1:. ”(Ana 5. .9 stwkmwmfi 1.! . 3.. aka. a. gig innufiw in? . . st 5...... . gum»? {gun Ammm I w...“ a , . 2,3 . Benin .3. 2 91.2.3.3; 2:: . fit it) If; , .52 .3. .l a 70. :9 . 05‘?- :..§_.,..n._u 2% $2. «is?! 5;! rent‘iz. s. u Emu} 1:”. Estate“ 2 . You: . xi . stifunvfiwgrwu r... 7.§!\‘2u..~ 5.431.1’51... t}. .. '23:... .3. I”. avlug. ‘ v.2. . . v.8. .flfl. 11.17 $5,551.21} . .1 :l git-115.119. » 5 r2 . ’ A. ti 3. . .., . )‘u.lu5..Y t‘Nl’kv.‘ . .1 I!!! x A 4:. . .J 7"“ LIBRARY goo; Michigan State , University 6-7 6; 3 0 77—75 This is to certify that the thesis entitled THE USE OF ENHANCED PCR TECHNIQUES FOR NUCLEAR DNA ANALYSIS OF COMPROMISED FORENSIC SAMPLES presented by LISA M. RAMOS has been accepted towards fulfillment of the requirements for the MS. degree in FORENSIC SCIENCE -— o-u-o-I-0-0-C-O-n-o.oo—- v Major Professor’s Signature 527/ %/&6 Date MSU is an Affirmative Action/Equal Opportunity Institution —.-o-g.-¢_-.-A-A4 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE Fl? =1) 152C398 flit??? 2/05 p:/ClRC/DateDue.indd-p.1 THE USE OF ENHANCED PCR TECHNIQUES FOR NUCLEAR DNA ANALYSIS OF COMPROMISED FORENSIC SAMPLES By Lisa M. Ramos A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTERS OF SCIENCE School of Criminal Justice 2006 ABSTRACT THE USE OF ENHANCED PCR TECHNIQUES FOR NUCLEAR DNA ANALYSIS OF COMPROMISED FORENSIC SAMPLES By Lisa M. Ramos Biological samples encountered in the forensic laboratory are often received in compromised conditions. DNA analysis may be hampered by difficulties such as low- copy number (LCN) or degraded material. For these types of samples, standard PCR analysis may fail; therefore the use of enhanced PCR techniques for the amplification of nuclear DNA would be valuable. In this research a variety of compromised samples, including bone (nearly fresh to ancient), degraded blood, and touch-DNA, were examined using novel PCR techniques. Mini-STR primers were utilized as a means of amplifying smaller fragments of degraded DNA. Nested PCR was used with STRs and the sexing locus amelogenin, to determine if different primers and an increase in cycle number would amplify degraded DNA and to assist in anthropological sex determination. Amplification of the mini-STR loci was successful when using nested PCR on degraded blood and fresh bone, but not in other instances, such as with older skeletal material. The amelogenin nested PCR was successful to variable degrees with each of the sample sets, with the exception of 30 year-old bone, however allelic dropout due to stochastic effects was often observed. Overall, the enhanced PCR techniques were helpful with certain samples in amplifying degraded/LCN DNA and aiding in sex determination, and should be considered when dealing with compromised forensic DNA samples. This paper is dedicated to my father Steven, who would have been so proud. iii ACKNOWLEDGEMENTS I would like to thank thesis advisor, Dr. David Foran, PhD, Director — Forensic Science Program, School of Criminal Justice and Department of Zoology, Michigan State University for giving me the opportunity to work on this project, for guiding me through the research, and spending countless hours working on corrections for my paper. Thank you so much for your time and dedication, it is greatly appreciated. Thank you to Dr. Todd F enton and Dr. Vince Hoffman, for your time and input as my committee members. I would also like to thank the following individuals for providing the extracted samples: Lisa Misner, Virginia Clemmer, Amy Barber, Stephanie Rennick, and Michael Gehring. I would also like to thank Dr. Michael Coble, NIST, for his guidance with software and the use of their primer sets. Finally, I would like to thank my family and friends for their endless support and words of encouragement through my academic career. I could not have made it this far without each and every one of you. iv TABLE OF CONTENTS LIST OF TABLES ...................................................... vii LIST OF FIGURES ..................................................... viii INTRODUCTION ....................................................... 1 DNA Degradation ........................................................ 1 Bone Degradation ........................................................ 2 Sex Determination of Skeletal Material ....................................... 4 Sexing Skeletal Material Anthropologically .............................. 4 Sexing Bones Via Genetic Analysis .................................... 4 Mini-STR Primer Analysis ................................................ 7 Nested PCR ............................................................. 8 Contamination Issues with Compromised DNA ................................. 8 Description of Sample Sets Used in this Study ................................. 10 Skeletal Material .................................................. 10 Non-Skeletal Samples .............................................. 12 Research Goals ......................................................... 12 METHODS AND MATERIALS ........................................... 14 Research Sample Sets .................................................... 14 Nested PCR Using Amelogenin ............................................ 14 DNA Amplification of Samples ............................................. 15 Amelogenin Nested PCR Parameter Optimizations ............................. 16 Initial Nested PCR Experiments of Research Samples ........................... l6 Capillary Electrophoresis of Samples ........................................ 17 Mini-STR Experiments ................................................... l8 Parameter Optimization of Mini-STR Primers Using Control DNA ........... 18 PCR Amplification of Research Samples Using Mini-STR Primers ........... 20 Mini-STR Primer Set Allelic Ladder Preparation ......................... 21 Capillary Electrophoresis of Mini-STR Primer Experiments ................ 22 RESULTS ............................................................. 24 Sex Determination Using Amelogenin Nested PCR ............................. 24 Parameter Optimizations ............................................ 24 Voegtly Bone Samples .............................................. 28 Albania Bone Samples .............................................. 29 3O Year-old Bone Samples .......................................... 29 3 Week-old Bone Samples ........................................... 29 Blood Samples .................................................... 30 Pipe Bomb Samples ................................................ 3O Mini-STR Primer Experiments ............................................. 34 PCR Parameter Optimization with Control DNA 9947A ................... 34 Mini-STR Primer Analysis of Voegtly Samples ......................... 35 Mini-STR Primer Analysis of Albania Bone Samples ..................... 35 Mini-STR Primer Analysis of 30 year-old Bone Samples ................... 36 Mini-STR Primer Analysis of 3 Week-old Bone Samples .................. 36 Mini-STR Primer Analysis of Blood Samples ............................ 37 Mini-STR Primer Analysis of Pipe Bomb Samples ....................... 38 Capillary Electrophoresis on an ABIPRISM® 310 Genetic Analyzer ......... 39 Control DNA 9947A ......................................... 39 Mini-STR Primer Set Allelic Ladder ............................. 39 Albania Sample ............................................. 44 3 Week-old Bone Samples .................................... 44 Blood Samples ............................................. 44 Pipe Bomb Samples ......................................... 45 DISCUSSION .......................................................... 47 Analysis of Control Samples Utilizing the Amelogenin Nested PCR Technique ....... 47 Analysis of Control DNA 9947A Using Mini-STR Primers ...................... 48 An Overview of Degradation and Contamination within Research Samples .......... 49 Analysis of Voegtly Skeletal Samples ....................................... 51 Analysis of Albania Skeletal Samples ....................................... 53 Analysis of 30 year-old Bone Sample ........................................ 54 Analysis of 3 Week-old Bone Samples ...................................... 55 Analysis of Blood Samples ................................................ 57 Analysis of Pipe Bomb Samples ............................................ 58 CONCLUSION ......................................................... 60 REFERENCES ......................................................... 62 vi LIST OF TABLES Table 1. Mini-STR primers utilized in this study ............................... 19 Table 2. Nested PCR results .............................................. 31 Table 3. Panel created in GeneMapperTM ID software .......................... 40 vii LIST OF FIGURES Figure 1. Amelogenin Sequence from GenBank ............................... 6 Figure 2. Nested PCR diagram ............................................. 9 Figure 3. Primer activity in reagent blank lane ................................ 25 Figure 4. CEQ 8000 electropherogram of male known DNA ..................... 26 Figure 5. CEQ 8000 electropherogram of known female DNA ................... 27 Figures 6 and 7. Comparison of electropherograms from FW EDTA ............... 33 Figure 8. 2% agarose gel of control DNA amplified with mini-STR primer sets ...... 35 Figure 9. Blood samples amplified with nested STR PCR ........................ 38 Figure 10. Mini-STR primer set allelic ladder, 6 — F AM dye ..................... 41 Figure 1 1. Mini—STR primer set allelic ladder, VIC dye ......................... 42 Figures 12 and 13. Mini-STR primer set allelic ladder, NED and PET dyes ......... 43 Figure 14. Profile of blood sample D15 with all of the allele calls present ........... 46 viii Introduction In the realm of the forensic world, DNA can play an important role in casework, particularly in identification of victims and suspects. DNA can be obtained from numerous sources, such as blood and skeletal remains (Butler 2005, Arismendi et al. 2004), and if recovered in good condition, its analysis is generally straightforward. However, biological material is ofien found in less than ideal condition, and the DNA within may also be compromised. In many cases, a body or other evidence may not be recovered for many years, or found after it has been exposed to the elements. If the sample is very old or highly degraded, obtaining DNA results, particularly from more discriminating nuclear DNA, may be difficult or impossible (Butler 2005). When the samples encountered are compromised, there are a few different options for their analysis. For example, mitochondrial DNA (mtDNA) may be obtainable when nuclear DNA is not, as it exists in higher copy number in a cell, and is protected in the mitochondrion (Holland and Parsons 1999). However, in terms of identification, mtDNA will not produce a unique profile. It is maternally inherited, meaning all maternal relatives have the same sequence. There is also no way of determining sex using mtDNA. Nuclear DNA analysis is necessary for establishing both sex and a unique genetic profile from an individual or biological evidence. DNA Degradation DNA degradation can occur via chemical processes, including oxidation and hydrolysis. Oxidation can occur at both the nucleobases and the sugars of the DNA; this type of damage results in the DNA strands breaking at the sugar links or cross-linking to proteins (Christophersen et al. 1991). Hydrolysis is a water-based attack on the helix itself, either chemically or enzymatically. Chemically, while DNA maintains its stability in basic environments, it tends to break down in acidic conditions. The acids attack the phosphate ester bonds and also the N-glycosidic bond between the sugar and nucleotide (www.massey.ac.nz/~wwbioch/DNA/hydDNA/framset.htm). DNA is hydrolyzed enzymatically by endonucleases, enzymes (common in microorganisms) that cleave the DNA within the strand, and exonucleases, enzymes that cleave the ends of the DNA molecule. When nucleases attack the nucleosome, the DNA is released and exposed to materials that may cause further degradation. DNA degradation can also occur in laboratory settings. Examination of bones, such as with x-ray analysis, can further damage the DNA within. Surprisingly, there is no dependable correlation between the age of a sample, such as skeletal material, and the size of the DNA fragments remaining. Typically fragments only 100 — 200 base pairs in length are recovered from aged material (Kelman and Moran 1996, Opel et al. 2006). In contrast, DNA existing under favorable conditions, such as neutral pH and lower temperatures, may be stable for long periods of time. As important, DNA from ancient skeletal material may exist at very low copy numbers [LCN]. If the DNA is degraded, the larger target sequences typically yield less dependable results than smaller sequences. Bone Degradation Bone is made up of different components, such as minerals and proteins. Type I collagen is one of the most copious, comprising about 95% of bone proteins (Collins et al. 2002). According to these authors, chemical deterioration in bone can occur via three mechanisms: organic breakdown (usually affecting collagen), loss of bone minerals, and biodegradation. The first mechanism, organic breakdown, generally involves collagen being damaged via chemical hydrolysis. Collagen is a highly repetitive, water-insoluble very abundant protein. If damaged, the structure of the bone will be affected. The bone porosity may increase, up to 50%, causing it to be susceptible to microbial attack (Hedges 2002). The chemical hydrolysis of collagen can be affected by different factors such as temperature, pH and time. Proteins tend not to be stable at high temperatures, and therefore collagen will be lost at a higher rate when exposed to high temperatures. When the environment surrounding the bone is very alkaline, the pH of the bone tends to rise, and it can result in physical swelling or an acceleration of hydrolysis of amino acids within collagen. Mineral loss is another mechanism by which bone can degrade. For instance, if bones are found in or near water, the bone structure, such as weight and porosity, may become unstable and susceptible to degradation, whether it is biological or chemical. The porosity of the bones tends to increase, allowing the bone to become permeable to outside influences, such as microbial attack (Hedges 2002). As the minerals start to re- configure, collagen degrades, resulting in the organic breakdown (Collins et al. 2002). The final mechanism of bone degradation is microbial activity. Biodegradation typically occurs when the pH of the environment is nearly neutral. According to Hedges (2002), there is no set time frame as to the intensity of microbial attack after burial. Different environments have various microbes that attack bone, and studying the soil surrounding the bones may help to determine how their activity affects bone degradation. Very dry and very wet environments are both microbiological attack inhibitors (Hedges 2002). Sex Determination of Skeletal Material Sexing Skeletal Material Anthropologically When skeletal remains are found, it is generally the anthropologist who is called in to assist in identification. One important step is determining the sex of the remains. There are certain features within the skeleton that anthropologists use as a guide to estimating sex. The pelvis and skull are two of the best regions for sex estimation (Bass 1995). Within the pelvis, females have a U-shaped subpubic angle and a wide greater sciatic notch, while males are V-shaped and narrow. Two pelvic features are generally indicative of a female if they are present: the ventral arc and the well-developed preauricular sulcus. Examinations of adult skulls show that males tend to have a large mastoid process and supraorbital ridges. The skull of a female is generally small, smooth and gracile (Bass 1995). When the pelvis or skull are not present or are damaged, general bone size, while not highly reliable, may also be used to estimate sex. At times overall robustness is an indicator of the male sex (Ubelaker et al. 2003). Sexing Bones Via Genetic Analysis When using DNA as a means of determining sex, the amelogenin gene is often used (Butler 2005). Amelogenin is a gene that codes for a protein essential in the development of tooth enamel (Michael and Brauner 2004), but can be used in sex determination because the DNA sequence contains a 6-bp deletion on the X chromosome (Figure 1). This is not the only region where males and females differ however. Eng et al. (1994) detailed 19 regions of similarity between the X and Y chromosome. Within these regions are deletions that can be used to distinguish males from females. There are other markers that are found on the Y-chromosome that may also help to identify a male sample as well (Sinha et al. 2003), including numerous short tandem repeat (STR) loci that are located on the non-recombinant region of the Y chromosome. However, the use of these markers for sex determination, including amelogenin, is not infallible. It is possible that the Y-chromosome has polymorphisms that result in dropout (no amplification) of the Y allele, producing a female profile (Roffey et al. 2000). These authors speculated that this could have been due to genetic rearrangement that spanned the common primer binding sequences, or due to a deletion of one of the primer binding regions. In order to determine the sex of a sample using the genetic methods above, it first needs to be amplified, using a technique known as polymerase chain reaction (PCR). PCR allows a specific region of DNA to be copied over and over (Mullis and Faloona 1987). A piece of DNA template is combined with different PCR components such as Tris-HCl buffer, MgClz, dNTPs, primers and Taq DNA polymerase. There are multiple cycles, typically between 15 — 40, of DNA denaturation, primer annealing, and primer extension, in which the DNA becomes single stranded; the primers anneal to the single- stranded DNA, and the Taq polymerase extends the primers using dNTPs to copy the target region. During 30 cycles of PCR, over a billion copies can theoretically be made from one piece of DNA (Butler 2005). 161 201 281 C C 241 G _ G G C 321 +<>< »<:>< +<>< +<>< tax+<><<>< +<><+<>< . Promega Primers (X = 212bp, Y = 218bp) 0 British Primers (X = 106bp, Y = 112bp) Figure l. Amelogenin Sequence from GenBank. The top portion is the X chromosome sequence, while the bottom is the Y chromosome sequence. The outer most set of arrows represents ‘Promega’ primers and the innermost are ‘British’ primers (used in this research). The 6-bp deletion on the X chromosome can be observed in the bracketed region of the sequence. Standard PCR, for mtDNA or nuclear DNA, is not always successful when working with compromised DNA samples. There can be too few starting copies of the DNA available for successful PCR, or contaminants may be present that inhibit the reaction. These lead to a need for more cycles, which in turn may cause undesired regions of the DNA to be amplified. The ‘artifact’ bands that can be produced with high cycle number may make subsequent analysis impossible. Compromised DNA can also be degraded, meaning that primers that sit far apart may not successfully amplify the target region. Two techniques can be utilized for dealing with samples that are degraded or contain very little DNA: mini-STR primer sets (reduced size amplicon) and nested PCR. Mini-S TR Primer Analysis STRs are small (2 — 7 bp) segments of repeated DNA that are highly polymorphic (Butler 2005). Currently, they are the most commonly used markers for the analysis of forensic DNA samples. STRs are useful because, being relatively compact and having a PCR based analysis, only a small amount of DNA is required to obtain results (Kimpton et al. 1993). The methods for analysis are reliable, quick, and simple (Saferstein 2002). Most commercial STR kits amplify DNA regions from 100 to 450 bp. However, if a DNA sample is degraded, its largest length is generally reduced to about 100 — 200 bp (Kelman and Moran 1996, Opel et al. 2006). When DNA is degraded, mini-STRS can be utilized. The idea behind these markers is to move the primer positions closer to the ends of the repeat (Chung et al. 2004). This potentially allows for a more successful analysis of smaller fragments of DNA, which are often encountered in degraded samples. Nested PCR As detailed previously, a high number of PCR cycles using one set of primers can result in the amplification of non-specific regions of DNA. However, if two rounds of PCR and two different primer sets are used, this may aid in reducing or eliminating the occurrence of these artifacts. This technique is known as nested PCR (Strom and Rechitsky 1998) (Figure 2). The first set of primers (outer) is designed to amplify a desired region of DNA during one round of PCR. An aliquot of this DNA is then added to the reaction mix for the second round, which contains a set of inner primers. The inner primers will generally only amplify DNA within their region; if any unwanted DNA region was amplified in round 1, it will not amplify further during the second round. Thus nested PCR serves as a means to eliminate any non-specific amplification of unwanted product. Contamination Issues with C ompromised DNA Low copy number or compromised DNA is generally composed of small fragments. If the DNA exists in LCN, any contaminating DNA is more likely to be amplified. Since foreign DNA is a constant concern, precautions must be taken to ensure that only DNA from the sample itself is being amplified. According to Kaestle and Horsburgh (2002), one way to avoid contamination when working with LCN or ancient DNA is to conduct analysis separate from modern samples. It is also important to have separate equipment, such as pipetters, gloves, and reagents, for this type of DNA analysis. This may help to prevent DNA contamination resulting from the equipment that is being used on modern samples. It is important that the area where the samples are handled is clean and sterile, using bleach or UV-irradiation. Even with all of these precautions, contamination can still occur. In order to detect it, controls—positive, negative, and reagent blanks—should be used during the different steps of analysis. If a contaminant shows up in these controls, the analysis can be repeated from the step at which it was detected. It is also imperative the scientist conducts multiple repetitions of experiments on the samples, since the DNA may not be amplified readily during the initial experiments and also as a means of verifying the results that were obtained. First Set or Primers Target DNA /Flrst set or Primers (Out-r) \- (Outer) t First amicon *5 Second Set of Prlmen\ /Seeend Set of Primers (Inner) (Inner) i I Speciflc Amplification of the Target DNA Figure 2. Nested PCR diagram. The first set of primers (outer/extemal) amplifies a target region of the DNA. The second set of primers (inner/internal) amplifies a smaller region within the external set. This allows for specific amplification of the target DNA. (Figure obtained from http://www.wisconsinlah.com/nested_pcr.htm) Description of Sample Sets Used in this Study Skeletal Material When determining the samples that would be utilized in this research, the scientists who conducted the DNA extractions were consulted in regards to the quality of their samples. In general, skeletal material had been obtained, cleaned, ground, and the resultant bone powder weighed. The ground bone was then digested in buffer and proteinase K, followed by one or more phenol-chloroform extractions, which varied among samples (detailed in Misner 2004). After the extractions, the aqueous layer was precipitated, vacuum-dried, and resuspended in TE buffer, in a volume that was in concordance with bone weight. These DNAs were then amplified using primers designed for mtDNA. This basic protocol was followed by each of the analysts, with some small variations among sample sets. Samples in which previous analysts had successful mtDNA amplification were considered for analysis in this project. The oldest set of bone samples used in this research was retrieved from an archaeological site in Kamenica, Albania. The bones dated between the Bronze Age (1200 to 1150 BC) and the Early Iron Age (650 to 550 BC), making them 2,500 — 3,000 years old. A portion of the bone had been drilled and the powder collected. After digestion and organic extraction, the DNA was precipitated with sodium acetate and ethanol, dried, and resuspended in TE buffer. The next set of samples was collected from a former cemetery of the Voegtly Evangelical Lutheran Church, near Pittsburgh, PA, in conjunction with the Smithsonian Institution. The burials originated between 1833 and 1861, which meant that this set of samples was about 150 — 170 years old. The condition of samples ranged from the bone 10 surface having minimal flaking to being extensively fragmented (Ubelaker et a1. 2003). A portion of the bone was cut out and immersed in wash buffer (1% SDS, 25mM EDTA) and proteinase K and incubated as described in Misner (2004). It was then dried, ground, and extracted using the phenol-chloroform method. The DNA was precipitated in the same manner as previously discussed. The next bone samples were from a 35 year-old interment, originating from a body that had been prepared for burial with embalming fluid. Two different DNA isolation methods were conducted by another analyst. First, bone powder was obtained from the samples. A portion of this powder was digested in either 500 uL of digestion buffer and Sul proteinase K at 55°C overnight, or in 500 uL of 250mM EDTA, 5p] proteinase K with the same incubation. The samples digested in digestion buffer underwent an organic extraction, were placed in a Microcon YM-30 column (Millipore) and washed 4 times with TE. The samples digested in EDTA underwent only the wash technique described above, and were resuspended in TE buffer. The final bone samples were from a putrefied body exposed to the elements for approximately three weeks. The bones (femur and metatarsal) were subjected to various cleaning techniques in which they were boiled for 4 hours in either water, a sodium carbonate detergent mixture, or 25% bleach (Rennick et al. 2005). The samples were then digested using either digestion buffer or EDTA as above. However, the EDTA samples did not go through a Microcon column, and were organically extracted in the same manner as the digestion buffer samples. A phenol-chloroform extraction and sodium acetate/ethanol precipitation were conducted for these samples. 11 Non-Skeletal Samples A set of experiments was conducted on degraded human blood samples (UCHRIS IRB# 04-644). The first sample had been allowed to degrade at 37°C with no added humidity for 9 months. The second sample was placed in a 37°C humid environment for 15 days. Both samples were organically extracted using the phenol-chloroform method and precipitated as indicated above. The final samples analyzed in this project were obtained from detonated pipe bombs (Gehring 2004). The scientist had subjects take apart and put together a steel pipe bomb for 30 seconds, and the bombs were filled with smokeless gunpowder and detonated. The bomb fragments were collected and swabbed. This cotton swab was digested in digestion buffer and proteinase K, followed by a phenol-chloroform extraction. The samples were cleaned using a Microcon YM-100 column, as described for the 35 year-old samples. Reference buccal swabs had been obtained for individuals who participated in the study (UCHRIS IRB# 03-868). Research Goals The goals of this research were to determine if enhanced PCR techniques would be useful in amplifying and analyzing nuclear DNA markers, whether the DNA is old, present at low copy number, degraded, or all of these. Both the amelogenin nested PCR and mini-STR primer analysis methods were tested on a variety of samples that were assumed to be at different degradation levels. By examining these techniques on different types of samples, it is possible to gain an insight into what, if any, technique is most useful when compromised DNA samples are encountered. Note that these 12 techniques yield very different information. The amelogenin nested PCR technique would be useful in terms of sex determination of highly degraded and low copy number forensic samples, and could also be beneficial for anthropological research as an alternative means of sexing skeletal material. The mini-STR primers are more useful for individual identification. If a profile could be developed, and possibly matched to a known DNA sample, this would be helpful in cases where the samples are not in the best of conditions. Both techniques have different objectives—one aids in determining sex, the other in developing a STR profile—however, they would each be useful in forensic settings. 13 Materials and Methods Research Sample Sets The samples utilized in this research were ones in which the integrity of the DNA was thought to have been compromised. Four of the sample sets were from skeletal materials. The circumstances surrounding the conditions of the bones differed. The oldest set was from an archaeological site in Kamenica, Albania and dated from 2,500 — 3,000 years before present and the next oldest set was 150 — 170 years old from a burial site in Pennsylvania. The third set was from an exhumed body that had been embalmed and buried for approximately 35 years, and the final set of bone samples was from remains that were exposed to the elements for about three weeks. Two additional sample sets were also tested— degraded blood samples (9 months and 15 days) and DNA obtained from detonated pipe bombs. These samples were genetically analyzed using two techniques: nested PCR and mini-STR primers. Nested PCR Using Amelogenin Amelogenin primer sets were designed based on the amelogenin sequence from Genbank (Sullivan et al. 1993). The sequence featured primers that were labeled as ‘Promega’ and ‘British’ (Figure 1). The smaller amplicon producing set, the British pair, was chosen as the external primers for nested PCR experiments, and internal primers were created within the British primer sites. The British primers were: (forward) BF 5’ CCCTGGGCTCTGTAAAGAATAGTG 3’ and (reverse) BR 14 5’ ATCAGAGCTTAAACTGGGAAGCTG 3’. The internal primers were: (forward) LF 5’ AAGAATAGTGTGTGGATTCTTTATCCCA 3’ and (reverse) LR 5’ GGAACTGTAAAATCGGGACCACTTGAG 3’. The LP primer was also synthesized with a 5’ D4-PA dye (PrOligo) for detection on a Beckman-Coulter CEQTM 8000 Genetic Analysis System. The primer sets were received lyophilized and 1X TE buffer was added to bring the primers to a stock solution of 200 pM/uL (a 100X stock). The final amplicon sizes with the primer sets were: (male) BF/BR 112 bp and LR/LF 68 bp, and (female) BF/BR 106 bp and LR/LF 62 bp. DNA Amplification of Samples Primer concentrations and cycling parameters were optimized using a known DNA sample (Promega Human Genomic DNA: Male). Amplification reactions contained 1X buffer (Promega), MgC12 (2.5mM, Promega), dNTPs (0.2 mM of each dNTP, Promega), 1X (2 pM/ uL) extemal/internal forward primer and 1X extemal/intemal reverse primer (external for round 1 and internal for round 2), deionized sterile water (volume variable), and two units of Promega Taq DNA Polymerase in a total volume of 10 uL. All amplification reactions were set up using pipetters and aerosol barrier pipette tips that were UV-irradiated for 300 seconds between each preparation. Round 1 reactions were prepared in the pre-PCR room, as were round 2 reactions, except for the addition of the round 1 PCR product, which was added in the post-PCR room. Positive and negative controls were run each time an experiment was conducted. To look for any artifact bands that might have resulted from primer combinations, amelogenin primers were paired together in the forward/reverse combinations of BF/BR, 15 BF /LR, LF/LR, and LF/BR. These were amplified on an Eppendorf Mastercycler or a Perkin Elmer GeneAmp PCR System 2400 using the following conditions: denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 1 min, and extension at 72°C for 30 sec, with a final 5 min extension at 72°C. PCR products were electrophoresed on a 3% agarose gel to determine the presence of amplification product. A melogenin Nested PCR Parameter Optimizations Using the above conditions, the primer amounts were optimized by testing three different concentrations (0. 1 X, 0.2X, 1X) using the control DNA. Based on results, the 1X concentration was used for all subsequent experiments. The next parameter optimized was cycle number. The primer sets of LF/LR and BF/BR were amplified for 20, 25, 30, 35 and 40 cycles using conditions previously detailed. Based on the results of these experiments, the amount of DNA required for successful amplification was determined. Ten ng, lng, 100 pg, 10 pg, 1 pg, and 0.1 pg of DNA were amplified using nested PCR with a 35 cycle first round and a 30 cycle second round. Initial Nested PCR Experiments of Research Samples Amelogenin nested PCR was initially tested utilizing the 150 year-old Voeglty bone samples. DNA from these had previously been extracted, and mtDNA successfully amplified. One uL of this extract was added to 9 uL of TE buffer to make a 1:10 dilution. Each of the samples was amplified using the aforementioned reaction volumes l6 and thermal cycling parameters. The final extension differed between rounds with the first having a 5 min extension at 72°C, while the second was extended for 45 min at the same temperature to ensure addition of a terminal adenine residue. After amplification, the samples were electrophoresed on a 3% agarose gel and examined for the presence of bands. The influence of potential PCR inhibitors was explored by adding bovine serum albumin (BSA) to the first round of amplification. A solution of BSA (2X (2 mg/mL) final concentration in reaction mix) was added to samples that did not amplify using the optimized parameters. Another factor that was tested was the use of a hot-start Taq. Based on results, the hot start method was then used for all nested PCR experiments. These reactions included Eppendorf 1X HotMaster Taq Buffer with 2.5 mM Mg2+, dNTPs (0.2mmol/L of each dNTP), 1X external/internal forward primer and 1X external/internal reverse primer, de-ionized sterile water (variable), and two units of Eppendorf HotMaster Taq DNA polymerase. The same thermal cycling conditions and 3% agarose gel electrophoresis were followed as above. Capillary Electrophoresis of Samples Samples that appeared to have an amplification product on an agarose gel were analyzed on a Beckman-Coulter CEQTM 8000 Genetic Analysis System. During the preliminary trials, a 1:100 and 1:200 dilution of each PCR product was made. A 400 bp size standard was used in initial experiments, but the size standard 80 was subsequently chosen due to the basepair size of the peaks that were produced. One uL of each dilution was mixed with 38.5 uL of sample loading solution (SLS, Beckman) and 0.5 uL CEQTM 17 DNA size standard 80, and transferred into a well of the sample plate. A drop of mineral oil was placed in each well. Using the Frag-3 short method (capillary temperature 50°C, denature at 90°C for 120 sec, inject at 2.0kV for 30 sec, and separate at 6.0kV for 25 min), the samples were electrophoresed. Resulting data were analyzed in the Fragment Analysis window using the SNP—SSSO analysis parameter set (project — default; dye mobility calibration — no correction; size standard — size standard 80; model — linear). Resultant electropherograms showed that the 1:200 dilution gave the best result, thus this dilution was used for subsequent experiments. CEQTM runs that failed (no data present on an electropherogram) led to a set of experiments which revealed that both the amount of primer and dNTPs could be cut in half without having an adverse effect on amplification. This primer/dNTP adjustment was made and used for all further analysis. Mini-S T R Experiments Parameter Optimization of Mini-ST R Primers Using Control DNA The mini-STR primers used in this study were created and supplied by Drs. Michael Coble and John Butler of the National Institute of Standards and Technology (N IST). These included 6 individual primer pairs and one mini A1 primer set, which contained a multiplex of all of the primers. Table 1 lists the CODIS loci included in the set and additional information for each. The research samples analyzed with the mini- STR primers were chosen based on successful amplifications from the amelogenin nested PCR experiments and in some cases, on previous analysts’ ability to obtain an mtDNA sequence. Each of the 6 primers and the mini A1 multiplex were initially tested on 18 control DNA 9947A (Applied Biosystems). The PCR master mix contained: 1X PCR Buffer II (Applied Biosystems), MgC12 (2.5mM), 2 units of AmpliTaq Gold DNA polymerase, dNTPs (0.2mmol/L of each dNTP), and water (volume variable) to bring the reaction up to 10 uL. Thermal cycling parameters consisted of denaturation at 95°C for 10 min, followed by 28 cycles of denaturation at 94°C for l min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, with a final extension of 60° for 45 min. The PCR product was electrophoresed on a 2% agarose gel, and upon examination the absence of bands indicated that there were not enough cycles to produce a PCR product, thus the number of cycles was increased to 35. STR Locus Allele Allele Product Size Mini-STR Size Size (Dye Label) Range Spread (STR Kit) Reduction FGA 12.2—51.2 156 bp 196—352 bp 125—281bp 71 bp (6-FAM) THO] 3—14 44 bp 160—204 bp 51—98 bp 105 bp (6-FAM) D168539 5— 15 40 bp 233—273 bp 81 — 121 bp 152 bp (NED) D1885] 7—27 80 bp 264—344 bp 113—193bp 151bp (VIC) D2S1338 15 — 28 52 bp 288 - 340 bp 90 — 142 bp 198 bp (PET) Amelogenin X, Y 106 — 114 bp (6-FAM) Table l. Mini-STR primers utilized in this study. (6—FAM=blue, NED=yellow, VIC = green, PET=red); Allele range is the repeat numbers that are found within the loci; allele spread is the number of base pairs the locus spans; product size is the original size of the IdentifilerTM STRs; mini-STR size is the size of the STRs examined in this study; size reduction is the number of base pairs that the STRs were reduced from the original kit.) Information from Butler et al. 2003. 19 PCR Amplification of Research Samples Using Mini-S TR Primers DNA from an anthropologically-sexed male (167 femur) and female (529a rib) from the Voegtly cemetery were amplified using the primers listed in Table 1, except for THOl and FGA. The PCR reaction conditions with the control DNA were used, while the number of cycles was increased to 40. Next, the use of the mini-STR primers and nested PCR was explored. The first round PCR reaction mixture contained: 4 uL AmpFiSTR® PCR reaction mix, 2 uL AmpF ESTR® IdentifilerTM primer set, 0.5 uL (two units) AmpliTaq Gold DNA polymerase, DNA (1 uL sample DNA, or 4 uL control DNA 9947A) and water to bring the reaction up to 10 uL. Thermal cycling parameters were: denaturation at 95°C for 10 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 56°C for 1 min, and extension at 72°C for 1 min, with a final extension of 5 min at 60°C. For the second round of PCR, 1 uL of PCR product from the first round was added to the master mix for round two containing the mini-STR primers. The second round had the same thermal cycling parameters with a final extension of 45 min at 60°C. The DNA was electrophoresed on a 2% agarose gel and examined for the presence of bands. This nested PCR technique, incorporating the IdentifilerTM primer set as the external primers and the mini-STR primers as the internal primers, was then utilized on samples that were successfully amplified using the amelogenin nested PCR technique, i.e., those samples which produced amelogenin peaks on an electorpherogram. The 3-week old femur and metatarsal samples from the putrefied remains were analyzed with nested STRs. The samples were first amplified using the IdentifilerTM Kit and the mini-STR primer set separately, using the optimal conditions for each as indicated in the IdentifilerTM User’s Manual (Applied Biosystems) and Butler et al. 20 (2003), respectively. The number of cycles for each reaction was increased from 28 to 50 to determine if any amplification was possible. A nested PCR cycling parameter test was also conducted using different combinations of cycles ((20,20),(30,30),(20,30),(30,20)). Following this, the samples were amplified using the nested PCR technique described above, with two 30-cycle amplifications. If PCR product was observed on a 2% agarose gel, the samples were set aside for analysis on an ABIPRISM® 310 Genetic Analyzer. The same method was used for the degraded blood samples and the 30 year-old bone samples. DNA isolated from the pipe bombs was tested with the mini-STRs in the same manner as previously discussed, along with reference samples that were also collected. The pipe bomb samples were amplified with the ldentifilerTM Kit for 50 cycles using the parameters found in the User’s Manual. The reference samples were amplified with both the IdentifilerTM Kit and the mini-STR primer set as per the standard protocols (User’s Manual and Butler et al. 2003). Two different volumes of DNA, 0.5 uL of pure extract and 1 uL of a 1:10 dilution of the extract, were both examined due to the fact that the amount of DNA present in the sample was unknown. For each set of amplifications, a 2% agarose gel was used to determine if the samples would be further analyzed using an ABIPRISM® 310 Genetic Analyzer. Mini-S TR Primer Set Allelic Ladder Preparation A mini-STR primer set allelic ladder was created as instructed in Butler et al. (2003). A 1:1000 dilution of AmpFESTR® IdentifilerTM Allelic Ladder was made and 10 uL was added to a PCR reaction mix containing: 10 uL buffer, 6 uL MgC12, 10 uL mini 21 A] primer set, 10 uL dNTPs, 1 uL AmpliTaq Gold DNA Polymerase, and water to bring the volume up to 100 uL. This reaction mix was then amplified for 15 cycles using the same thermal cycling parameters used for the research samples. The PCR product was run on an ABIPRISM® 310 Genetic Analyzer, using luL and 5 uL of product. Using the data produced from this run, a new panel (set of bin definitions for one or more markers) and bin set (basepair range and dye color that define an allele) were created in GeneMapperTM ID Software Version 3.1, using the Panel Manager, according to the protocol in the GeneMapperTM ID User’s Guide (Applied Biosystems). The raw data were used as a means to determine the allele basepair range of the locus, and each of the peaks that appeared within this range was assigned an allele call, according to the known alleles in the IdentifilerTM panel. The new panel and bin set were saved and used for analysis of mini-STR primer samples. Capillary Electrophoresis of Mini-S T R Primer Experiments Samples that produced mini-STR primer amplification products, visible on an agarose gel, were prepared for capillary electrophoresis on an ABIPRISM® 310 Genetic Analyzer. The analysis methods used with the multiplex STR and mini-STR primer experiments varied slightly. In samples in which nested PCR was conducted or only the mini-STR primers were used, each tube contained 19 uL formarnide, 0.75 uL GeneScan- 500 LIZ Size Standard (Applied Biosystems) and l uL of PCR product. As detailed in Butler et al. (2003), the samples were placed on the machine without denaturation, with a 5 sec injection at 15 kV and a 24 min separation at 15 kV. The temperature of the run was 60°C. A 47-cm x 50-um capillary was used with Genetic Analyzer POP-4TM 22 polymer and 1X Genetic Analyzer Buffer with EDTA (Applied Biosystems). For the samples that were amplified using the IdentifilerTM Kit, each tube contained 24.5 uL formamide, 0.5 uL of GeneScan-SOO LIZ Size Standard, and 1.5 uL of PCR product or AmpFCSTR® IdentifilerTM Allelic Ladder. These were heat denatured for 3 min at 95°C and cooled on ice for 3 min, then placed on an ABIPRISM® 310 Genetic Analyzer. The parameters on the instrument were the same as for the mini-STR primer samples, with a separation time of 28 minutes. The samples were analyzed using GeneMapperTM ID. The mini-STR primer set allelic ladder was utilized to analyze the mini-STRs, and the AmpFCSTR® IdentifilerTM Allelic Ladder was used for the IdentifilerTM samples. The GeneMapperTM ID analysis method for the mini-STRs was the microsatellite default method, with the HID__Advanced method for the Identifiler samples. The allele panel for the Identifiler samples was the AmpFLSTR_Panels_vl: Identifiler_vl , while the panel that was created, named mini primer panel, was used for the mini-STR samples. Both sets of samples were analyzed using the size standard setting CE_G5_HID_GSSOO (5-dye filter set using a 500 basepair size standard) and D-33 matrix standard file. 23 Results Sex Determination Using Amelogenin Nested PCR Parameter Optimizations In order to conduct experiments on the various samples, it was first necessary to optimize PCR parameters using known DNA (Promega: Male). The primers were tested in different combinations (LF/LR, BF/BR, LF/BR, BF/LR) using increasing cycle numbers (20,25,30,35,40). A PCR product was observed on a 3% agarose gel for each of the primer combinations at 30, 35 and 40 cycles. The primers were also tested at three different concentrations, 0.1X, 0.2X, 1X, with the best product resolution appearing at 1X. The next round of experiments consisted of testing different concentrations of DNA (lOng, lng, 0.1ng, 100 pg, 10 pg and 1 pg). DNA was successfully amplified down to 1 pg. The nested PCR technique itself was then tested using BF/BR as the external primers and LF/LR as the internal primers, at the different DNA concentrations. The same thermal cycling conditions were used as indicated in the nested PCR experiments (see Methods), however, the first round had 35 cycles and the second round had 30 cycles. Bands were present on a 3% agarose gel using starting DNA amounts down to 10 pg. The optimal parameters consisted of 30 cycles of PCR for both rounds of PCR, with a 1X concentration of primers, and 10 pg of control DNA. In the reagent blanks and negative controls that were run with each reaction, there were small bands observed on an agarose gel. One of these samples was run on a CEQTM 8000, and there were no peaks present on the electropherogram. Given this result, the bands were interpreted as background primer activity, as seen in Figure 3. However, not 24 all reactions contained primer activity, which was an indicator that they were possibly inhibited. Reactions that contained bands beyond those resulting from primer activity were used for subsequent analysis. Figure 3. Primer activity in reagent blank lane. The top arrow indicates primer activity. The bottom arrow indicates a band beyond the primer activity. If a band appeared in this area, this sample was sent on for further analysis. Lanes 2, 3, and 4 were sent on for analysis. Lane 7 is the known (positive control) DNA sample. Lane 8 is the reagent blank. A successful amplification was one that resulted in an electropherogram that clearly indicated whether the sample was male or female, utilizing the amelogenin primers. For instance, when the known samples were run, the resultant electropherograms produced a male profile with 2 peaks, one at 65 — 66 bp and the other at 71 — 72 bp, while a female would only have one peak at 65 — 66 bp, as seen in Figures 4 and 5, respectively. 25 .30me 2:82:25 > 2: 35839.. Eut 2: .5 Beta 2: can Amhmev 98:352.? X 2: 35339. fio. 2: .5 Beta 25. Ema.— one: a one <75 28.: as..-” 2:. .Gnfifiag 9.8.. ~ 5? £2: 585. 2:: a Baggage.» 83 Geo ... 95»: .. 88m Rm. nu . 8:3 . cocoa . 889 9598 8 8 E 8 8 9. 8 a 9 a _ A P o . . qiql «Irr— rlld‘ q d — .— d u 8 89.8 as???“ u was” an; 8.3 . u /./.\<. r 28 l 85 88 3.8. 3;...“ U 8.83.8 8.2 x I y 2.8 .....1 ocean m. ocean 8.2 ‘ m 83. 2.8 n o > . n. Baa a s 2 H m. r U - e. $ .1; 88° N U 26 Afimw «a :3.— oEemeEP—ao X 2: 953:2: mm 39:: 2:. 2:3 2.2. E on: <75 9327. Earn 25. .3568 :3.— 28 :3 Jim— oiEou 555— .? anemone—.3583 =25 GHQ .m Bang 95 £3 8 8 s. 8 mm 3.8 on. NM 0 F G IIl111LllllllllLlJllLLllllLLlllllllllllllLLlllllllllllllllllllllll r T 82: goon 88m 989» 880 825 82:. 8:8 083 . 889 can: 808— 808— irufiis Mo 27 Voegtly Bone Samples Voegtly bone samples were chosen for analysis based on the previous analysts’ mtDNA amplification success (Misner 2004). The PCR parameters that were optimized using the control DNA were then tested on anthropologically sexed male (burial 111 femur) and female (burial 448 femur) skeletal samples. The same parameters were optimized using these samples, and it was determined that the parameters for the known DNA were also applicable to the Voegtly samples. However, the volume of DNA used was variable among samples, generally being 1 uL of a 1:10 dilution of extract. Of the 68 samples analyzed, 12 produced an amelogenin profile. Originally, 27 of the bone samples were amplified using a standard Taq polymerase, of which 4 (167 femur, 545 femur, 111 rib, and 164 femur) appeared to amplify based on agarose gel electrophoresis. These samples were analyzed on a CEQTM 8000, and when no peaks appeared on the electropherograms, a primer and dNTP reduction experiment was conducted. Altogether, the amount of primer and dNTPs was cut in half without having any adverse effects on the amplification. The rest of the 21 Voegtly bone samples were then amplified using this reduction and the standard Taq polymerase of which 2 (047 pelvis and 529a rib) produced an amplification product. The possibility of PCR inhibition was explored by adding BSA to the master mix. The samples that did not originally amplify were tested once again, resulting in the amplification of one additional sample (192 pelvis). The Voegtly teeth samples (18 total) were then amplified using the standard Taq polymerase, of which no samples resulted in successful amplification. BSA was also added to these samples, which did not result in any additional amplification products. Samples that did not amplify using the above 28 methods were then tested using a hot-start Taq, and 5 more samples (47 innominate, 030 femur, 448 femur, 328 crown and 545 crown) were successfully amplified. These results can be seen in Table 2. Albania Bone Samples Hot-start Taq was also used to amplify the 17 Albania bone samples analyzed. The samples were amplified using an undiluted extraction product and a 1:10 dilution of this product. The 1164 petrous 1:10, 1165 petrous 1:10, 234 tooth, and 233 radius all generated amplification product and resulted in an electropherogram profile, as listed in Table 2. 30 Y ear-old Bone Samples The 30 year-old bone samples were also amplified using the hot-start Taq. Neither of the 2 samples produced an amplification product with the nested PCR technique. 3 Week-old Bone Samples Twelve different samples were tested (see Methods) using the hot-start Taq, of which five successfully amplified (MW DB, F B EDTA, FS EDTA, FW DB, and FW EDTA). The F W EDTA was tested on 3 different occasions and had a different electropherogram result each time, generating 2 male and one female profiles (Figures 6 and 7). These results are listed in Table 2. 29 Blood Samples The degraded blood samples were both successfully amplified using the hot-start Taq. The resultant electropherograms produced consistent male profiles, as indicated in Table 2. Pipe Bomb Samples There were two separate sample sets analyzed from the pipe bombs. The first were obtained from the pipe bombs that were detonated. Of the 20 samples that were tested using hot-start Taq, 9 were successfully amplified, as indicated in Table 2. Since there could have been bomb residues extracted along with the DNA, the possibility of PCR inhibition was addressed with the addition of BSA to the samples that were not successfully amplified. Three additional samples produced a profile. The second set of samples analyzed were the 9 reference DNAs (5 were not available). Of the reference samples, which were assumed to be uncompromised DNA, two samples did not amplify. The results from both sets of experiments can be seen in Table 2. 30 Table 2. Nested PCR results. The samples that produced amplification products, along with a profile, are listed under their respective subsets. The peak position(s) are indicative of the basepair size on an electropherogram. The sex determination was conducted after the electropherograms were examined, based on the number of peaks (1 or 2) and their location(s). (MW = metatarsal in water; FB = femur in bleach; FS = femur in sodium carbonate; FW = femur in water; DB = digestion buffer) Samples Peak Position(s) Sex Determination Voegtly Samples 030 femur 71.62 Male 047 pelvis 72.33 Male 111 rib 65.64 Female 164 femur 65.72 Female 167 femur 65.13, 71.99 Male 192 pelvis (BSA) 65.36 Female 448 femur 65.62 Female 47 innominate 65.51 Female 529a rib 65.55 Female 545 femur 65.28 Female 328 crown 64.98 Female 545 crown 65.31 Female Albania Samples 1164 petrous 1:10 65.32 Female 1165 petrous 1:10 65.38 Female 234 tooth 65.41 Female 233 radius 65.39 Female 3 Week-old Samples MW DB 65.44 Female F B EDTA 72.18 Male FS EDTA 65.66, 72.35 Male F W DB 65.47 Female FW EDTA (1) 65.52, 71.97 Male FW EDTA (2) 65.39 Female FW EDTA (3) 72.3 Male Blood Smles Blood D15 65.59, 72.26 Male Blood 37C 65.35, 72.23 Male Pipe Bomb Samples 26 (BSA) 72.41 Male 27 66.0 Female 29 65.84 Female 30 65.74 Female 31 (BSA) 72.49 Male 33 65.79 Female 36 (BSA) 65.78 Female 41 65.86 Female 42 65.92, 72.51 Male 32 45 65.69 Female 46 65.95 Female 47 65.74 Female Pipe Bomb References 110 65.73, 72.36 Male 11 1 65.71 Female 1 12 65.71 Female 1 13 65.73 Female 1 14 64.89 Female 115 65.86 Female 1 18 65.81 Female 72.3 65.37 I 65.3? 04.09 64.00 62.91' 81.68l r i r i 4.201 r r L j I ‘1 4 ppm L J r 50 60 7'0 Size (m) 50 BO 70 80 9i“ ("3) Figures 6 and 7. Comparison of electropherograms from FW EDTA. Figure 6, on the left, has one X peak present in sample FW EDTA at 65.37, while Figure 7, on the right, shows one Y peak at 72.30, also from sample FW EDTA. 33 Mini-ST R Primer Experiments PCR Parameter Optimization with Control DNA 994 7A The initial rounds of mini-STR primer experiments were conducted using control DNA 9947A (AmpFESTR® IdentifilerTM PCR Amplification Kit, Applied Biosystems). The first sets of primers (each of the primers listed in Table 1, along with the mini Al combination) were amplified with the control DNA for 28 cycles (Butler et al. 2003). No bands were seen on a 3% agarose gel. However, when the number of cycles was increased to 35, amplification was successful (Figure 8). The control DNA 9947A (100 pg/uL) was also amplified with the AmpFiiSTR® IdentifilerTM PCR Amplification Kit (Applied Biosystems), since these primers would be used in subsequent experiments (see below). When the DNA was amplified according to the manufacturer’s instructions (IdentifilerTM User’s Guide), there were no bands present. However, when the number of cycles was increased to 50, the sample appeared to have over-amplified, based on bands appearing in size ranges that were outside of the normal amplicon range. The 9947A DNA was then amplified using the nested PCR technique (two 30-cycle rounds), with the IdentifilerTM primer set for round 1 and mini A1 for round 2. This produced bands on a 2% agarose gel. These samples were set aside for further analysis on an ABIPRISM® 310 Genetic Analyzer. 34 Figure 8. 2% agarose gel of control DNA amplified with mini-STR primer sets, along with a 100bp ladder (on right). Each individual locus should have one band (if homozygous), or two close hands (if heterozygous) in each lane, and there should be multiple bands in the mini Al lane. The only lane without a band is locus D281338. The bands also appear to be between 100 and 200bp in size, which corresponds to their expected amplicon sizes. Mini-STR Primer Analysis of Voegtly Samples Two of the Voegtly bones (167 femur and 529a rib), successfully amplified using the amelogenin nested PCR technique, were tested with the mini-STR primer sets. These samples were initially amplified using the individual primer sets (Table 1), along with mini A], for 40 cycles, but no amplification products were present on a 2% agarose gel. The nested PCR technique was then tested, this time using IdentifilerTM primer set for round 1, and D1885], D16S539 and the mini A1, for round 2 (30 cycles each round). No bands were seen on a 2% agarose gel. Mini-STR Primer Analysis of Albania Bone Samples Albanian bone samples that were successfully amplified using the amelogenin nested PCR technique were tested with the mini-STR primers. Samples 1164 petrous 35 1:10, 1165 petrous 1:10, 233 radius 1:10 and 234 tooth were amplified using the nested technique and 30 cycles for both rounds of PCR. When these samples were run on a 2% agarose gel, the tooth from individual 234 produced bands. This sample was then re- analyzed using the same primer set for round 1, while using the amelogenin mini-STR primer set in round 2. A small band was produced on the yield gel and this sample was set aside for analysis on an ABIPRISM® 310 Genetic Analyzer. Mini-S TR Primer Analysis of 3 0 year-old Bone Samples The 30 year-old bone samples were amplified using the nested PCR technique with mini-STR primers, utilizing the IdentifilerTM primers in round 1 and the mini A1 primers in round 2 for 30 cycles per round. One sample produced a band on a yield gel, although it was not in the size range appropriate for this amplicon. Mini-ST R Primer Analysis of 3 week-old Bone Samples The femur and metatarsal cleaned in water (FW and MW) were the first 3 week- old bone samples analyzed, which were each amplified for 40 cycles using both the IdentifilerTM and mini A] primer sets separately. The DNAs amplified using both primer sets, producing bands on a 2% agarose gel. The samples that were cleaned in sodium carbonate (F S and MS) underwent the same experiment, using 30 cycles, resulting in no bands on the yield gel. The next experiments were conducted on the samples that were extracted in either DB or EDTA and all involved the nested STR technique, with the IdentifilerTM primers for round 1 and mini A1 for round 2. Samples FS EDTA, FS DB, FW EDTA, and FW DB were all amplified using the following cycling combinations: 36 (20,20), (20,30), (30,20), (30,30). The (30,30) and (30, 20) cycling combinations resulted in amplification for all of the samples, as did F W EDTA and FS EDTA at the (20,30). These samples were set aside for analysis on an ABIPRISM® 310 Genetic Analyzer. Samples FS EDTA, FS DB, FW EDTA, and F W DB were then tested using only one round of PCR, at both 28 and 50 cycles. IdentifilerTM and mini A] primer sets were both tested in this manner. After 28 cycles, there were no bands present on a 2% agarose gel for either primer sets. After 50 cycles, bands were present for both primer sets, except for FW DB amplified with IdentifilerTM and FS DB amplified with mini A1. These samples were also set aside for analysis on an ABIPRISM® 310 Genetic Analyzer. Samples were then amplified using nested PCR (30 cycles per round) with IdentifilerTM primer set in round 1, and the second round with individual mini-STR primer sets THO], D18S5], and D16SS39. Samples FB EDTA, FW DB, FW EDTA, MS DB, and MW DB were analyzed using this method, and bands were present for each sample on a 2% agarose gel. Once again, these samples were set aside for capillary analysis. Mini-S TR Primer Analysis of Blood Samples The blood samples, D15 and 37C, were analyzed using the nested STR technique. The first set of experiments involved nested PCR with IdentifilerTM primers for round 1 (30 cycles) and mini A1 (30 cycles) for round 2. Bands were present for both samples on a 2% agarose gel. The samples were then amplified again, but with each of the primers listed in Table 1 for round 2. The amplification products can be seen on the 2% agarose gel in Figure 9. Since each of these samples had bands, they were set aside for capillary analysis. 37 Figure 9. Blood samples amplified with nested STR PCR (individual primer sets), with the top bands (block arrow) being amplification products from round one. The lower band(s), one example indicated by small white arrow, are products from round 2 amplification with individual primer sets. Mini-STR Primer Analysis of Pipe Bomb Samples The pipe bomb samples were unique because they had reference samples, therefore, there was a means to compare profiles that were obtained. Using 50 cycles, samples 11, 33, 40, 44, 46, 47, 48, and 50 were amplified, each with the IdentifilerTM and mini A1 primers. They were also amplified using nested PCR, for 30 cycles each round. Both the nested and standard PCR amplifications resulted in amplification product, for both the IdentifilerTM and mini A1. Some samples, such as 33, 40, and 50, had variable bands present, indicating that amplification was not successful at all of the loci. Although some of the samples appeared to have amplified, they were not sent for further analysis due to time constraints and instrument complications within the research. The reference samples were then analyzed using 0.5 uL of pure extract and 1 pL of a 1:10 dilution of pure extract. The samples were amplified for 28 cycles with the IdentifilerTM primer set and mini A] primer sets. Both sets of samples were run on a 2% agarose gel, 38 and amplification products were observed for both experiments. The 1:10 dilution appeared to have amplified with a better, clearer product, so this dilution was set aside for capillary analysis. Reference samples 102, 113, and IIS did not amplify using the mini A1 primers. All amplified products from the reference samples were set aside for analysis on an ABIPRISM® 310 Genetic Analyzer. Capillary Electrophoresis on an ABIPRISM® 310 Genetic Analyzer Control DNA 994 7A The control DNA 9947A amplified with the IdentifilerTM primer set was run on an ABIPRISM® 310 Genetic Analyzer, however, a clear profile could not be developed. The control DNA 9947A that was amplified with the mini A1 produced a profile for all loci, consistent with the known profile. However, the profile had a few peaks that were unidentified (locus D18851) and several off-ladder alleles at all of the loci, except amelogenin. Mini-S TR Primer Set Allelic Ladder The mini-STR primer set allelic ladder also had to be analyzed on an ABIPRISM® 310 Genetic Analyzer. Two different amounts of ladder were added, lpL and 5 uL, and it was determined that 5 pL was optimal for good peak height and resolution. The results from this run were used as reference data to create a panel and bin set in the GeneMapperTM ID software. The details of this panel can be viewed in Table 3. Examples of the electropherograms produced by the mini-STR primer set allelic ladder can be seen in Figures 10 — 13. 39 STR Marker Dye Min Max Control Marker Specific Ladder Name Color Size Size Alleles Repeat Stutter Alleles ' Ratio 17 — 26, 26.2, 27 ~— 30, 30.2, 31.2, 32.2, FGA Blue 135.0 287.0 23, 24 4 0.15 33.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 50.2, 51.2 THO] Blue 57.0 102.5 8, 9.3 4 0.05 4 — 9, 9.3, 10, 1], 13.3 Amelogenin Blue 102.7 113.0 X 0.0 X, Y 7, 9, 10, 10.2, 11 — D1885] Green 104.0 190.0 15, 19 4 0.17 13, 13.2, 14, 14.2, 15 — 27 D16SS39 Yellow 73.0 123.0 11,12 4 0.1 5, 8— 15 D2Sl338 Red 87.0 150.0 19, 23 4 0.11 15 — 28 Table 3. Panel created in GeneMapperTM ID software. Headings denote the marker, or locus, name, dye color, minimum and maximum size in base pairs, control alleles (known for control DNA 9947A), marker repeat (number of repeats), STR specific stutter ratio (obtained from known IdentifilerTM panel; percentage of peak heights acceptable as stutter), and ladder alleles (all possible allele calls within the specific locus). 40 Figure 10. Mini-STR primer set allelic ladder, 6 — FAM dye, loci/markers TH01 (4 — 9, 9.3, 10, 11, 13.3), Amelogenin (X,Y) and FGA (17 — 26, 26.2, 27 — 30, 30.2, 31.2, 32.2, 33.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 50.2, 51.2), with all allele calls present. 41 mun-ll 1 110 120 130 140 150 160 170 180 190 .. WWm DMMEEEEEMIEEIEIEEEEEBEEIEJE Figure 11. Mini-STR primer set allelic ladder, VIC dye, locus/marker D18851 (7, 9, 10, 10.2, 11 — 13, 13.2, 14, 14.2, 15 - 27), with all allele calls present. 42 J1. .. small 100 100 15 1? 20 23 25 2? 16 19 21 24 26 28 18 22 Figures 12 and 13. Mini-STR primer set allelic ladder, NED and PET dyes, loci/markers D16SS39 (5, 8 — 15) and D281338 (15 — 28), with all allele calls present. 43 Albania Sample One sample, a tooth from individual 234, from the Albania skeletal material appeared to be successfiilly amplified. The sample was electrophoresed on an ABIPRISM® 310 Genetic Analyzer. There were no alleles detected when the electropherogram was examined. 3 Week-old Bone Samples The 3-week old bone samples produced amplification products for some of the samples using the mini-STR primer sets. The products from the cycling parameter experiments ((20,20),(30,30),(20,30),(30,20)) were analyzed on an ABIPRISM® 310 Genetic Analyzer. A full profile was not produced for any of the samples; however, three loci had consistent peaks among samples, which resulted in a partial profile. Sample FW EDTA had one X allele at the amelogenin marker for PCR combination (30,30), and had two alleles (19,20), at locus FGA for cycling parameters (20,30), (30,30), and (30,20). Samples FS DB and FS EDTA both had 2 alleles (4,6), at locus TH01 for the (30,30) cycling set. Blood Samples The blood samples, D15 and 37C, were from a known male, whose profile had been previously obtained. The blood samples produced consistent profiles, including TH01 (4), Amelogenin (X,Y), FGA (18,19), D1 8851 (23,24), and D2S1338 (22,23) (Figure 14). 44 Pipe Bomb Samples Alle1e(s) observed for the reference samples at the amelogenin locus were: 102 (X), 110 (X,Y), 11] (X), 112 (X), 113 (X), and 114 (X), which correctly correspond to the sex of the subjects involved. 45 .352: 252 a 8.2... 93583.. .2. 2...: 22: .835 mamas E; 5.3 Ema:— "312:2.93 £53.95, "2:355: .3 55 "10:33:. 2; 2:9:— w=_>5=8 2:. Ace—9:3 328.:— E3 29:: 2.: .8 =a .53 m:— 0383 953 me oEP—h .3 95m:— 7 114 <4: : . I: 5.: llsimwllfllfimfimtiang Im VA allilalll 38333:.L illlill llqllltilrhlllhlil.l.lllx 46 Discussion The goal of this research was to use enhanced PCR techniques, such as nested PCR and mini-STR primers for STR analysis, to amplify compromised nuclear DNA samples in which standard techniques often fail. By discerning if forensic samples can be successfully analyzed using these methods, scientists can determine which type of analysis can best be used on compromised samples that they may encounter. Analysis of Control Samples Utilizing the Amelogenin Nested PCR Technique A male, human genomic DNA control sample was utilized to optimize different parameters of the amelogenin nested PCR technique. As little as 10 pg of DNA produced a PCR product using this method, which was promising because not only was the DNA in this research assumed to be degraded, but if there was still nuclear DNA present, it was thought to be in very minute amounts (LCN). Increasing the cycle number from 30 to 40 allowed more DNA to be amplified, but also resulted in non-specific amplification of areas outside the target region (Figure 2). Based on the yield gels, it appeared that the primers were binding to each other (primer dimer) after 40 cycles, and these bands were beginning to overshadow the target DNA. Nested PCR aided in reducing primer dimer by using different primers (BF/BR and LF/LR) in two 30-cycle rounds of PCR, thus eliminating amplification of areas outside of this region (e.g., Figure 3). The nested PCR technique was then used with a known female sample (9947A, Applied Biosystems) and both control male and female samples were electrophoresed on a yield gel, with bands present in the target size range. Initially, the samples were analyzed on a CEQTM 8000 using both a 1:100 dilution and 1:200 dilution of PCR 47 product, along with a 400 bp size standard. The 1:200 dilution worked best based on the amount of background peak reduction that occurred (Figures 4 and 5). The 400 bp size standard was determined to be too large for accurate sizing; since the target amplicon size was less than 75 bp, an 80 bp size standard was utilized instead. The control male sample electropherogram had two peaks, one at 65 — 66 bp (X chromosome) and the other at 71 — 72 bp (Y chromosome), while the female had one peak at 65 — 66 bp (X chromosome). Analysis of Control DNA 994 7A Using Mini-S T R Primers Based on the parameters in Butler et al. (2003), the control DNA was amplified for 28 cycles using the mini-STR primers, but there were no bands present on a 2% agarose gel; when the cycle number was increased to 35, amplification products were seen. In these experiments the reaction volumes were decreased from 25 to 10 pL, along with the amount of input DNA, from 1 ng to 0.4 ng. Since the amount of input DNA was reduced, it appears that more cycles were necessary for the control DNA to be amplified. It is also possible that smaller reaction volumes lead to non-optimal concentrations of reagents and DNA, making amplification more difficult using a low cycle number. The control DNA was also tested using the nested STR technique. Two 30-cyc1e rounds of PCR were conducted, and bands were present on a 2% agarose gel. When the product was analyzed on an ABIPRISM® 310 Genetic Analyzer, a profile that was consistent with the known (9947A) was produced; although, not all of the loci had identifiable peaks. There were numerous off-ladder alleles, most likely from the first round of amplification with the IdentifilerTM primers. With the mini-STR primers, amplification occurs over 6 loci while the IdentifilerTM kit amplifies 16 loci. The off- 48 ladder alleles generally occurred beyond the target size of the mini-STRs, falling into regions of IdentifilerTM loci, such as CSF 1 PO and VWA. Although they were not analyzed using the IdentifilerTM allelic ladder, they do correspond, in terms of size and location, to alleles produced from the first round. Since the primer sets (mini A1 and IdentifilerTM) have never been tested together, it may be beneficial to future analysts to clean up the product from the first round using a filter device, such as a Microcon, to eliminate the first round primers and products. An Overview of Degradation and Contamination within Research Samples The samples utilized in this research were all subjected to conditions that facilitate DNA degradation. The decay of post-mortem DNA can result in strand breaks, generally due to hydrolytic damage, which may fragment the DNA into smaller pieces. Crosslink formation can occur from alkylation, which in turn prevents endogenous template molecule amplification; oxidative damage to the DNA may result in lesions that block the DNA polymerase, while hydrolytic damage of bases produces miscoding lesions resulting in the incorporation of incorrect bases during amplification (Willerslev and Cooper 2005). When bones or other biological material are collected from a burial or crime scene, additional matter found with these items may also be removed. For instance, when bones are recovered from soil, there is generally a certain amount of soil carried along with them. When the bone is ground and the DNA from within extracted, components within the soil, such as humic acid, are extracted along with the DNA and can act as inhibitors of PCR. According to Tsai and Olson (1992), a mere 1 nL of a “humic-acid- 49 like extract” (concentration not stated) is enough to completely inhibit the polymerase activities or binding of the primers in a 100 uL reaction mixture, regardless of large amounts of initial template added. In this research, when primer activity was not present on an agarose gel, the possibility of inhibition was examined. The addition of BSA, which is thought to bind the inhibitors and may even protect the T aq polymerase (Kreader 1996), as a component in the PCR reaction successfully reduced the inhibition in certain samples (192 pelvis and pipe bomb samples 26, 31 and 36). Since the BSA did not make a difference with the remainder of the samples, it appears that inhibition was not the problem. Inhibition is not the only obstacle to overcome with degraded samples. Contaminating DNA is problematic in any forensic setting. Since DNA from compromised samples is often highly degraded, generally no larger than 200 bp (Kelman and Moran 1996, Opel et al. 2006), and the target fragments are in low copy, it is easy to see how any trace amount of contaminating DNA could overshadow the amplification of these types of products. Ladd et al. (1999) found that small amounts of DNA can be transferred from a person to an object yielding interpretable DNA profiles. In dealing with compromised samples, whether they are degraded or LCN, the occurrence of any contaminating DNA, located on tubes or other items used during the amplification preparation, can be detrimental to the experiment, resulting in false positives or mixtures. Pipe bomb sample 42 is an example in this research where a male profile was developed, when the bomb handler was a female, presumably resulting from contaminating DNA. As a safeguard, it is ideal to have the profiles of everyone who may come into contact with the samples on hand, although this may be difficult, if not impossible in some cases. 50 Analysis of Voegtly Skeletal Samples The Voegtly bones, which were the second oldest of the skeletal samples, were studied most extensively. Initially, the objective was to determine the sex of the bones, and confirm or refute the anthropological conclusions about sex. However, not all of the DNAs from the samples successfully amplified, thus the goal became to determine what technique, if any, worked best for sexing the material. Since the bodies were buried in wooden coffins that deteriorated over time, this left them exposed to microorganisms and different elements within the soil. When the bones were recovered, many of them had either black deposit or green discoloration (Ubelaker et al. 2003); the black deposit was determined to be manganese from constant contact with the soil, while the green discoloration was most likely due to contact with different copper artifacts that were found within the gravesite. Copper has the ability to bind to phosphate in the DNA backbone and also the bases (Eichhorn and Shin 1968), which causes breaks in the DNA or prevents the primers from annealing during PCR. Manganese can be either beneficial or detrimental to DNA depending on whether it is found in high or low concentrations. At low concentrations, it can stabilize DNA conformation and can interact electrostatically with the phosphate backbone; however, at high concentrations, it destabilizes DNA secondary structure and disturbs the hydrogen binding of base pairs because it binds to bases with high affinity (Ma and Bloomfield 1994). Manganese can also affect the fidelity of DNA replication by modifying the activity of the DNA polymerase, interfering with the extension phase (Gerber et al. 2002). This staining may have had an effect on the state of the DNA in the bones in this research, whether it caused mutations, or interfered with the PCR process. 51 DNA from 32 individuals (skeletons), with multiple bones from each (68 samples total) was analyzed. Twelve of these samples successfully amplified (Table 2), generating 3 male and 9 female profiles. Of these profiles, 5 corresponded to the sex estimated by the anthropologists, and 7 contradicted their estimation. Sample 47 (pelvis/innominate) was amplified twice and produced 2 different results, one female and one male, with the electorpherogram for the male profile having only one peak at 72.33 bp. If this sample is indeed male, then there appears to be dropout of both the X and Y chromosome in the two different experiments. Results such as these simply reinforce the importance of repeating experiments to ensure that the profiles obtained are authentic. According to the anthropologists, the remains were from a child, had black staining, and female sex was suggested based on an earring that was recovered with the remains (Ubelaker et al. 2003). Since there was black deposit on the bones, it is possible that manganese interfered with the extension phase, causing one chromosome to dropout in one amplification and the other to dropout in the next. However, the results may also be due to stochastic effects. The addition of BSA to Voegtly DNAs that did not originally amplify only resulted in one additional amplification (192 pelvis), indicating that BSA did not aid in eliminating inhibition. Since previous mtDNA amplification was successful in all samples tested for this study, it appears that inhibition was not a factor. The use of hot- start Taq increased the successful amplification rate, resulting in five more amplifications. However, two of the samples that typed as male (030 femur and 047 pelvis) only had a peak in the 71 — 72 bp area. Since the Y chromosome peak occurs in this area, these samples were included with the successful amplifications. The allelic 52 dropout of the X chromosome peak is most likely due to stochastic effects. The sex estimation of these samples was based on large bone size, general pelvic and skeletal morphology, skull features (mastoid process and supraorbital ridges) and artifact recovery (Ubelaker et al. 2003). Since general features and large bone size are not very reliable techniques for anthropological sex estimation, it is quite possible that the anthropologists’ estimations were incorrect. Amplification of the Voegtly bones with the use of mini-STR primers was then attempted; however, no product was observed on any of the yield gels. If mutations occurred in the primer binding regions, then this affected the ability of the primers to anneal to the template. The bones themselves were very weathered, and the DNA within was almost certainly degraded to the point where nuclear amplification with STRs was impossible. Analysis of Albania Skeletal Samples The oldest of the skeletal samples analyzed in this study were obtained from a tumulus in Albania. The four samples that produced viable amelogenin nested PCR electropherograms were anthropologically sexed as a 15 — 17 yr old male (1164), a 14 — 16 yr old female (1165), a young adult female (234) and an adult male (233). According to the anthropologist (T. Fenton, personal communication), the bones that are typically used to estimate sex were either missing or damaged, making it very difficult to determine if remains were male or female, thus leading to the prospect that the estimation was inaccurate. The four samples that produced a genetic profile all typed as female. The bones were introduced to factors that promote degradation, such as microbial attack 53 in the soil, which may have reduced the length of the DNA to less than 70 bp, and since the larger allele (Y) occurs between 71 — 72 bp, it did not amplify. If the anthropological sex estimation was correct, it appears that the DNA was basically too degraded to completely amplify. The tooth from individual 234 was the only Albanian sample that may have amplified using the mini-STR primers through nested PCR, based on the location of a band on the yield gel. However, when this sample was analyzed on an ABIPRISM® 310 Genetic Analyzer, no alleles were detected at any of the loci. The age of these samples, having been buried for over 2,000 years, leads to the possibility that there is no nuclear DNA remaining in the bone material. If there is any nuclear DNA present, it has been degraded, possibly by oxidative or hydrolytic damage, beyond the point of amplification. As stated previously, it may also have mutations that essentially prevented the primers from annealing to the primer binding region. Analysis of 3 0 Y ear-Old Bone Sample No results were obtained from the 30 year-old bone sample; neither the amelogenin nested PCR nor the mini-STR primers produced an amplification product. The bone was obtained from an exhumed body, and it is plausible that the embalming fluid that was used over 30 years ago had an effect on the degradation of the DNA in the bone, although mtDNA had been amplified from it previously. MtDNA generally has a higher success rate in terms of amplification because it is robust, located within the protective mitochondrion (Foran 2006), and plentiful in number, with typically more than 1000 copies per cell (Holland and Parsons 1999). PCR did not appear to be inhibited, 54 since primer activity was observed. Embalming fluid does contain formaldehyde, a known mutagenic agent, which at high concentrations can change the transforming activity of DNA; according to Zamenhof et al. (1953), formaldehyde has the ability to break hydrogen bonds and reacts with primary amino groups, causing a decrease in the molecular weight or even the DNA to collapse altogether. Formaldehyde can also increase the action of denaturing agents, such as heat, by lowering the melting temperature; after the DNA undergoes denaturation during PCR, the interaction with the formaldehyde prevents it from renaturation (Feldman 1973). Analysis of 3 Week-Old Bone Samples The 3 week-old bone samples provided some of the most variable results. A femur and metatarsal were obtained from a body that had been exposed to the elements for approximately 3 weeks, and then cleaned, and the DNA extracted using both high EDTA and normal digestion buffer in organic preparation. The different methods in which the bones were cleaned and the DNA extracted also introduced additional DNA recovery factors. According to Rennick et al. (2005), the bleaching technique reduced the yield of mtDNA, while the detergent/carbonate method produced the greatest yield of mtDNA. In this research, DNA was successfully amplified from MW DB, FB EDTA, FS EDTA, F W DB, and FW EDTA. There was no observable correlation between the cleaning technique and amplification success using either nested or mini-STR primer technique, since DNAs from each of the cleaning techniques successfully amplified. The extraction method, DB or EDTA, was also not a factor, with DNA from both methods amplifying. 55 The FB EDTA was cleaned with bleach, and when electrophoresed, produced a male profile, with one peak at 72.18 bp (Y). Since stochastic effects are common with degraded DNA, it appears degradation did occur, but it cannot be determined as to whether it was during exposure to the elements, or during the cleaning process. Rennick et al. (2005) indicated that the sodium carbonate might aid in protecting the mtDNA within the bone from further degradation; however, in terms of nuclear DNA experiments, only one sample, FS EDTA, resulted in amplification. There was a clear male profile developed, with both peaks present, which could indicate that the sodium carbonate protected the DNA from further degradation during the cleaning process. Since the bones cleaned in water (FW DB, F W EDTA, and MW DB) represented 60% of amplification success, this could be considered the optimal cleaning technique, however, there were inconsistencies in the sex determination of these samples as well. FW EDTA was amplified three times using the nested PCR amelogenin technique, and each time different electropherogram results were produced. This particular sample typed as a female (X), a male (X,Y) and a male with one peak (Y), indicating possible allelic dropout of the X and Y peaks, most likely due to stochastic effects. The femur and metatarsal samples were also amplified using the mini-STR primers and nested PCR. A full profile was not obtained for any of the samples. Bone F W EDTA produced an (X) at amelogenin and (19,20) at FGA; samples FS DB and FS EDTA both had 2 alleles (4,6) at locus TH01. If the data obtained from these profiles were combined, then 3 of the 6 loci had reportable alleles. This result is unique in itself, due to the fact that these are both on opposite ends of the scale in terms of amplicon size. The FGA locus is the largest amplicon produced with the mini-STR primers, while TH01 56 and amelogenin are two of the smallest. Based on the size of the fragments obtained in their commercial STR v. mini-STR experiments, Opel et al. (2006) suggested that the degradation cut-off length of the DNA template generally occurs around 200 bp. It is possible that the FGA region amplified by chance (preferential amplification) in the early rounds, resulting in callable alleles at this locus. The 3 samples that produced a partial profile were cleaned in water or sodium carbonate. The mini-STR primer technique did not produce any results with the bleach-cleaned bone, which could indicate that the bleach had an adverse effect on the DNA during cleaning, although this did not appear to be the case when using the amelogenin nested PCR technique. It is also worthy to note that results were only obtained from the femur and not the metatarsal, although it is not known why this is the case, since the size of the pieces that were cleaned were similar (Rennick et al. 2005). Analysis of Blood Samples The degraded blood samples were tested using both the amelogenin nested PCR technique as well as a single round of 40 cycles. Upon examination of the electropherograms from blood samples D15 and 37C, a male profile was obtained for both. Since the samples were not exposed to extreme heat conditions, it appears that their analysis would not require more involved PCR procedures. At 40 cycles, there was non- specific amplification that produced background peaks, but when nested PCR was conducted, these background peaks were eliminated. The nested mini-STR primer technique was also successful on the blood samples, which produced a profile that was consistent with that of the known donor. However, 57 when examining the electropherograms, extra peaks were observed. Most of these were outside of the mini-STR allele size range. As seen in Figure 14, the extra peak positions in the D1 885] area around 230 bp (homozygous peak), 275 bp and 350 bp (both heterozygous peaks) are consistent with alleles at ldentifilerTM loci D13S317, D16S539 and D2S1338, respectively. Therefore it seems that these peaks are product from the first round. Some of the others were simply stutter or pull-up. For instance, in this same figure, there is pull-up in the blue spectra (top frame) from the (23,24) alleles in the green spectra (second frame). Analysis of Pipe Bomb Samples When analyzing the DNA obtained from the pipe bombs, DNA degradation undoubtedly resulted from the extremely high heat generated by the smokeless gun powder, which according to Esslinger et al. (2004) can be in excess of 2000°C. The samples were analyzed using the amelogenin nested PCR technique in an attempt to amplify DNA that may or may not be present after a detonation. Compared to the results obtained from the skeletal materials, this technique was fairly successful. Nine of the 20 samples produced a profile, only one of which did not correspond with the sex of the subject who handled the bomb. As previously stated, pipe bomb 42, which was handled by a female, produced a male profile, which was most likely the result of contaminating DNA. Three additional samples were amplified with the addition of BSA (26, 31, and 26). Two of the 3 BSA samples resulted in a male profile; however, samples 31 and 26, both assembled by males, had only a Y chromosome peak (71 — 72 bp) on the 58 electropherogram. The BSA appeared to have removed the inhibition; however, stochastic effects still occurred, with the dropout of the X peak. The bombs were only handled for 30 seconds (Gehring 2004), indicating that if there was DNA present, it was in LCN. The analysis of LCN DNA may also result in allelic dropout, as seen in samples 31 and 26, with the dropout of the X chromosome peak. Another stochastic effect, peak imbalance, is also typical with LCN, since there is so little DNA present, one allele may be amplified by chance in the early rounds, causing the height of one allele to be much larger than another. The advantage with these samples was that the actual sex of the handler was known. Therefore, if only an X peak was obtained from a sample that was handled by a male, allelic dropout of the Y peak was evident. The DNAs obtained from the pipe bombs were also amplified using a single round of PCR (40 cycles). The single round amplification (Figure 8) resulted in high background noise (non-specific amplification) in sample 26, which was eliminated when the sample was amplified using the nested PCR technique with the addition of BSA (Figure 9). However, along with the removal of the non-specific amplification, stochastic effects arose with allelic dropout of the X peak. Based on the data compiled in this experiment, it appears that nested PCR is the most usefiil technique for analyzing DNA samples obtained from pipe bombs, which is consistent with the findings of Gehring (2004), who had to use semi-nested PCR in order to successfully amplify mtDNA from the pipe bombs. 59 Conclusions The results of this study indicate that nested PCR and mini-STR nuclear DNA testing techniques can be useful for analysis of forensic samples when standard methods fail, but perhaps only to a limited degree. The success of the techniques was variable among the sample sets. The amelogenin nested PCR method should be utilized when dealing with skeletal samples, regardless of whether they are merely a few weeks old or thousands of years old, as it showed the ability to aid in sex estimation for ancient burials. However, different artifacts may arise with this method. Stochastic effects were a factor with the majority of the samples that did successfully amplify. There was a constant concern of allelic dropout, which made it difficult to accurately determine the sex. Five of the twelve samples that produced a male profile had allelic dropout of the X peak. Allelic dropout of the Y peak can also occur, which is problematic since an electropherogram with only an X peak would be considered a female profile. Peak imbalance was another frequent occurrence in the electropherograms. The pipe bomb DNAs, being touch samples, were LCN and the DNA was exposed to excessive temperatures, a condition that is favorable for degradation. With these DNAs, if both the X and Y peaks were present, the X was much higher than the Y, which indicated that the smaller allele (X) was more successfully amplified, causing the height of the X peak to be larger than the Y. These artifacts need to be taken into consideration when dealing with compromised samples, whether they are degraded and/or present in LCN. As long as multiple amplifications are conducted and consistent results obtained, then the results can be deemed authentic. 60 The mini-STR primers were not successful at amplifying any of the older skeletal materials, so this technique would most likely not be useful when dealing with ancient DNA. The success of the mini-STRs with the degraded blood samples would lead one to believe that this method should be explored when encountering DNAs exposed to small increases in temperature that can occur when forensic samples are improperly stored, such as in property rooms where the temperature can vary. In cases of mass disasters, such as the World Trade Center bombings, only minimal human remains are generally found, and these can be severely degraded from excessive heat exposure, therefore this technique could be very useful in making positive identifications. The mini-STRs Butler et al. (2003) and Opel et al. (2006) experimented with produced amplicons no larger than 300 bp. Butler et al. (2003) had amplification success on blood samples that were stored at room temperature for 14 to 15 years, while Opel et al. (2006) successfully amplified DNA from human skeletal remains that were exposed to different conditions for varying lengths of time. The use of nested PCR with mini-STRs has never been attempted before, and with amplification success amongst some of the samples in this research, it potentially offers a new method of STR analysis on compromised DNA. Further research involving this method should be explored. 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