, Peele AG, Hannah K, Cooper DML. 2013. Variation in osteocyte lacunar morphology and density in the human femur a synchrotron radiation micro - CT study. Bone . 53(1):126 32. 266 Casalla DA, Moore MK. 2012. High soil acidity associated with near complete mineral dissolution of recently buried human remains. Proceedings of the American Academy of Forensic Sciences. 18:400 1. Cattaneo C, Smillie DM, Gelsthorpe K, Piccinini A, Gelsthorpe AR, Sokol RJ. 1995. A simple method for extracting DNA from old skeletal materi al. Forensic Sci Int . 74(3):167 74. Clarke B. 2008. Normal bone anatomy and physiology. Clin J Am Soc Nephro. 3:S131 S9. Collins MJ, Nielsen - Marsh CM, Hiller J, Smith CI, Roberts JP, Prigodich RV et al. 2002. The survival of organic matter in bone: A re view. Archaeometry. 44(3):383 94. Craig EA. 1995. Intercondylar shelf angle: A new method to determine race from the distal femur. J Forensic Sci. 40:777 82. Crow P. 2008 . Mineral weathering in forest soils and its relevance to the preservation of the buried archaeological resource. J Archaeol Sci. 56:475 9. Dent BB, Forbes SL, Stuart BH. 2004. Review of human decomposition processes in soil. Environ Geol. 45(1):576 85. Di Bennardo R, Taylor JV. 1979. Sex assessment of the femur: A test of a new meth od. Am J Phys Anthropol. 50:635 8. Edson SM, Ross JP, Coble MD, Parsons TJ, Barritt SM. 2004. Naming the dead confronting the realities of rapid identification of degraded skeletal remains. Forensic Sci Rev . 16:63 90. Eglinton G, Logan GA, Ambler RP, Boo n JJ, Perizonius WRK. 1991. Molecular preservation. Philos T Roy Soc B . 333:315 28. Eilert KD, Foran DF. 2009. Polymerase resistance to polymerase chain reaction inhibitors in bone. J Forensic Sci . 54(5):1001 7. Eriksen EF, Axelrod DW, Melsen F. 1994. B one histomorphometry . New York, Raven Press, pp 1 12. Fisher DL, Holland MM, Mitchell L, Sledzik PS, Wilcox AW, Wadhams M, et al. 1993. Extraction, evaluation, and amplification of DNA from decalcified and undecalcified United States Civil War bone. J For ensic Sci . 38(1):60 8. Foran DR. 2006. Relative degradation of nuclear and mitochondrial DNA: an experimental approach. J Forensic Sci . 51(4):766 70. Fu Q, Li H, Moorjani P, Jay F, Slepchenko SM, Bondarev AA et al. 2014. Genome sequence of a 45,000 - year - old modern human from western Siberia. Nature. 514:445 - 50. 267 Gardner, E. and Gray, D. J. 1970. The prenatal development of the human femur. Am J Anat. 129:121 40. Gordon CG, and Buikstra JE. 1981. Soil pH, bone preservation, and sampling bias at mortuary sites. Am Antiquity. 46(6):566 71. Götherström A, Collins MJ, Angerbjörn A, Lidén K 2002. Bone preservation and DNA amplification. Archaeometry . 44:395 404. Gray, H. 1918. Anatomy of the human body . Philadelphia: Lea & Febiger; Bartleby.com, 2000. http:/ /www.bartleby.com/107/59.html . [18 May, 2015]. Hagelberg E, Gray IC, Jeffreys AJ. 1991. Identification of the skeletal remains of a murder victim by DNA analysis. Nature . 352:427 9. Hagelberg E, Clegg JB. 1991. Isolation and characterization of DNA from archeological bone. P Roy Soc Lond B Bio. 244:45 50. Hebda LM. 2013. DNA isolation and analysis from skeletal remains: Evaluating the utility of Hebda LM, Foran DR. 2015. Assessing the utility of soil DNA extraction kits for increasing DNA yields and eliminating PCR inhibitors from buried skeletal remains. J Forensic Sci . 60(5):1322 30. Hedges RE. 2002. Bone diagenesis: An overview of processes. Archaeometry . 44:31 9 28. Hedges RE, Millard AR. 1995. Bones and groundwater: Towards the modeling of diagenetic processes. J Archaeol Sci. 22:155 64. Hickman MJ, Hughes KA, Strom KJ, Ropero - offices, 2004. Bureau of Justice S tatistics Repot. NCJ 216756, Washington, DC: United States Department of Justice, Bureau of Justice Statistics. Hilz H, Wiegers U, Adamietz P. 1975. Stimulation of proteinase K action by denaturing agents: Eur J Biochem . 56:103 8. Hochmeister MN, Budowle B, Borer UV, Eggmann U, Corney CT, Dimhofer R. 1991. Typing of deoxyribonucleic - acid (DNA) extracted from compact - bone from human remains. J Forensic Sci. 36(6):1649 61. Horneck DA, Sullivan DM, Owen JS, Hart JM. 2011. Soil test interpretation guide. Oregon State University Ex tension Services. 1 8 . 268 H ö ss M, Jaruga P, Zastawny TH, Dizdaroglu M, P ää bo S. 1996. DNA damage and DNA sequence retrieval from ancient tissues. Nucleic Acids Res . 24(7):1304 7. Hughes MA, Jones DS, Connolly RC. 1986. Body in the bog but no DNA. Nature . 3 23 (6085) :208. Hunter RL, Agnew AM. 2014. Nonuniform osteocytic lacunae distribution across the femoral cortex. [Poster presentation] 83 rd Annual Meeting of the American Association of Physical Anthropologists. Calgary, Canada. Iborra FJ, Kimura H, Cook P R. 2004. The functional organization of mitochondrial genomes in human cells. BMC Bio . 2(9):1 14. International Committee of the Red Cross. Missing people, DNA analysis and identification of human remains, second edition, ICRC, Geneva, 2009. Janaway RC. 1997. The decay of buried human remains and their associated materials . Studies in crime: An introduction to forensic archeology, Chapter 4, 58 85. Hunter RJ, Martin A, Eds. Routledge, London. Johnson M. 2013. Detergents: Triton X - 100, Tween - 20, and more. Mater Methods . 3(1):163. Kemp BM, Monroe C, Smith DG. 2006. Repeat silica extraction: A simple technique for the removal of PCR inhibitors from DNA extracts. J Archaeol Sci. 33:1680 9. Kline MC, Duewer DL, Redman JW, Butler JM. 2005. Results from NIST 2 004 DNA quantitation study. J Forensic Sci. 50(3):571 8. Kreader CA. 1996 . Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl Environ Microb. 63(3):1102 1 6. Kuntz M. 1950. Crystalline Desoxyribonuclease: II . Digestion of thymus nucleic acid (desoxyribonucleic acid) the kinetics of the reaction. J Gen Physiol. 33:363 73. Lange TB. 2008. The effects of soil chemistry on skeletal preservation at the Voegtly cemetery [ Thesis] . East Lansing (MI): Michig an State University. Latham KE, Madonna ME. 2013 . DNA Survivability in Skeletal Remains . Manual of Forensic Taphonomy, Chapter 15, 403 26. Pokines J & Symes SA, Eds. CRC Press, Boca Raton FL. Lee HC, Pagliaro EM, Berka KM, Folk NL, Anderson DT, Ruano G et al. 1991. Genetic markers in human bone: 1. deoxyribonucleic acid (DNA) analysis. J Forensic Sci . 36:320 30. 269 Leney MD. 2006. Sampling skeletal remains for ancient DNA (aDNA): A measure of success. Hist Archeol. 40(3):31 49. Lindahl T. 1993. Instabilit y and decay of the primary structure of DNA. Nature . 362:709 15. Lindquist CD, Evans JJ, Wictum EJ. 2011. Developmental validation of feline, bovine, equine, and cervid quantitative PCR assays. J Forensic Sci . 56(S1):S29 S35. Loreille OM, Diegoli TM, Irw in JA, Coble MD, Parsons TJ. 2007. High efficiency DNA extraction from bone by total demineralization. Forensic Sci Int Gen . 1(2):191 5. Martin RB, Burr DB. 1989. Structure, function and adaption of compact bone . Raven Press, New York. Martinson HG. 1973 . Nucleic acid - hydroxylapatite interaction. II. Phase transitions in the deoxyribonucleic acid - hydroxylapatite system. Biochemistry . 12:145 50. Mashiba T, Turner CH, Hirano T, Forwood MR, Johnston CC, Burr DB. 2001. Effects of suppressed bone turnover by bisphosphonates on microdamage accumulation and biomechanical properties in clinically relevant skeletal sites in beagles. Bone . 28(1):524 31. Matthews LS, Hirsch C. 1972. Temperatures measured in human cortical bone when drilling. J Bone jt. Surg. 54:2 97 308. Michaud CM, Foran DR. 2011. Simplified field preservation of tissues for subsequent DNA analysis. J Forensic Sci . 56:846 52. rates of nuclear short tandem repeat typing from different skeletal elements. Croat Med J . 48:486 93. Misner LM, Halvorson AC, Dreier JL, Ubelaker DH, Foran DR. 2009 . The correlation between skeletal weathering and DNA quality and quantity. J Forensic Sci. 54(x):822 8. M ü ller A, Hinrichs W, Wolf WM, Saenger W. 1994. Crystal structure of calcium - free proteinase K at 1.5 - Å resolution. J Bio Chem . 269(37):23108 11. Mun dorff AZ, Bartelink E, Mar - Cash E. 2009. DNA Preservation in skeletal elements from the World Trade Center disaster: Recommendations for mass fatality management. J Forensic Sci. 54(3):739 45. Mundorff AZ, Davoren JM, Weitz S. 2013. Developing an empirica lly based ranking order for bone sampling: Examining the differential DNA yield rates between human skeletal 270 elements over increasing post mortem intervals. Final Technical Report submitted to National Institute of Justice, April. Mundorff AZ, Davoren JM. 2014. Examination of DNA yield rates for different skeletal elements at increasing post mortem intervals. Forensic Sci Int Gen. 8(1):55 63. Murphy WA, Spruill FG, Gantner GE. 1980. Radiologic identification of unknown human remains. J Forensic Sci. 25(4 ):727 35. Nielsen K, Mogensen HS, Hedman J, Niederstätter H, Parson W, Morling N. 2008. Comparison of five DNA quantification methods. Forensic Sci Int Gen . 2:226 30. Opel KL, Chung D, McCord BR. 2010. A study of PCR inhibition mechanisms using real tim e PCR. J Forensic Sci . 55(1):25 33. Parsons TJ, Weedn VW. 1996. Preservation and recovery of DNA in postmortem specimens and trace samples . Advances in forensic taphonomy: The fate of human remains, pg. 109 38. Haglund W & Sorg M, Eds. CPR Press, New York NY. Pearson K. 1917 1919. A study of the long bones of the English skeleton I: The femur. University of London, University College, Department of Applied Statistics, Company Research, Memoirs, Biometric Series X, Ch. 1 4. Pretty IA, Sweet D. 2001. A l ook at forensic dentistry part 1: the role of teeth in the determination of human identity. Br Dent J . 190(7):359 66. Price PA, Liu TY, Stein WH, Moore S. 1969. Properties of chromatographically purified bovine pancreatic deoxyribonuclease . J Biol Chem. 244:917 23. Price PA, 1975. The essential role of Ca 2+ in the activity of bovine pancreatic deoxyribonuclease. J Biol Chem. 250:1981 6. Prinz M, Carracedo A, Mayr WR, Morling N, Parsons TJ, Sajantila A et al. 2006. DNA commission of the International S ociety for Forensic Genetics (ISGF): Recommendations regarding the role of forensic genetics for disaster victim identification (DVI). Forensic Sci Int Gen. 1:3 12. Privé GG. 2007. Detergents for the stabilization and crystallization of membrane proteins. Methods . 41(1):388 97. Rennick SL, Fenton TW, Foran DR. 2005. The effects of skeletal preparation techniques on DNA from human and non - human bone. J Forensic Sci . 50(5):1 4. Robin ED, Wong R. 1988. Mitochondrial DNA molecules and virtual number of mitoc hondria per cell in mammalian cells. J Cell Physiol . 136(3):507 13. 271 Roger M, Hotchkiss RD. 1961. Selective heat inactivation of pneumococcal transforming deoxyribonucleate. P Natl A Sci USA. 47(5):653 69. Rogers HJ, Weidmann SM, Parkinson A. 1952. Studie s on the skeletal tissues. 2. The collagen content of bones from rabbits, oxen, and humans. Biochem J . 50(4):537 42. Rozen S, Skaletsky HJ. 2000. Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S, editors. Bioinf ormatics methods and protocols: methods in molecular biology. Totowa, NJ: Humana Press, 365 86. functional adaptation. Am J Phys Anthropol. 129:484 96. Salamon M , Tuross N, Arensburg B, Weiner S. 2005. Relatively well preserved DNA is present in the crystal aggregates of fossil bones. Proc Natl Acad Sci USA . 102(39):13783 8. Satoh M, Kuroiwa T. 1991. Organization of multiple nucleoids and DNA molecules in mitocho ndria of a human cell. Exp Cell Res :196(1):137 40. Scholz M, Giddings I, Pusch CM. 1998. A polymerase chain reaction inhibitor of ancient hard and soft tissue DNA extracts is determined as human collagen type I. Anal Biochem . 259(2):283 6. Scientific Wor king Group for Forensic Anthropology (SWGANTH). 2010. Personal Identification. http://swganth.startlogic.com/Identification%20Rev0.pdf. Seo SB, Zhang A, Kim HY, Yi JA, Lee HY, Shin DH, Lee SD. 2010. Technical note: Efficiency of total demineralization an d ion - exchange column for DNA extraction from bone. Am J Phys Anthropol . 141(1):158 62. Shapiro SS, Wilk MD, Chen HJ. 1968. A comparative study of various tests for normality. J Am Stat Assoc . 63(324):1343 72. Silver A. 1963. The ageing of domestic anim als . Science in archeology: A comprehensive survey of progress and research, Chapter 26, 250 68. Brothwell D, Higgs E, Clark G, Eds. Basic Books, New York. Simpson EK, James RA, Eitzen DA, Byard RW. 2007. Role of orthopedic implants and bone morphology i n the identification of human remains. J Forensic Sci . 52(2):442 8. Smith CI, Chamberlain AT, Riley MS, Stringer C, Collins MJ. 2003. The thermal history of human fossils and the likelihood of successful DNA amplification. J Hum Evol. 45:203 17. 272 Stea dman DW, DiAntonio LL, Wilson JJ, Sheridan KE, Tammariello SP. 2006. The effects of chemical and heat maceration techniques on the recovery of nuclear and mitochondrial DNA from bone. J Forensic Sci . 51(1):11 7. Stewart TD. 1962. Anterior femoral curvatur e: Its utility for race identification. Human Bio . 34:49 62. Stewart TD. 1979. Essentials of forensic anthropology . Springfield, Illinois: Thomas, 120. Trotter M, Gleser GC. 1952. Estimation of stature from long bones of American whites and Negores. Am J Phys Anthropol . 10:463 514. Trotter M, Gleser GC. 1958. A re - evaluation of estimation based on measurements of stature taken during like and of long bones after death. Am J Phys Anthropol . 16:79 123. Turner - Walker G. 2008. The chemical and microbial de gradation of bones and teeth. Advances in human paleopathology, Chapter 1, 1 29. Pinhasi R, Mays S, Eds. John Wiley & Sons. Vaananen HK, Zhao H, Mulari M, Halleen JM. 2000. The cell biology of osteoclast function. J Cell Sci 113:377 81. White TD, Folken s PA. 2005. The human bone manual . San Diego: Elsevier Academic Press, 31 46. Yamaguchi M, Ma ZJ, Suzuki R. 2003. Anabolic effect of wasabi leafstalk ( Wasabia japonica MATSUM) extract on bone components in the femoral - diaphyseal and - metaphyseal tissues o f aged female rats in vitro and in vivo. J Health Sci . 49(2):123 8. Yamaguchi M, Yamaguchi R. 1986. Action of zinc on bone metabolism in rats: increases in alkaline phosphatase activity and DNA content. Biochem Pharmacol . 35(5):773 7.