2.3:. s§‘t¢o lltlzz$019 x ullzhtttcvl I» I . 3. ii... If . .. butaax l‘ t‘rH.V . x 5...“... ...i 3 Snug-‘1‘...» 5.3:} 1 I. 3... A .5... I “W¢% D 1.5.9:! I... , ‘22:)..33, .\4. 1.3.3153. u I 3.1!), «:5 :3... fa. lbs”? «an: a... . in! 0 .$>:5: . . firth“. LDIADt 5“ to! Era . .r... 8». itwnmajh.“ u Vkmflfl .41. .223). i . .. .539: , ’5‘ -. 3.. 1.... x . v. t . .2 3.3..‘.52....... .. gr...- . . Fl.ulo.»:§al.a . v . .11... a! .ll’l v. ‘1 . I Hi; ii‘.‘ afi§ittfi .2 p! 1131.1... . . ‘ .. Ram. .uawfiréwkémfinfiz gas... 7 This is to certify that the thesis entitled USING STR ANALYSIS TO DETECT PRIMARY DNA TRANSFER presented by Tara Ann Reinholz has been accepted towards fulfillment of the requirements for \MLdegree in W03 with Specialization in Forensic Science Date b l 0-7539 M50 is an Affirmative Action/Equal Opportunuy Institution Ll 63AM Mich i9 ear; State U n iversity l E11 I BOX to remove this checkou I: from _y PLACE m R AVRng FINES return on or before TO M our l‘ECOrd. Clare due i f"(i‘QIJesterL DATE DUE DATE DUE VV‘ DATE DUE 6/01 cJCIRClDateDue.p65.p_15 “x USING STR ANALYSIS TO DETECT PRIMARY DNA TRANsFER BY Tara Ann Reinhol z A THESIS Submitted to Michigan State University in partial fulfillment of the rec“l for the degree of irEmente MASTER OF SCIENCE School of Criminal Justice 2002 ABSTRACT USING STR ANALYSIS TO DETECT PRIMARY DNA TRANSFER BY Tara Ann Reinholz In this study, the primary transfer of DNA from a person’s hand to an object was explored. A hammer with an unfinished wood handle was used as a test object, and holding times were varied l) to determine if a relationship existed between holding time and the amount of DNA recovered and 2) to evaluate the sensitivity of the testing nethod. An AmpFlSTR®>Profiler Plus Amplification Kit and the ABI Prism®>310 Genetic Analyzer, equipped with Genescan®>2.0.2 and Genotyper®>2.0, were used to generate DNA profiles. In addition, two new procedures were developed in an attempt to increase the sensitivity of the testing method. The results Show that primary transfer of DNA to this type of surface is common. It is possible to generate complete DNA profiles from holding times as low as 5 seconds, and no apparent connection exists between holding time and the amount of DNA recovered. The new Procedures proved useful in increasing allele detection but, occasionally, heterozygote peak imbalances of true alleles were observed. ACKNOWLEDGMENTS First, I would like to thank my professor and advisor Dr. Jay A. Siegel. His knowledge, guidance, and commitment to forensic science have Shaped me into the scientist that I am today. This project would not have been possible if not for his extraordinary patience and support. Second, I would like to thank the entire Biology Unit at the Michigan Department of State Police Forensic Laboratory in Northville, Mi. Lynne Helton, Heather Spillane, Leslie Nixon, Dorothy Martus and Guy Nutter all played an instrumental part in my research from the very beginning. Without them, I would not have gained the knowledge or had the resources needed in order to carry out my project. I want to offer a special thanks to Heather Spillane. Heather’s suggestions and ideas created the backbone of my project, and her time, knowledge, experience and incredible patience allowed me to successfully carry it out. Without her, and the rest of the unit, none of this would have been possible. Third, I need to thank all of the individuals who were a part Of my sample population. Without the donation of their time, the Significance of the results obtained in this study would have been severely compromised. iii TABLE OF CONTENTS List of Tables v List of Figures vi List of Abbreviations vii Introduction 1 Review of the Literature ' 9 Materials and Methods 12 Results and Discussion 39 Conclusions 64 Suggestions for Further Research 65 References 66 Appendix A - Electropherograms generated from decontamination testing 72 Appendix B — Electropherograms generated from known DNA samples 76 Appendix C — Electropherograms generated from 5, 15, and 30 second samples 90 Appendix D — Electropherograms generated from samples containing 3ul of PCR Product 136 Appendix E - Electropherograms generated from samples containing 3ul of concentrated PCR product- 152 Appendix F - Electropherograms generated from 168 ladders and quality control samples ......... iv LIST OF TABLES DNA . marl Table 1 - Applied BlOSYStemS Quant iBlOt® H” QuantitatiOn Klt Contents. Profiler plus PCR Table 2 —— Applied Biosystems AmpFlSTR® Amplification Kit: contents. . iler Plus Table 3 — Applied Biosystems AmpFlSTR® PIGi loci. Table 4 - 310 run parameters. Table 5 — Stutter percentages. Table 6 _. First round quantiblot IesLths __ Second round quantiblot Table 8 — DNA profiles Of known Samples Table 9 — DNA profiles of 5, 15, and 30 &%Q 1e8- Table 10 — Profile types of 5, 15, and 30 0nd Sam? Table 11 — DNA profiles of samples USing 3 econd samples. PCR product. K11 of Table 12 — DNA profiles Of Samples using 3 of concentrated PCR product, Lll Table 13 - Profile types of samples using 3111 PCR product and 3ul of concent:bat8dc>;C product. R Figure 1 Figure 2 - Figure 3 - Figure 4 ~ Figure 5 — Figure 6 — Figure 7 — LIST OF FIGURES DNA struc : ure. Polymerasez chairl reaction- Capillary electrophoresis- Yield gel interpretation- Quantiblot interpretat i on - - ers- Genescan® analysst paramep Laboratory deSign. vi ‘ .m' l._\“ w 1.“ MIA ,. l p f o .;. "\ A-S '- A-lS - A- 3 O - AS CLD — CE _ DNA __ FBI - ILC — LCN —— MSP — ng - PCR —- QA _ QB _ QC _ RFU STR SWGDAM - TAE TE TWGDAM _ ul - LIST OF ABBREVIATIONS l ndi'v'iailual . . n i letter deSlgnation “A" glven to a who took part in the study individual “A” / 5 second hOld individual. “A” / 15 second hold individual. “A” / 30 secol’ld hold tor Dire American Society of Crirne Labora capillary electrOphores i5 dOXyribonucleiC acid Federal Bureau of Investigatiofl iI'Iternal laboratory contrOl low copy nurnber . 08 Michigan Department of State POl )— nanOgram Polymerase Chain react ion picogram quality assurance gnantiblot Quality ContrOl relative fluorescent Lll‘iit short tandem repeat Scientific working grOL1p on DNA anal methods YSI Q trig/acetate/EDTA tris/EDTA Technical working group on DNA '31") 31y . 81 microliter S meth (Dds vii 11' I NTRODUCT I ON DNA (Deoxyribonucleic acid) represents one makeup. Located in the nucleus of cells! for how the human body funCtions, as Well as DNA it i5 Forensicalxy tec‘fiflo this 0 ' 5% di f ferenCe that is import: ant _ TOéaY' ‘ 3x30 «“3 appearance . Approximate 1y 9 9 . 5 % o f the identical 111 all human rpeifnas [131 makes it possible to detect these di 0 . . . . ne indiVidual to be d1. fferentiated from a .96 . (,6 ,0 6 Common sources of nuclear DNA lnClude Why 0 A36 cells .300 Cf, r spermatona, tissues and hair rootS. 9 O 9' biological con - eé OI stituents are commonly encount: €335 COOXi D16 scenes, DNA has proven to be an eEritrernely va lag the criminal justice system. Source. _ . . A hammer Wlth an unfinisth wood ha d1 5’ DNA b a ' - . , e w Y n individual for a SpeCific amQunt of tin Q sample Wa e’ a!) field S Collected from the handle Th' t f . lS tYpe a ran S Er, DNA from ones hand to an Object i of I S as a ‘ I‘ primary transfer. Before the next ind . eferred l jdu t a1 held Onto the handle, it was decontaminated in O eliminate a bGEr my DNA left over from the previQ to Lls hOlder. Choosin 9 t0 decontaminate before each hold $11- m e J‘I‘lated the A]? 161 ant. need to use separate hammers for each part p 0 W' for C - - hosen unfinished wood surface Was speCificallY c on m com - many reasons. One, this substrate 18 found Or1 on5 household items that could be used as weap d knives, baseball bats, etc. Two, un’fln oecu . mote rougher surface Which could p0881bly pro . {a 8 of DNA transfer. A DNA profile was gene P» 019 fl . . , know sample and compared to the indiVidualS . 0V8 "O o the two profiles matched: this would Pr transfer of DNA to this type of subst . 0 8 This reSEEarch has the potentlal to p . 6 a“ a Knj’fi 0‘9 area of forensic evidence. For example: . . w“ dany crime scene exhibiting only a partial finger? 559 roc could not be identified, WOUld nOt usually be P further_ BY incorporating the procedures pr Cposed Jill HHS study, the knife could become a very probati—Ve pieQ . G evidence. The appearance Of the partial prL rut Pro Of an individual did, in fact, touch the handle of U3 88 “Pat Q BY C01lecting a sample from the handle, a DNA PrOf knife 1'16 ' be generated and a possible SUSPeCt could b3 , co lden , “Id In addition to opening a new area of fgr ied. Ens. . 1c ev1dence, this study explores the need to acid res . . . s . of contami nation The POSSlblllty Of detth. the lSsue lng Dr imary DNA transfers reinforces the importance of weabi r19 gloves 7. T176 during the collection and handling of all eVldenCe- tefltial - . - ' the PO forenSlC seience community needs to reallze ‘tivitY eflsl for contamination to Occur. Wlth the lncre cies t agen an of the current testing methods, law en «161 o e . t and laboratories need to fOllow the higheg oi the in order to insure the reliability a testing performed. PolymeraseW ‘ €569 6” _ C DNA 18 a double st: randed mo]_EBC2Llle tha 6 itfi nucleus of the cell. It: is composed of 4 d 6 Adenine (A) , Thymine (T) , Guanine (G) I It is the specific order of these bases along 66 ps3 strand that dEtermineS one’S genetic makeup- one Figure 1’ two of these bases Specifically at: Each to another to fOrm a base pair [19] ~ A binds with T binds with C, It is estimated that the humaI—j genome Consists of over three bill ion of the Se 13 ‘38 [15] . pairs AS mentioned earlier: apprOXimatelY 0~ 3 9.. Q of ' . t bllllon baSe pairs differ from one person to he 3 t: be next_ and Polymorphi Sm is a short: tandem repeat, or ST. One tYpe R These differences are called p01ym0rphisms, 0f 7 . ‘ 8 base pair repeats thalt Occur along a DNA TRS are 2‘ trand [4] . Structure of DNA backmniese Base .hala ”#6‘ ?T\\__._~\N‘_ Hydrog e :n ban “is Figure 1 _ DNA structure- M ' e the Forensically, the tetranuCleotlde repeats ar current areas of intereSt. For example. core ‘5 or locus, on a DNA strand, an STR c20111d exl exist alleles, to repeat unit of TAGC. Different forms. or amongst the population, thus allowing f0 be differentiated from another. One p8 ext '0 30 allele (TAGC repeated 30 times) 40 allele (TAGC repeated 40 times) ° . m. rue longer than the other, this type of referred to as a length polymorphism- 096 9 as two pu‘ 6&6 The POlYmerase chain reaction h (0 forensic DNA analysis. One, is to ampllfy' 01; millions of. copies of the particular STR Of doing this, DNA that has become decomposed due to environmental insults has the pOssibilitY viable sample. Also, very small Samples can be €115l Two, is to attach a fluorescent label to the STR fQ . detection by a laser- By using Primers iii-199% d with ‘5 different Colored labels, many STR locations DNA strand can be amplified at one time, thus ’ much higher power of discrimination. FigurQ the process of pCR [22] Ca illar Electro hores iSQCE) of Dec: M Polymerase Chain Reaction ‘. firegion 0f irzeresz. D131 .‘5 dena tured. 9r;.-.:r MLLL toeach Strand. Pr Lnev. B213: strand is syn hes; zed uehm n31 primers an e1 :h ....r: 2:: srari. - ”I. I'll-“III IL" l-uulilflflllll 0"“ . V 1. do Another roundztrlé is Esther Eclmdimiip- is Ana: he: roundi: r3: denatured, {gr-mfg are denature" , primrs are d: natured, p." .1: 3:5 atzached, and he attached: and :he G333 iE-d and the. nut-her of [3:92, nutter oi Eli-‘4. number of Jr’s-"A \’ Strands are doubled. strands are d‘mmd' 3-8170, 5555‘ d3 11b .. ‘)Jd ‘(jw 50 " '.“‘fl r. &; L‘\ . ,; f?‘ " "Nur‘g ngw" ‘ch‘m‘tc IC 111165 of arr.c-Lif;ca:;cr. 5‘... "I 91““ " Large number s of 34.1..." ~. fraqmerfis' Each -t---ha, r M? ' . " "3315' -' Lagma- 301211325 the Emmi-CE “i r S Figure 2 \ Polymerase chain reaction. 6 H) Electrophoresis is defined as a technique used for the separation of molecules by means of electric current [1]. Molecules that have similar charges but differ in size can be separated using this technique. Since DNA is negatively charged and the STRs vary in length from one person to another, this makes capillary electrophoresis a perfect technique to use for STR analysis. As diagrammed in Figure 3, a sample is pulled up into the capillary and the applied electric current allows the negatively charged DNA fragments to travel from the negatively charged cathode to the positively charged anode [6]. As the fragments travel through the capillary they are separated by size. The smaller sized STRs reach the anode first, followed by the larger fragments. When referring to the example given earlier, the 30 allele would reach the anode in front of the 40 allele because it is a smaller fragment. As they pass through a detector window, a laser excites the fluorescent tag that was attached to he STR during PCR, and a CCD camera collects the emitted fluorescence. Computer programs analyze the data and a genetic profile is generated for that particular sample. Com uteI P in» I _____j Data in has .wav. -— __ [— laia .J L filial} L {I Kata m; t 1.. pin» 1 awry '1' Big EbLé-n pin 9-!8' j-Lit Buffer R , Cathode I.“ i-xIlOdE-B “1} + Time {min} Figure 3 — Capillary electrophoresis. REVIEW OF THE LITERATURE In 1997, Roland A.H. Oorschot and Maxwell K. Jones were the first to explore the possibility of using PCR/STR technology to detect primary and secondary DNA transfers [24]. Although the holding times were lengthy (up to 20 minutes), it was proven that the possibility to transfer DNA from ones hand to an object (primary transfer), as well as from an object to ones hand (secondary transfer) does exist. The substrates tested included leather, plastic, and glass. The human hand itself also proved to be a successful substrate. After a one minute handshake, the palm of one participant revealed both DNA profiles. Oorschot and Jones’s study set the stage for all future research in this area. Colleagues at the Connecticut State Police Forensic Laboratory duplicated the Oorschot/Jones study, but concentrated more on secondary transfers [16]. Skin to skin to object and skin to object to skin were the two modes of secondary transfer that were explored. In contrast to Oorschot and Jones study, the handling times were greatly decreased, thus resulting in significantly lower yields of DNA. The results of their study indicate that primary DNA transfer is possible but not always detectable, and, unlike Oorschot and Jones study, secondary transfers were not able to be detected. In research conducted by R.A. Wickenheiser of the Royal Canadian Mounted Police (RCMP), PCR/STR technology detected the transfer of DNA to over 15 different substrates [28]. Because of these findings, this approach was implemented into everyday casework, and a recent murder case was solved. The handle of a knife recovered at a crime scene revealed the DNA profile of the suspect [27]. P. Wiegand and M. Kleiber of the Martin-Luther— Universitat in Germany used PCR/STR technology to detect the transfer of DNA from the hands of the suspect onto the neck of the victim in cases of strangulation [29]. 16 suspect/victim combinations were used and the success rate was over 70% for the three STR loci tested. Colleagues at the University of Gent in Belgium performed DNA profiling on physical fingerprints left on glass and wooden plates [23]. Good results were obtained from both substrates when at least 5 physical fingerprints were present. At the 1999 proceedings of the National Commission on the Future of DNA evidence, Lynne Fereday of the Forensic Science Service in England offered a summary of the past research in this area [7]. In this summary, many of the 10 studies conducted by the authors mentioned above were discussed. In addition, the Forensic Science Service is currently conducting their own DNA transfer studies. These studies were briefly described, but have yet to be published. One experiment involved determining the most recent driver of a vehicle by detecting DNA transferred to the steering wheel by the driver’s hands. 11 MATERIALS AND METHODS Decontamination Testing Before an individual was to touch the handle of the hammer, it was decontaminated in order to insure that DNA had not been left over from a previous holder. The handle was soaked in a bleach/distilled water solution for 20 minutes, wiped off with a paper towel, rinsed in distilled water, wiped off with a paper towel, placed in a heated oven to dry, and allowed to come to room temperature. Both a 10% and 20% bleach/distilled water solution were tested and the 20% solution proved to be the most effective. This entire procedure was extensively tested in order to insure that no DNA carry over was taking place. A male, who consistently transferred DNA, held onto the handle of the hammer for 2 minutes. The handle was decontaminated, followed by a 2 minute hold by a female. Procedures, that will be explained in more detail shortly, were followed in order to generated DNA profiles from the female samples. The absence of a male/female mixture in the female samples will assure that the decontamination process is working properly. 12 First Round of Sample Collection Initially, samples were collected from 32 Caucasian males and females, 16 years and older. In order to give a true representation of primary DNA transfer occurring in this type of population, the hands of the individuals were not controlled in any way. Not controlling the hands refers to not requiring any special procedures, such as washing the hands or rubbing them together, before coming into contact with the handle. By encouraging normal, everyday behavior by the test subject, evaluating the rate at which primary transfer occurs can be more accurately determined. After decontamination, an individual held onto the handle of the hammer for 2 minutes. Forcefully gripping onto the handle, swinging the hammer around, and switching hands were all encouraged, thus mimicking actions that could occur during the commission of a crime with this type of weapon. After 2 minutes, a sample was collected from the handle by using a technique known as the double swab technique [20]. First, a sterile cotton tipped swab was dipped into distilled H20 and rubbed over the entire handle of the hammer. Second, a dry swab was used to do the same. Both swabs were allowed to air dry before placed in a storage tube. This storage tube was given a letter 13 designation in order to identify the individual who contributed the sample. All samples were stored in a -20°C freezer until time to be extracted. Organic Extraction For each sample, a scalpel was used to remove the cotton tip from each swab and both were placed in a single extraction tube. Six—hundred microliters of stain extraction buffer (10 mM Tris-HCL, 10 mM EDTA, 50 mM NaCl, 2% SDS, pH 7.5) was added and the tube was placed in a centrifuge and spun down (5 minutes/15,000 9). Thirty microliters of Protinase K (10 mg/ml) were then added. The tube was vortexed to mix, spun down(5 minutes/15,000 g) and placed in a 56°C oven for an overnight incubation. The next day, the cotton tips were removed from the extraction solution and placed in a basket insert. The basket was inserted into the original extraction tube, and and this was spun down (5 minutes/15,000 g). The basket insert was removed from the extraction tube and thrown away. Five—hundred microliters of a Phenol /Chloroform / Isoamyl Alcohol solution (25:24zl) were added and the tube was vortexed to mix for 5 - 10 seconds. After being spun down (5 minutes /15,000 g), the DNA extract (top layer), was removed and placed into a new microcentrifge tube. 14 Next, Amicon®>centricon concentrators were used to purify the DNA extract. Five-hundred microliters of Tris/EDTA (TE) Buffer (10 mM Tris-HCL, 0.1 mM EDTA, pH 8.0) were added to the centricon filter and it was spun down (500 g/10 minutes). The rinse filtrate in the centricon reservoir was discarded. One milliliter of TE Buffer and the entire DNA extract were added to the centricon filter and spun down (500 g/30 minutes). This procedure was performed three times. After the third wash, the centricon filter was removed from the reservoir and inverted into a vial. This was spun down (3 minutes/1000 g) and the filtrate was removed from the vial and placed into a new microcentrifuge tube. The purified DNA extract (~ 30 ul) was stored at ~20°C until further testing. Yield Gel A yield gel can be used to determine the quality of DNA and determine how much DNA is present in an extract. A submarine gel electrophoresis unit was used to carry out this procedure. The running buffer, 25 ul of ethidium bromide (0.5 ug/ml) in 250 ml of Tris/acetate/EDTA (TAE) buffer (40 mM Tris—acetate, 1 mM EDTA, pH 8.3), was poured into the apparatus, and a 1% agarose gel (1.3 grams agarose/130 ml TAE buffer), containing two columns of 14 15 wells, was placed into the buffer. Wells # l - 7 in the first column were reserved for standards to which the samples are compared. Three microliters of Lambda Hind III/Eco R1, a mixture of standards, were added to well #1 in columns 1 and 2, and 6 ul of the following DNA standards were added to wells #2-7 in column 1: 500 ng, 250 ng, 125 ng, 63 ng, 31 ng, and 15 ng. For each sample, 4 ul of the purified DNA extract were added to 2 ul of 6x loading buffer (bromophenyl blue/glycerol) and this 6 ul mixture was added to a well. Electrophoresis was carried out for 12 minutes at 175 volts. An ultraviolet (UV) light box was used to obtain a photograph of the gel. The ethidium bromide that was added to the running buffer binds to the DNA strand and fluoresces in the presence of UV light. Therefore, the DNA in the gel can be visualized when exposed to ultraviolet light. The picture was then used to evaluate the DNA sample. When interpreting a yield gel (Figure 4), the samples are compared to standards in order to determine the quality and quantity of the DNA sample [2,17]. A solid band represents high molecular weight, or good quality, DNA and a smear represents degraded, or poor quality, DNA. Quantity 16 Solid, bright band = large quantity of high molecular weight DNA Solid, light band = small quantity of high molecular weight DNA Smear = degraded DNA Figure 4 - Yield gel interpretation. 17 is determined by picking a standard that most closely resembles the intensity of the sample band. The brightness of the band is directly proportional to the quantity of DNA. If a band is not seen, a more sensitive quantitation method will need to be performed. Applied Biosystems QuantiBloUO Human DNA Quantitation Kit A QuantiBloUO kit was used as a more sensitive way to quantitate the purified DNA extract (Table 1). Yield gels detect from 125 ng of DNA/ul to 3.75 ng of DNA/ul, while the QuantiBlotC>detects from 2 ng of DNA/ul to 0.03125 ng of DNA/ul. This test is based on the hybridization of a biotinylated, primate—specific probe (D17Z1 locus) to DNA samples immobilized on a nylon membrane and chemiluminescent detection of the bound probe. The procedures for hybridization, chemiluminescent detection, and interpretation of results printed in the product insert were followed [5]. Just as in a yield gel, the samples were compared to standards (10 ng/5 ul to 0.15 ng/S ul) to in order to determine the concentration of DNA present in the samples. When interpreting the quantiblot (Figure 5), the size and intensity of the bands on the X—Ray film were evaluated [3]. 18 Table 1 - Applied Biosystems QuantiBloUO Human DNA Quantitation Kit contents. Reagent VOlume Description 1 vial containing 1pmole/ul _ biotin lated DNA oli onucleotide QuantiBlotC> 220 ul y “g D17Z1 P b probe of the follow1ng sequence: r0 e 5’—biotin-TAGAAGCATTCTCAGAAACTA CTTTGTGATGATTGCATTC-B’. 1 bottle containing Horseradish Enzyme . . . . Perox1dase—Streptav1din (HRP-SA) Conjugate: 2 0 ml . . . conjugate supplied in buffer HRP-SA . . with preservative. Bromothymol 1 vial containing 0.04% . 200 ul . Blue Solution Bromothymol Blue in water. QuantiBlot®>DNA. 250 ul 1 vial containing 2 ng/ul human Standard A genomic DNA in TE buffer. QuantiBloU® DNA. 100 ul 1 vial containing 0.7 ng/ul Calibrator 1 human genomic DNA in TE buffer. QuantiBloU® DNA 100 ul 1.vial containing0.1 ng/ul human Calibrator 2 genomic DNA in TE buffer. 19 10ng 2.5 1.2 0.3 0.15 Figure 5 - QuantiBlot® interpretation Hill! C» '“‘- 11 Ill 20 | Y I 10ng ()1 2.5 1.2 0.6 0.3 0.15 Second Round of Sample Collection The results generated from the quantiblot procedure allowed the samples to be put into groups according to the amount of DNA present in the purified DNA extract. Because of time and resource limitations, approximately half of the individuals from each group were randomly chosen to take part in the second round of sample collection. Three samples, one at 30 seconds, 15 seconds and 5 seconds, were collected from each person. A buccal sample (cells from the inside of the mouth) was also collected from each participating individual so a known DNA profile could be generated. These samples were exposed to all of the same procedures mentioned above. The letter designations previously given to the individuals were used to identify the buccal samples. The letter designation followed by a dash and the handling time in seconds, was used to identify the timed samples. Applied Biosystems AmpFlSTR® Profiler PlusTM PCR Amplification Kit PCR was carried out using the Profiler Plusm‘Klt (Table 2) and the Applied Biosystems GeneAmp®>2400 PCR Instrument System [3]. This kit will co—amplify nine STR loci and a segment of the amelogenin gene for gender 21 Table 2 — Applied Biosystems AmpFlSTRC>Profiler Plusm'PCR Amplification Kit contents. Kit Component Volume Description AmpFlSTR® PCR Reaction Mix 1.1 ml/tube Two tubes each containing MgClL deosynucleoside triphosphates (dATP, dCTP, dGTP, dTTP), bovine serum albumin (BSA), and 0.05% sodium azide (NaN3) in buffer and salt One tube of locus—specific 5—FAM— , JOE-, and NED-labeled and unlabeled primers in buffer to AEPFISTRC) amplify the STR loci D3Sl358, Pr0f¥1er PlusTM 1‘1 ml vWA, FGA, 0881179, 021811, Primer SEt 018851, 058818, 0138317, and D78820, and the gender marker amelogenin AmpliTaq GoldC> Two tubes of enzyme with an DNA Polymerase 50 Ul/CUbe activity Of 5 U/Ul One tube containing 0.10 ng/ul human cell line DNA in 0.05% NaN3 and buffer. The enot e of this AmpFlSTRC) female DNA is D3SI358 15,15; vWA contrOI DNA 0'3 ml 17,18; FGA 23,24; 0881179 13,13; 9947A 021811 30,30; 018851 15,19; D58818 11,11; D13S317 11,11 and D78820 10,11 Mineral oil 5 ml One dropper bottle One tube of AmpFlSTRC>Blue AmpFlSTRC>B1ue 25 1 Allelic Ladder containing the Allelic Ladder u follOWing amplified 5-FAM-labeled alleles: D3S1358 12-19, vWA 11- 21, FGA 18-30 (including 26.2) One tube of AmpFlSTwO Green II Allelic Ladder containing the following amplified JOE-labeled AmpFlSTw® Green alleles: amelogenin X and Y, II Allelic 25 U1 D881179 8-19, D2181l 24.2-38 Ladder (including 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2), D18851 9-26 (including 10.2, 13.2, 14.2) One tube of AmpFlSTRC>Yellow AmpFlSTR®> Allelic Ladder containing the Yellow Allelic 25 ul following amplified NED-labeled Ladder alleles: D58818 7—16, D138317 8— 15, and D78820 6-15 22 identification (Table 3). Prior to amplification, the DNA samples were diluted to the appropriate concentration. According to studies done by the Michigan Department of State Police (MSP), approximately 1.0 ng of DNA is required for optimum amplification to occur. The purified DNA extract was diluted in Milli-Q®>water to a final concentration of 0.1 ng/ul, and 10 ul of this DNA stock solution would be added to the PCR reaction. If the DNA extract did not contain a high enough concentration of DNA, 10 ul of straight extract was used. Each PCR reaction required 10.5 ul of reaction mixture, 5.5 ul of primers, and 0.5 ul of AmpliTan>Gold DNA polymerase. After the number of samples to be amplified was determined, a master mix of these reagents was prepared in a separate microcentrifuge tube. Fifteen microliters of this master mix was added to a PCR reaction tube, followed by 10 ul of the 0.1 ng/ul DNA stock solution prepared earlier. This gave a final reaction volume of 25 ul. PCR was then carried out using the following parameters [3]: 1. Initial Denaturationzx3> 95°C/1l minutes Denature = 94°C/1 minute 2. Step Cycles:>:>:>:>:>:>:> Anneal = 59°C/l minute (28 cycles) Extend 72°C/1 minute 23 Table 3 — Applied Biosystems AmpFlSTR®>Profiler Plusm‘loci. Locus Chromosome Common Sequence 128;: ea Dye Designation Location Motif (88?“ Label TCTA (TCTG) 1-3 - - F D381358 3p (TCTA)n 114 142 5 AM TCTA(TCTG)}4 _ 7 _ _ VWA 12p12 pter (TCTA)n 15 197 5 FAM (TTTC) 3TTTTTTCT FGA 4q28 (CTTTWnCTCC(TTC 219—267 S-FAM C) 2 . X: p22.1-22.3 - 107 Amelogenin Y: p11.2 _ 113 JOE D881179b 8 (TCTR);C 128—168 JOE [321811 (TCTA)n(TCTG)n[( 21 TCTA)3TA(TCTA)3T 189-243 JOE CA(TCTA)2TCCATA ] (TCTA) n D18SSI 18q21.3 (AGAA)n 273-341 JOE D58818 5q21—31 (AGAT)n 135—171 NED D13S317 13q22-31 (GATA)n 206—234 NED D78820 7q11.2l—22 (GATA)n 258-294 NED a. The size range is the actual base pair size of sequenced alleles contained in the AmpFlSTR®>Profiler PlusTM Allelic Ladders. The sizes in the table include the 3’ A nucleotide addition. b. In some literature references, as D6SSO2 c. R can represent either an A or G nucleotide. 24 this locus is designated 3. Final Extension2>2>::>: 60°C/45 minutes 4 . Final Step:>:>:>:>:>:>:>:> 2 5°C/ forever ABI PrismC>310 Genetic Analyzer The 310 Genetic Analyzer was used to perform capillary electrophoresis on the amplified samples. First, a capillary electrophoresis reaction mixture was prepared. This was composed of 24 ul of deionized formamide and 1 ul of GeneScan—SOO [ROX] internal lane size standard for each sample. Just as in PCR, the number of samples to be run on the instrument was determined and a master mix was prepared in a separate microcentrifuge tube. Twenty—five microliters of master mix were added to a 310 sample tube, followed by 1 ul of amplified product. The ladder sample was prepared by adding 3 ul of.AmpFlSTRC>Profiler PlusTM allelic ladder to 25 ul of the master mix. This ladder consists of DNA fragments of known sizes to which the unknown samples will be compared to. Samples were then incubated at 96°C for three minutes and cooled in a benchtop cooler for three minutes. After appropriate electrophoresis parameters were selected, the samples were loaded onto the instrument and the run was started (Table 4). Following separation, GenescanC>2.0.2 and GenotyperC>2.0 software were used to 25 Table 4 — 310 run parameters. Injection Time 5 seconds Injection kV. 15.0 Run kV. 15.0 Run Temperature 60°C Run Time 24 minutes Matrix File Profiler plus Size Standard ROX GS 500 Instrument Configuration Pop-4TM polymer with.1—nd syringe Instrument Module GS STR POP4 (1ml) F 26 analyze the raw data and electropherograms, or genetic profiles, were generated. Figure 6 illustrates Applied Biosystems recommended GenescanC>analysis parameters [3]. For this study, the peak amplitude thresholds illustrated in Figure 6 were changed from 150 to 50 RFU’s (relative fluorescent units) in an attempt to detect more alleles. When interpreting the genetic profiles, the following guidelines were followed: 1. Alleles of a genetic profile with an RFU value of 150 to 4500 are declared true alleles [3]. 2. Alleles of a genetic profile with an RFU value of 50 to 149 are declared active. 3. Alleles of a genetic profile that fall below 50 RFU’s are declared undetectable. 4. For alleles from a heterozygous individual at a particular locus, heterozygote peak ratios are determined by dividing the peak height of the allele with the lowest RFU value, by the peak height of the allele with the largest RFU value and multiplying this value by 100 to obtain a percentage. This value must be 3 70% for a heterozygote to be declared. Any ratios below 70% should be interpreted with caution. 5. A sample can be considered to have originated from a single source if 1) only one or two alleles are present at all loci examined, and 2) the peak height ratios of heterozygous individuals at a locus are within the expected range. 6. A sample can be considered to have originated from multiple (two or more) sources if 1) more than two alleles are present at two or more loci, 27 * _ “ “ 7 ‘ I ' ‘ :5 H n a" '3'! S I S P a [a m B t e rs gig;1;."iii-2::Li';:'.:.:.§" a; .— «a v onto”... --—.~—..-‘ ... .~. .0..-.- .. .- ——flnalgsis Range —-Size Call Range E MultiComponent ——Smooth Options C) None Ci Light (E) Heavu 0 Full Range 0 an Sizes 6) This Range (Data Poi nts) © This Range (Base Pairs) Start: Min: Stop: Max: ——Data Processing —Si2e Calling Method — E Baseline 0 2nd Order Least Squares 0 3rd Order Least Squares 0 Cubic Spline Interpolation (Q) Local Southern Method 0 Global Southern Method —Peak Detection Peak Amplitude Thresholds e- <81 '5 1:,- Min. Peak Half Width: [_3—___| Pts —$plit Peak Correction — (E) None 0 GENESCAN 2500 O Left Most Peak 0 RightMost Peak Correction Limit: Data Pts. I Cam 1 Figure 6 - Genescan® analysis parameters. and/or 2) the peak height ratios for heterozygotes fall outside the expected range. 7. Stutter is an expected minor product peak that is four base pairs, or one repeat unit, shorter than the main allele [26]. Table 5 was used as a guideline in determining the maximum % stutter allowed at a particular locus. Peaks in the stutter position that exceed the maximum % stutter value may be designated as a true allele. 8. A complete genetic profile is declared if all 10 loci exhibit alleles with an RFU value of 150 to 4500, and the heterozygote peak ratios are acceptable. 9. A partial genetic profile is declared if at least two out of the ten loci exhibit alleles with an RFU value of 150 to 4500, and the heterozygote peak ratios are acceptable. 10. An active genetic profile is declared if 1) only one out of the ten loci exhibits alleles with an RFU value of 150 to 4500 and the heterozygote peak ratios are acceptable, and/or 2) loci exhibit alleles with an RFU value of 50 to 149. 11. An undetectable profile is declared when all alleles fall below 50 RFU’s. New Procedures In an attempt to increase the percentage of complete genetic profiles, new procedures, not previously utilized by the manufacturer, were developed. One of the new procedures developed was to add more PCR product to the capillary electrophoresis reaction mixture. Instead of adding 1ul of PCR product to the 25 ul of formamide/ROX solution, 3 ul were added. Another 29 Table 5 — Stutter percentages. DYE COLOR LOCUS CHROMOSOME % STUTTER* 5-FAM Blue D3S1358 3 15 5—FAM Blue VWA 12 15 S-FAM Blue FGA 4 15 JOE Green D881179 8 12 JOE Green D21S11 21 15 JOE Green D18851 18 18 NED Yellow D58818 5 12 NED Yellow [m38317 13 12 NED Yellow D78820 7 12 *Acceptable values established by Applied Biosystems [3] and confirmed by Michigan State Police validation studies. 30 procedure developed was using Millipore microconC> centrifugal filter devices to concentrate the PCR product. First, the filter was washed by adding 100 ul of TE buffer and spinning down (500 g/15 minutes). All of the PCR product was then added to the filter and spun down (500 g/6 minutes). The filter was removed from the tube, inverted, placed into a new tube, and spun down (1000 g/3 minutes). This tube now contained the concentrated PCR product. Three microliters of this concentrated product were then added to the 25 ul of formamide/ROX solution. Both procedures developed involve direct manipulation of the PCR product generated from the Profiler PluS”‘kit. Since I chose to use the reagents supplied in this kit and the protocols established by the Michigan State Police laboratory, manipulating the actual PCR reaction in hopes of generating more PCR product was not an option. If it were an option, varying reagent concentrations, manipulating PCR cycles, and developing a nested PCR reaction [21] would have all been explored. Quality Control (QC)/Quality AssuranceProfiler PlusTM PCR Amplification Kit and the ABI PrismC>310 Genetic 33 EVIDENCE HANDLING WORK AREA DNA EXTRACTION WORK AREA Evidence examination Sample digestion {DNA-quandtatidn-(do-t Bio-t pr-dc-ed-ui'el ] Differential lysis Organic extraction“ microconce ntration Stains. specimens divided for DNA extraction Storage oi extracted DNA samples and AmpFISTR Profiler Plus Kit components (except AmpFrSTR' Chelex extraction AHQHC Ladders) Evidence ir Midget-op; 1 photography L ------ 4 ' PCR SETUP WORK AREA Microscopy PCR reagent and DNA sample additions AMPLIFIED DNA WORK AREA DARK ROOM r----—-:—f-—‘1 Photography l DNA quantitation i DNA Thermal Cycler 480. . Developed OuantiBlot- : (slot blot procedure) : GeneAmp’ PCR System 9600. and membrane e ........... a GeneAmp PCR System 2400 Film Development - Chemiluminescent DNA quantitation Amth’STR Profiler Plus detection QuantiBlot membrane - I - lot (p08! 8 0t b ‘) - ABI Parser 377 and ABI PRISM 310 instruments ~ Gel pouring Storage of amplified DNA Waste disposal of amplified samples and Allelic Ladder products Figure 7 — Laboratory design. 34 Analyzer have been subjected to rigorous validation studies. These studies, designed by TWGDAM and the DAB, are necessary in order to prove the testing method reliable validation studies on both the kit and the instrumentation. for forensic DNA casework [12]. This re-inforces the fact that manipulating the PCR reaction in forensic casework is not an option. The current cycling parameters and reagent concentrations have been established by TWGDAM and the DAB and must be upheld. Changing any of these established parameters and concentrations is a lengthy and time consuming process which requires a consensus of the forensic scientific community employing the particular method. In addition, the Michigan State Police laboratory has conducted their own validation studies on both the kit and the instrumentation. As a result of these studies, the Michigan State Police has documented specific procedures that must be followed when using this kit and instrumentation in forensic DNA casework. All of these procedures were followed when conducting this research. The technique of the scientist performing the testing also plays a critical role in preventing contamination. The following is a list of precautions that Applied Biosystems would like all practicing forensic DNA analysts to consider [3]: 35 10. ll. Examine and sample from pieces of evidence at a separate time from the handling and sampling from known samples. Use disposable gloves at all times and change them frequently. Always use a clean cutting surface and clean scissors when collecting evidence samples. Perform DNA extraction from samples containing high levels of DNA separately from samples containing low levels of DNA. Perform the DNA extraction of evidence samples at a separate time from the DNA extraction of known samples. If possible, PCR reactions should be setup in a dedicated PCR hood equip with a UV light source. Use sterile, disposable, hydrophobic filter— plugged pipet tips and microcentrifuge tubes. Always change pipet tips between handling each sample. Store reagents as small aliquots to minimize the number of times a given tube of reagent is opened. Cap all tubes before beginning the addition of DNA. Only open the tube to which DNA is being added. Do not store reagents close to samples containing high levels of DNA. All of these recommendations were followed in order to minimize the possibility of contamination. Running appropriate quality control samples is also very important in assuring that contamination is not occurring and to guarantee the testing methods ability to 36 produce accurate and precise results. In this study, QC samples, positive and negative controls, were introduced into the testing method at three very distinct points of analysis. First, was during the extraction process. An internal laboratory control (ILC) and a substrate control were both extracted in parallel with research samples. The ILC has a known DNA profile and is used by the Michigan State Police as a positive control. The substrate control was used as a negative control. This sample was collected randomly after the handle was disinfected but before the next individual came into contact with it. A negative result for this sample assures that the disinfection process is working properly to prevent DNA carryover from occurring. Second, was during the PCR setup process. Again, both a positive and negative control were setup in parallel with research samples. AmpFlSTR®>Control DNA 9947A, provided in the Profiler Plusm‘kit, was used as a positive control. This control DNA has a known DNA profile, as established by Applied Biosystems. The negative control was PCR master mix without the addition of DNA. Third, was during the capillary electrophoresis setup process. An additional negative control was run in 37 parallel with research samples. This sample consisted of 310 master mix without the addition of PCR product. A total of two positive controls and three negative controls were run with every batch of samples that went through analysis. If unexplainable discrepancies exist with any one of the control samples, results generated from that particular batch of research samples are deemed invalid and need to be re-tested. 38 RESULTS AND DISCUSSION Decontamination Testing QuantiBlotC>results confirmed that the male consistently transferred DNA to the handle of the hammer. Since male/female mixtures were not seen in any of the DNA profiles generated from the female samples, the decontamination process was working effectively. First Round of Sample Collection Yield Gel Results No visible bands were observed in any of the 32 sample lanes, which represents a concentration of DNA less than 3.75 ng/ul (15 ng/4ul). All results obtained from QC samples were acceptable. QuantiBlot® Results DNA concentrations ranged from 0.125 ng/ul to 0.03125 ng/ul, and the results generated from QC samples were acceptable (Table 6). Most important, the substrate control sample did not show a visible band in its slot. For quality control purposes, this sample was randomly collected after the handle had been decontaminated but before the next individual 39 Table 6 - First round quantiblot results. Quantiblot Results Sample (ng of DNA/ul) 0.125 HH DD* ILC (2) 0.0625 G 0.03125 < 0.03125 A BB* II Substrate Control *The individuals who supplied these samples were chosen to take part in the second round of sample collection. 40 held onto it. This was used to assure that the decontamination process was working properly to prevent DNA carryover from occurring. Second Round of Sample Collection Yield Gel Results No visible bands were observed in any of the 5, 15, or 30 second sample lanes, and all results generated from QC samples were acceptable. The known buccal samples showed solid, bright bands in their respective lanes. QuantiBlot®>Results For all 5, 15, and 30 second samples, the highest amount of DNA recovered was 0.0625 ng/ul (Table 7), and the known buccal samples yielded between 0.25 and 0.125 ng of DNA/ul. Results obtained from QC samples were all acceptable. It should be noted that in both rounds of sample collection, a relationship did not appear to exist between the amount of DNA quantatied and the gender of the individual who supplied the sample. Not only does this data show the possibility to quantitate DNA from holds as low as 5 seconds, it also 41 Table 7 — Second round quantiblot results. Quantiblot Results (ng/ul of DNA) Sample Amount of DNA to be amplified 0.25 FF—KNOWN M-KNOWN U-KNOWN DD—KNOWN 1.0 ng* 0.125 AA-KNOWN J-KNOWN O-KNOWN Q-KNOWN BB—KNOWN ILC N-KNOWN X-KNOWN 1.0 ng 0.0625 Q-30 0.625 ng 0.03125 FF-5,15 L—30 J—15,30 F-15,30 H-15 0.3125 ng <0.03125 AA-5,15,30 FF-3O L-S,15 J—5 F-5 M-5,15,30 0-5,15,30 U—5,15,30 Q—5,15 BB-S,15,30 H—5,30 DD—5,15,30 N—5,15,30 X-5,lS,30 W-5,15,30 Substrate Control <0.3125 ng * Amount required for optimum amplification to occur, as determined by Michigan State Police validation studies. 42 shows that there is no apparent relationship between handling time and the concentration of DNA recovered from the handle. For a true proportional relationship to exist, there should be an increase in the amount of DNA recovered from the 5 second hold up to the 30 second hold. The data generated in this study does not show this to exist, as one might expect. For example, samples FF—5 and FF-15 each gave 0.03125 ng of DNA/ul but FF—30 gave < 0.03125 ng of DNA/ul. Also, sample H—5 gave < 0.03125 ng of DNA/ul, H-15 went up to 0.03125 ng of DNA/ul, and H-30 dropped back down to < 0.03125 ng of DNA/ul. Data from the initial 2 minute holds also plays a part in evaluating this relationship. After 2 minutes, individual Q transferred < 0.03125 ng of DNA/ul to the handle. At a later date, a sample was collected from individual Q after only 30 seconds. From this sample, 0.0625 ng of DNA/ul were recovered. Since time does not seem to be related to the amount of DNA recovered, the condition of the test subjects hands appears to be an important factor. DNA from skin cells shed directly from the hands (epithelial cells), can contribute to the DNA recovered from the surface of the handle. One 43 previous study suggested that DNA transfer is highly dependent on the individual handling the object [14]. Those that shed skin cells more readily than others were categorized as “sloughers”. Natural human variation can account for one being categorized as a “slougher” or a “non-slougher. For example, those who naturally have dryer hands might shed skin cells at different rates than those who do not. Cells shed directly from the surface of the hands can contribute to the amount of DNA transferred to the handle, but it does not necessarily represent the entire amount. DNA transferred to the hands from other parts of the body can also contribute. Before coming in to contact with the hammer, the subject could have coughed or sneezed into their hands, scratched their head, rubbed their eyes, or touched any other part of their body that contained large amounts of nucleated cells. Each of these instances can transfer many nucleated cells to the hands which in turn could be transferred to the handle. In this particular study, this was a possibility because the hands of the individuals were not controlled in any way prior to coming into contact with the handle. To get a true representation of primary DNA 44 transfer occurring in the sample population, it is essential that the hands not be controlled. In many instances, violent criminal activity is an extremely random event. Controlling the hands would not produce “true—to—life” results, thus compromising the significance of the results. This lack of control over the individuals hands, along with natural human variation, is what accounts for the reason why some samples were able to be quantitated while others were not. STR Results All known DNA profiles generated from the buccal samples were complete and consistent with originating from a single source (Table 8). Table 9 shows the genetic profiles generated from each 5, 15 and 30 second sample. Out of the 45 total samples, alleles were detected in 44. This shows that primary DNA transfer to this type of surface is very common in this particular population. After each profile was subjected to the interpretation guidelines listed previously, its DNA profile type was determined (Table 10). The majority of the samples, 20 out of the 45, produced partial DNA 45 cocmflanwumo >Hmsofl>oud one: moaflwoud «zo czocxae «H.0H Ha.oa Ha.m ma.ea om.om ea.ma x.x N.N~.ma oa.ea ea.oa eem execs oa.m mH.HH Ha.oa aa.ma N.om.om mH.HH x.x am.mm ma.ea oa.ma are means ma.a NH.HH ma.~a ma.ma om.mm Ha.m x.x mm.mm ma.ma ea.oa seq geese HH.HH Hates ma.aa ma.ma om.em ea.ea x.x em.mm ma.ma oa.ma eee seeps Na.ma HH.HH NH.HH ea.ma om.mm ma.aa ».x em.em ma.ma oa.oa mm paces Ha.oa Na.m ma.aa ma.ma Hm.om ma.ma x.x mm.em ea.aa ma.ma o seeps Ha.m Ha.m NH.HH Hm.wa m.am.mm ma.ma ».x om.am ea.ma ea.ea 8 cases NH.NH NH.HH ma.a ma.ma Hm.em ea.m x.x om.em ma.ma ea.ma o esoem aa.ea NH.HH ma.aa ma.ma om.om aH.HH x.x mm.mm aa.ea oa.oa .2 seems HH.HH NH.HH aa.aa ea.ma mm.mm ma.oa x.x Hm.am aa.ea oa.ea a axons NH.HH NH.HH ma.oa ma.ma em.em ea.ma w.x em.am ma.ma ma.ma ea seeps NH.oa Ha.m HH.HH ea.ea om.mm ma.ea ».x am.mm ma.ea oa.ma we eeoem oa.oa Ha.oa ma.a mH.NH am.mm ma.ea s.x mm.mm matea ma.ma e seeps oa.a NH.HH NH.Ha ea.ma N.om.mm ma.oa ».x mm.ma ea.oa ma.oa x macaw «H.0H HH.HH NH.HH ea.ma om.mm ma.aa x.x om.mm ma.ma oa.oa 2 seems oa.e NH.HH HH.HH ea.ma am.mm aa.ma w.x mm.am ea.oa oa.ma on ozone oummpo 5339 333 Romeo 333 2.3me an 4mm 65 $3me 3 395m .moaeooom 42o execs i m magma 46 nauseouonm 3w: on pomOQXo mums memEmm paom ouduxfle n no oocopfl>o pesosm moHQEmm ee Am.aun mea-omv wmamaam o>fiuo¢ 4 or 30 o o OHDMH wuom>noumuom « 4x . miz HH.HH NH.HH «A.HH ea.ma mmtmm ma.oa xix Hm.am mH.eH oH.eH om-e HH.HH NH.HH ea:aa ea.ma mm.mm ma.oa x.x Hm.am ma.ea oa.ea mH-e ‘HH «Eff: awataaa 4¢HKNH mmtmm gmafoa “TN HNtHN «mHHH wail” mih. ema.eaa ema.eaa ema.eoa mH.mH amm.aem ea.ma w.x em.em ma.ma mH.mH om-mm mataa mataa mated malma mm.em ea.ma w.x em.em matma ma.mH ma-mm NH.HH NH.HH ma.oa ma.ma mm.em «Hima w.x em.em ma.ma mH.mH m-mm ‘OH 239% HHJH .x emm.emm «matema ema.ema m-z eoa.em amateaa NH.HH eatama eem.om.mm ema_oa w.x mm.ma ma.oa ma.ma om-x eoa ema ema.xaa exea.ama em.om.emm MH.OH w.x emm.4ma ema.eoa ma.ma mars «oatem ema.eHH ema.eHH eeeateMH «m.om.mm mH.oa w.x mm.ma aema.oa ma_oa m-x «somiz awe mH-z semiz camataaa aHtHH 4MH «mm.amm eava_ama M.x ‘Hm matma ema.‘ma omiDQ oatm NH.HH HH.HH ea.ma mm.mm eH.mH w.x mm.am mH.mH ma.ma mH-oo oa.m mH.HH HH.HH eatma mm.mm eatma w.x mm.am ma.ma matmfl m-oo enemas bananan mammmn Hmmman Hamaaa meaammo MN «on ¢z> mmmammn nH odesom .monEmm Ucoomm om me.mH.m i m magma 47 moedcououm 30c 00 pomomxm onoz mmHQEMm paom annexes a mo oommcfi>o posocm oHQEmm «e Am.DwH m¢Hiomv mwfiofifim m>aoo¢ 4 Moe 30Hon HHWMIcHDMH ouom>monooom e _ «tomim oH.m MH.HH Ha.oa ea.ma m.om.om mH.HH x.x em.mm mH.eH oa.ma warm “teal aaaw eHH.eoa ama em.om.xom oema.aa x.x aem.emm eama.eea oa.ma m-m aeomrq ¢ mmmammn nH oaaaom .Ae_oeoov m oanme 48 Table 10 — 5,15,and 30 second profile types. Type of DNA profile 5 seconds 15 seconds 30 seconds Complete DD-S FF—15 W-30 FF-S H-15 J-3O DD—15 J-15 Partial X-S X—lS DD—3O J—S W-lS X-30 Q—5 M—15 AA—3O BB—5 Q—15 FF—30 F-5 BB-lS O-30 H—5 F-15 Q-30 L—lS BB-30 Active W—S N—15 U-3O AA-S AA-15 M—5 O-15 0—5 U—15 Uhdetectable U—5 Mixture N-S L-5 49 profiles. Of the remaining 25 samples, nine gave active profiles, eight gave complete profiles, one profile was undetectable and seven profiles showed evidence of a mixture. In mixtures, alleles belonging to the primary handler are present, but additional alleles, not belonging to the handler, are also detected. Again, it should be noted that a relationship did not appear to exist between the type of DNA profile generated and the gender of the individual who supplied the sample. These results are extremely significant when evaluating the sensitivity of the testing method. With one exception, all of the extracts contained DNA concentrations 5 0.03125 ng/ul. Therefore, the DNA amounts introduced into the amplification reactions were 5 0.3125 ng (312.5 pg). This is much lower than the 1.0-1.25 ng recommended by the manufacturer [3]. Generating complete and partial DNA profiles from samples containing such low copy numbers (LCN) of target DNA, as this study did, illustrates the extreme sensitivity of PCR/STR technology. As the DNA profiles were being interpreted, two issues surfaced which need to be addressed. The first issue is the occurrence of heterozygote peak 50 imbalances. The STR interpretation guidelines, based on results generated from Michigan State Police validation studies, state that the heterozygote peak ratios must be 3 70% for a heterozygote to be declared at a particular locus. When looking at the 38 samples that did not show evidence of a mixture, imbalances (ratios <70%) were seen in 45% of them. Of this 45%, 27 separate instances occurred with the majority seen at the VWA and D18S51 loci. Of the 27 instances, only one instance involved heterozygote alleles 3 150 RFU’s. The Michigan State Police interpretation guidelines states that true alleles are declared if they fall between 150 and 4500 RFU’s [3]. In this study, a new guideline declared active alleles if they fell between 50 and 150 RFU’s. Without this guideline in place, imbalances would have occurred in only 3% of the samples. Explanations as to why these imbalances occurred include primer binding site mutations and low copy number DNA [25]. Given that this study directly involved the analysis of low copy number DNA, this appears to be the reason why imbalances occurred. With low levels of input DNA, it is possible that two alleles of a heterozygote will amplify unequally. 51 This phenomena is known as stochastic fluctuation [13]. It should be noted that not all laboratories use the same heterozygote peak ratio guideline. In studies conducted by the Federal Bureau of Investigation, ratios 3 60% were deemed acceptable [18]. If this guideline were used to interpret the DNA profiles generated in this study, only 16% of the samples would have shown imbalances. The second issue involves the seven samples (16%) that showed evidence of a mixture. No more than three loci in any one sample showed additional alleles, and no more than one additional allele was seen per loci. Again, it should be recognized that if the active allele guideline was not incorporated into this study, only one sample would have showed evidence of a mixture. Previous studies have suggested that when dealing with low copy number DNA, mixtures are commonly encountered [8]. There are two explanations as to why the mixtures could have occurred. One is DNA contamination and the other is artifacts produced by the electrophoretic and/or PCR system itself. In regards to DNA contamination, heterozygote peak 52 imbalances at the Amelogenin loci (XzY) are typical of a male/female mixtures [9]. Since this imbalance was seen in three of the mixed samples, DNA carryover, laboratory contamination and secondary transfer all need to considered as possible sources of DNA contamination. If the procedure explained earlier to decontaminate the handle of the hammer was not working efficiently, this would cause DNA carryover to occur. This would result in a mixture of alleles belonging to the primary handler and the individual who handled the hammer just before. Since the decontamination process was extensively tested prior to beginning the study, DNA carryover does not seem to be the likely reason as to why mixtures were seen. Research conducted by Peter Gill of the Forensic Science Service in the United Kingdom has shown that low copy number DNA amplification is prone to sporadic contamination [8]. Just as in this study, strict QA/QC guidelines were followed in order to prevent contamination from occurring, but additional alleles were still detected [8,11]. Possible sources of contamination include extraneous DNA, cross 53 contamination from other samples processed in the laboratory and plastic-ware contamination by the manufacturer [8]. In Gills research, contaminants were typically associated with low molecular weight loci which included Amelogenin, D38158 and D8S1179 [11]. In this study, 67% of the additional alleles were seen at these loci. The occurrence of a secondary transfer is another possible source of DNA contamination [16,24]. As explained earlier, the hands of the individuals were not controlled in any way prior to coming into contact with the handle of the hammer. Therefore, the mixtures seen in this study offer proof that it could be possible for one individual to transfer another individuals’ DNA to an object and have it be detected. When explained in terms of criminal activity, it could be possible for a criminal to transfer an innocent person’s DNA onto a weapon of this type, thus connecting him/her to the crime. More extensive research needs to be conducted in this area in order to prove this possibility to be true. Artifacts produced by the electrophoretic and/or PCR system itself is the second explanation as to why the mixtures could have occurred. One type of 54 artifact is stutter. Stutter peaks are one repeat unit shorter than the main allele and are caused by slippage of the DNA polymerase enzyme during PCR [26]. Studies conducted by Applied Biosystems and confirmed by the Michigan State Police showed that stutter peak heights are typically 12% to 18% of the main allele. This range exists because different loci show different intensities of stutter. When low copy numbers of DNA are introduced into the PCR reaction, stutter peaks can actually be equivalent to the size of the main allele [9]. In this study, four samples contained additional alleles that were in the stutter position. Non-specific artifacts can also be produced by the electrophoretic and/or PCR system. This type of artifact occurs as a result of non—specific priming from fragments of bacterial or degraded human DNA [9,10]. The results of Peter Gill’s studies show that appropriate guidelines need to be established for interpreting DNA profiles generated from low copy number DNA [8,11]. As recommended by Gill, an allele should only be reported if a duplicate result can be obtained from two or more replicate samples [11]. 55 Only 4 out of 1225 comparisons showed the same spurious allele when replicates were compared. This “duplication guideline” was applied to a statistical theory which proved this to be a reasonable approach to interpreting low copy number DNA profiles. Unfortunately, the results of Gill’s studies were not acquired until after the research had been completed in this study. Otherwise, an honest attempt to replicate all samples showing evidence of a mixture would have been made. Tables 11 and 12 show genetic profiles generated from 15 samples after exposure to the two newly developed procedures. Profile types were determined and compared to each other, and to those generated from the samples exposed to the original procedure (Table 13). When using 3 ul of PCR product in the 310 reaction, two active profiles became partial, while the remaining four showed increased activity but remained active. One partial profile became complete, while eight showed increased activity but remained partial. Two out of the nine partial profiles would have been declared complete if heterozygote peak ratios would have fallen within the 56 Amtdwh mediomv moHoHHm o>fluu¢ d woe SOHmn Hamw oflumu muom%nouwuwm a ma.ma ma.ma om.mm aa.m x.x mm.mm ma.ma oa.oa mare NH.HH eeoa.¢ma om.mm ma.ea ».x eom.aem wa.ma oH.oH m-mm ma.ma Hm.om ma.ma x.x mm.em ea.ea ma.ma ma-o . eema.ama mmmammn” oH meadow .oosaond mom no Ham i Ha canoe 57 meduxflE M MD monopfl>® UwBOnm moHQEdm ea Amldwh mvHiomV mmamgm o>floo< a wooa. 26an Sow Capos muomxamoteouom a ma_m NH.HH ma.ma ma.ma om.mm Helm x.x mm.mm ma.ma oa.oa ma-q matma HHtHH NH.HH eoa.ma om.mm ma.ea ».x om_em ma_ma oa.oa m-mm HH.OH ma.m ma.aa ma.ma Hmtom ma.ma x.x mm.em ea.ea ma.ma ma-o eeom-o samHiD «smiO eoa NH.HH ama.¢aa eama.ma om.om ea.HH xtx mm.mm ema.¢eax HH.HH «A.HH ea.aa ea.ma mmtwm ma.oa x.x Hm.Hm melee oa.ea m-e NH.HH ma.aa ma.oa matma mmtem ea.ma wtx em.em ma.ma ma.ma om-mm ma.oa Ha.m HH_HH halos om.mm ma.ea w.x em.mm ma_ea oa.ma om-4¢ emieoH «Ha.m HH.HA ea.oa eom.mm emH.eH w.x em.mm ema.ea eoa.ma m-<< oa.oa Ha.oa ma.m ema.ma mm.wm ma.ea w.x emm.mm wa.oa ema.oa ma-z oa.m NH.HH NH.HH eeatma m.om.mm ma.ea w.x mm.ma ema.oa mH.mH ma-x ma.aa NH.HH ea.ma m.om.mm ma.ea w.x mm.ma «wa.oa ma.ma m-x r. a .3 Ha enigma :omtamm Jami”; xtx «mmlemm «ma matma maiz ommmeo hammman memmmo Hmmmao HHmHNn meaammn ex «on ¢z> mmmammn nH madamm .DUSUOMQ mum commencwucoo wo dam i We canoe. 58 Table 13 — New procedure profile types. Sample ID Quantiblot Original 3ul of 3ul of results Protocol amplified concentrated (ng of (lul of product amplified DNA/ul) amplified product product) N-lS < 0.03125 Active Partial Partial AA-S < 0.03125 Active Partial Partial M-S < 0.03125 Active Active Partial O-5 < 0.03125 Active Active Mixture 0-15 < 0.03125 Active Active Mixture U-30 < 0.03125 Active Active Mixture Q-15 < 0.03125 Partial Complete Complete X-S < 0.03125 Partial Partial* Partia1* W-IS < 0.03125 Partial Partial* Partial* X-15 < 0.03125 Partial Partial Partia1* AA—30 < 0.03125 Partial Partial Complete BB-S < 0.03125 Partial Partial Partial* L-IS < 0.03125 Partial Partial Complete FF-30 < 0.03125 Partial Partial Complete J_5 < 0.03125 Partial Partial Complete *All 10 loc1 70%. 59 L exhibited alleles with an RFU value of 150 to 4500, but the heterozygote peak ratios at some loci were below acceptable range. Heterozygote peak imbalances were seen in 60% of the samples. Of this 60%, 20 separate instances occurred with the majority seen at the VWA, D21811 and D18S51 loci. In comparison to the same samples from the original procedure (1 ul of PCR product), the same nine samples showed imbalances but six more instances were seen. Originally, no more than two loci in any one sample showed imbalances. Adding 3 ul of PCR product generated DNA profiles with up to five loci exhibiting imbalances. When comparing DNA profiles generated from the original procedure to those generated in this procedure, two major differences were seen. One is the peak heights of the alleles showing imbalances. Of the original 15 samples, only active alleles (50-149 RFU’s) were involved. When more PCR product was introduced into the 310 reaction, 25% of the instances involved alleles with peak heights in the acceptable range (150-4500 RFU’s). Another difference is the severity of the imbalances. Originally, the majority of the imbalances involved alleles with peak height ratios that fell between 60% and 70%. With this procedure, the majority of the 60 ratios fell between 40% and 60%. When using 3ul of concentrated PCR product in the 310 reaction, three out of the six active profiles became partial, but the remaining three showed evidence of a mixture. Five partial profiles became complete, while the remaining four would have been declared complete if the heterozygote peak ratios had fallen within the acceptable range. Heterozygote peak imbalances were seen in 58% of the samples that did not show evidence of a mixture. Of this 58%, 16 separate instances occurred with the majority seen at the VWA and D18851 loci. Up to six loci in one sample showed imbalances and one sample that did not exhibit imbalances with the original procedure showed imbalances at two loci. The majority of the peak height ratios fell between 50% and 70%, and 75% of the instances involved alleles with peak heights in the acceptable range. When compared to the 3 ul of PCR product that was not concentrated, the ratio imbalances were less severe but the percentage of instances involving peak heights in the acceptable range increased by 50%. Adding 3 ul of concentrated PCR product to the 310 reaction generated three DNA profiles that showed 61 evidence of a mixture. A total of 32 additional alleles were seen. Up to eight loci in one sample exhibited additional alleles and up to four additional alleles were seen at a single locus. Of the alleles belonging to the primary handler, 84% exhibited peak heights within the acceptable range. In contrast, alleles not belonging to the primary handler were in the acceptable range only 16% of the time. Both procedures were successful in increasing the ability to detect alleles, but adding 3ul of concentrated PCR product to the 310 reaction generated more complete profiles. Unfortunately, results from both procedures need to be interpreted with extreme caution. In regards to the original 15 samples, all heterozygote peak ratio imbalances involved active alleles and mixtures were not seen. The new procedures produced true alleles (150—4500 RFU’s) with imbalances and three samples showed evidence of a mixture. Explanations as to why imbalances and mixed samples occurred are the same as those explained earlier. In addition to the two procedures developed to increase allele detection, interpretation guidelines established for low copy number DNA analysis could 62 have been implemented into this study and evaluated as a more reliable method [8, 9, 10, 11]. Unfortunately, since the results of these studies were acquired after the research was completed, the guidelines could not be used to interpret DNA profiles. This is due to the fact that duplicates were not run on samples exhibiting imbalances and/or evidence of a mixture. QA/AC Results Acceptable results were obtained from all quality control samples. This assures that all results generated from test samples are accurate. 63 CONCLUS IONS In a population of Caucasian males and females over the age of 15, the following conclusions can be made regarding primary DNA transfer to an unfinished wood surface: 1. Primary transfer is extremely common. 2. No apparent relationship exists between handling time and the amount of DNA recovered from the substrate. 3. Complete DNA profiles can be generated from holds as low as 5 seconds where < 0.3125ng of DNA is amplified. 4. Adding 3ul of PCR product to the 310 reaction slightly increases the ability to detect alleles, but imbalances in heterozygote peak ratios of true alleles can occur. 5. Adding 3ul of concentrated PCR product to the 310 reaction significantly increases the ability to detect alleles, but imbalances in heterozygote peak ratios of true alleles can occur. 64 SUGGESTIONS FOR FURTHER RESEARCH Since DNA transfer has only recently become a topic of interest, conducting further research is going to be critical in fully understanding its forensic significance. It is crucial that this significance be determined in order to assess just how much of an impact it will have on the criminal justice system. In parallel with this study, the following is a list of variables that could be changed in order to gain additional knowledge in the area of DNA transfer: 1. Use a test object made of a different substrate (plastic, glass, finished wood, etc.). 2. After the object is handled for the allotted time, expose it to a variety of storage conditions before collecting the sample in order to determine if the transferred DNA will be able to withstand its environment. 3. Allow the test subjects hands to come into contact with another individual before handling the object. By doing this, the ability to detect secondary transfers can be further studied. 4. Attempt to recover the amount of DNA required for optimum amplification by using Millipore microconC> centrifugal filter devices to concentrate the DNA extract. This could eliminate the need to concentrate the PCR product and allow for less heterozygote peak imbalances. 65 REFERENCES 66 [l] [2] [3] [4] [6] [8] [9] REFERENCES Access Excellence. Desktop Electrophoresis Lab. [Online] Available http://www.accessexcellence.org/AE/AEPC/WWC/l993/movin g.html, September 18, 2000. Agarose gel fractionation of RSA l digested genomic DNA. [Online Image] Available http://www.biotech.ufl.edu/~etcl/mc_picture_gallery.ht m, December 31, 2001. Applied Biosystems Corporation. AmpFlSTRC>Profiler Plus User’s manuals. Foster City, CA 2000. Applied Biosystems Corporation. Introduction to Microsatellite Analysis. [Online] Available http://www.appliedbiosystems.com/molecularbiology/abou t/dna/377/377a2a.html, September 18, 2000. Applied Biosystems Corporation. QuantiBloU® Human DNA Quantitation Kit package insert. Foster City, CA 1996. Capillary Electrophoresis Theory and Background. [Online Image] Available http://www.ceandcec.com/cetheory.htm, December 18, 2000. Fereday, L. Technology Development: DNA from Fingerprints. [Online] Available http://register.aspensys.com/nij/dnamtgtrans6/trans- i.html, August 31, 2000. Gill, P. “Application of Low Copy Number DNA Profiling.” Croatian Medical Journal 42 (2001): 229- 232. Gill, P., Sparkes, 8., Clayton, T. M., Whittaker, J., Urquhart, A., Buckleton. “Interpretation of Mixtures Based on Peak Area — Identification of Genetic Anomalies, Stutters and Other Artefacts.” [Online] Available http://www.promega.com/geneticidproc/eusymp2proc/16.pd f, February 1, 2002. 67 [10] [12] [l3] [14] [15] [l6] [17] [18] [19] Gill, P., Sparkes, R., Kimpton, C. “Development of guidelines to designate alleles using an STR multiplex system.” Forensic Science International 89 (1997): 185-197. Gill, P., Whitaker, J., Flaxman, C., Brown, N., Buckleton, J. “An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DN .” Forensic Science International 112 (2000): 17-40. Holt, C. L., Bunocristiani, M., Wallin, J. M., Nguyen, T., Lazaruk, K. D., Walsh, P. S. “TWGDAM Validation of AmpFlSTRm PCR Kits for Forensic DNA Casework.” Journal of Forensic Sciences 47 (2002): 66-96. Inman, K. & Rudin, N. An Introduction to Forensic DNA Analysis. Boca Raton, Fl: CRC Press, 1997. Kisilevsky, A. & Wickenheiser, R.A. (in press). “DNA PCR Profiling of Skin Cells Transferred Through Handling.” Canadian Society of Forensic Science. Kobilinsky, L. “Deoxyribonucleic Acid Structure and Function.” Forensic Science Handbook, VOlume III. Ed. R. Saferstein. Englewood Cliffs, NJ: Prentice-Hall, 1993, pp. 287-357. Ladd, C., Adamowicz, M. S., Bourke, M. T., Scherczinger, C. A. & Lee, H. C. “A Systematic Analysis of Secondary DNA Transfer.” Journal of Forensic Sciences 44 (1999): 1270-1272. Lambda DNA/HindIII Marker, 2. [Online Image] Available http://www.fermentas.com/catalog/markers/marksmo101.ht m, December 30, 2001. Moretti, T. R., Baumstark, A. L., Defenbaugh, D. A., Keys, K. M., Smerick, J. B., Budowle, B. “Validation of Short Tandem Repeats (STRs) for Forensic Usage: Performance Testing of Fluroescent Multiplex STR Systems and Analysis of Authentic and Simulated Forensic Samples.” JOurnal of Forensic Sciences 46 (2001): 647-660. Structure of DNA. [Online Image] Available http://www.accessexcellence.com/AB/GG/structure.html, December, 18 2000. 68 [20] [21] [22] [23] [24] [25] [26] [27] [28] Sweet, D., Lorente, M., Lorente, J. A., Valenzuela, A. & Villanueva, E. “Improved Method to Recover Saliva from Human Skin: The Double Swab Technique.” JOurnal of Forensic Sciences 42 (1997): 320-322. Strom, C.M, Rechitky, S. “Use of nexted PCR to identify charred human remains and minute amounts of blood.” JOurnal of Forensic Sciences 43 (1998): 696- 700. The Polymerase Chain Reaction. [Online Image] Available http://www.acessexcellence.org/AB/GG/polymerase.html, December 20, 2000. Van Hoofstat, D. E. O., Deforce, D. L. D., De Pauw, I. P. H. & Van den Eckhout, E. G. “DNA Typing of Fingerprints using Capillary Electrophoresis: Effect of Dactyloscopic Powders.” Electrophoresis 20 (1999): 2870-2876 . Van Oorschot, R. A. H. & Jones, M. K. “DNA Fingerprints from Fingerprints.” Nature 387 (1997): 767. Wallin, J. M., Holt, C. L., Lazaruk, K. D., Nguyen, T. H., Walsh, P. S. “Constructing Universal Multiplex PCR Systems for Comparative Genotyping.” JOurnal of Forensic Sciences 47 (2002): 52-65. Walsh, P.S., Fildes, N.J. and Reynolds, R. “Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA.” NUcleic Acids Res. 24 (1996): 2807-2812. Wickenheiser, R. A. & Challoner, C. M. (in press). “Suspect DNA Profiles Obtained from the Handles of Weapons Recovered at Crime Scenes." lOw'Annual Symposium on HUman Identification. Promega, Lake Buena Vista, Florida, 1999. Wickenheiser, R. A., Roney, J. M., Hummel, K. H. J., Szakacs, N. A., MacMillan, C. E., Kuperus, W. R., Walker, T. J., Hrycak, T. L., Reader, L. J. V., Fenske, B. A., Hanniman, J. L., Faris, J. S., Lett, C. M., DeGouffe, M. J., Golin, M. & Jobin, R. M. “Unusual Exhibit Material Yielding Successful DNA Profiles 69 [29] Using PCR Str Typing.” 10Ch Annual Symposium on HUman Identification. Promega, Lake Buena Vista, Florida, 1999, Poster Presentation. Wiegand, P. & Kleiber, M. “DNA Typing of Epithelial Cells after Strangulation.” International JOurnal of Legal Medicine 110 (1997): 181-183. 70 APPENDICES 71 APPENDIX A Electropherograms generated from decontamination testing. 72 ABI Thesis data - contamination study licensed to Michigan State Police. DNA Unit Genotypem 2.1 r'lrlvrrfi'hrirrrr'riir“IVfrrrr'trwflrt 180 200 220 240 250 280 300 320 r—ITTTUTU"? 'VIVVrl 100 120 140 160 III‘|II.ICIICII'OII|III'IOI'IIC‘DIJ'III'OOI’IIU‘III too 120 :40 160 180 200 220 240 260 280 300 320 Aii-P (C) 7am P (o) A1 14’ (C) 800 oo zoo Ail-P (o) 7 Yellow P to) too . o B m 51:1. If} ED El E8 [Hi Ell HE Mi-P to) 7 M P (C) E800 800 L400 [200 73 east Thesis data - contamination chock, Licensed to Michigan State Police, DNA Unit Genotyporo 2.1 Ifiw 100 I | I I I 1 oo A9-L (0) A94- (0) AQ-L (C) NH- (C) r 120 I 120 ‘_T I I I I—T—I—I' l U I 140 160 I I I I I I I | 140 160 6 Blue L (C) sateen L (C) s‘fiflbw Lie) FE 6Red L(C) 180 200 220 240 180 200 220 240 T I' I r U I l I Y I I’ I I ‘I’ r, r I I f 280 300 280 300 ' III—l l 200 1 50 hIOO ~50 . 300 zoo Lioo 20 74 Thesis data - contamination check Licensed to Michigan State Police. DNA Unit Genotypew 2.1 Y—rT—IIT—IWII’IIII'IjI'fi'IIT'rIII'jjIITIj—ll‘ljfiriIll—IWUI[TU—l—I 0 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 0 80 100 I20 I40 160 180 200 220 240 260 280 300 320 340 360 A7-HiC) 5 Blue HIC) 4o 30 20 to A7-H(C) 5 Green H(C) 20 EE AT-HlC) 5 Yellow HICI ‘— + 75 APPENDIX B Electropherograms generated from known DNA samples. 76 ABIA, PRISM Thesis date - Group 1 Licensed to Michigan State Police, DNA Unit GenotyporO 2.1 "IIIYTTIfirlI'Y—T‘FIIII‘U—I letjirfY'TrIrj‘l‘rYIIY—I—IT F0 100 120 140 160 180 200 220 240 260 280 300 'III’III’III’IIIIIII'IIIIIII‘III'lll'lIIIIII|I 0 100 120 140 160 180 200 220 240 260 280 300 BLKNOWNDD 98in. KNOWNDD 3000 2000 l 1000 is [E El 2689 HE] m [E 23 2317 m B4—KNOWNDD 96mm KNOWNDO r2000 -1000 “KNOWN DD i am 9 Red KNOWN DD 111 F ~2000 h ~1000 L 900 00 300 77 %_ Thesis data - Group 1 PR Licensed to Michigan State Police. DNA Unit Gonotypoio 2,1 VITfI’T-ITITrYTIIrIrI—TT—IrTj“[IITIVFII’T—IT‘T‘ITIVIIUIYI 100 120 140 160 180 200 220 240 260 280 300 320 340 lll'lll'llll'll IIIII'I II'III'III'Ill’ill'llO'IIIIIII' 100 120 I40 150 180 200 220 240 250 280 300 320 340 BIZ-KNOWNN 1380.10 KNOWNN 4000 3000 2000 1000 m m a": as was as: BiaKNOWN N 13 Green KNOWN N £4000 -2000 I it .AJL ’ 8 i3 re 5762 1866 [SE [B BIZ-KNOWN N 13 YOIIOW KNOWN N tISOO ~1000 I I -500 IE 39 Biz-KNOWN N 900 600 300 78 ABi.A. Thesis data - Group 1 (re-run) PRISM Licensed to Lynn. Helton, Michigan State Police GOHOWQIG 2. 1 TtIurrrIIIITr1r1{firjYWrYj—TITfifiY1‘TVI—[rltl’I‘fI—‘fiY—I‘tr“: 100 120 140 160 180 200 220 240 260 280 300 320 340 350 llllll'Ill'llIllllllll'IIl‘lIlllllllilllll'lII‘lll‘lII'l I00 I20 I40 I60 I80 200 '220 240 250 280 300 320 340 360 CIT-KNOWN X 7 Blue KNOWN X 2000 1000 3000 2000 1000 1000 500 800 000 00 200 79 ABIA PRISM Thesis doll - Group 1 (rs-run) tat—mower w A I 14 IE “I” 12 Yellow KNOWN W 11 is m 12 Rod KNOWN w . Licensed to Lynn. Helton, Michigan State Police Genotypuc 2,1 lfi'llir'r'II—lrrj'llfIIT—TT—IIU‘IIIj—Yrilfi‘I'TIfI—IIIrIII 100 120 140 160 180 200 220 240 260 280 300 320 II‘IDIIIIIIIOI‘IOIIIIt'll!’lllllll'lIOIIll‘lllll 100 120 140 180 180 200 220 240 260 280 300 320 04—KNOWN W 12 Blue KNOWN W 2000 . RH 1000 E. m V a: an m. m 18 18 EB [Em D4—KNOWN W 12 Green KNOWN W 1000 500 800 00 200 80 ASIA Thesis data - Group 1 (re-run) PRiSM Licensed (0 Lynne Heiton. Michigan State Poiice GenotyperO 2_1 I VT 1 " I I I WI 1 I‘j I fT r I 1"] T I f l T I l I l I T—r 1—I fi‘ r T‘I— I ‘I r_l T I 100 120 140 160 180 200 220 240 260 280 300 320 I I ‘ I I I ' I I I I I I I | I I I ‘ I I «I ‘ I I I I I I I ‘ I I I ‘ I I I ' I I I l I I I I I 100 120 140 180 180 200 220 240 260 280 300 320 DIZoKNOWN AA 19 Blue KNOWN AA 3000 2000 A,jkl 1000 mg a; ’ EIIEi 115i! In a El!!! D12-KNOWN AA 19 Green KNOWN M 2000 i h 1000 mg 33 EE!!! [£11m IE 30 2 379 m BIZ-KNOWN AA 19 Yellow KNOWN AA P3000 Lzooo ;1000 *__J\.JL__ ' Ill Ell]! liflfli IIEIM IIIE! 01%KNCWWHAA waned KNOWNdAA 900 600 300 ABIA, PRISM YTIITTYT—rr'IrI’Y‘rI'l Thesis data - Group 2 Lcensed to Michigan State Police. DNA Unit Genotyper‘b 2. 1 200 220 C1-FF-KNOWN 14 Gwen FF-KNOWN ii IIII'III'III|IIIIIIIIDIIIIIIIIIIIIII'IIIIIII'III'III'Il 0 ‘00 120 ’40 ‘50 130 200 220 C1-FF0KNOWN 14 Blue FF-KNOWN 260 280 300 am 340 TVIrv—Y—rrr‘T—fYr'I'YTrTrrYYTIYr'1TV 0 100 120 I40 160 180 260 280 300 320 340 800 600 400 200 C1-F F-KNOWN 14 Yellow FF~ KNOWN (A m E U C Semi? C1-FF-KNOWN 14 Rod FF-KNOWN 8 [SEE RE HE W E :11 gm 647 300 200 100 900 600 300 82 ABIA_ Thesis data ~ Group 2 PRISNI Licensed to Michigan State Police, DNA Unit Genotypom 2,, Y’T’Y‘VrllirllIYTTIYYTTrT'I'TIIVTr I'— I‘lrerIlIITrth 0 100 120 140 160 100 200 220 240 260 230 300 320 340 IIII'III'III'III'IIIIIII'IIIIIII'IIIIIII'III'IIIlIIl‘II o 100 120 140 150 100 200 220 240 250 200 300 320 340 D4-J-KNOWN 21 Blue J-KNOWN 1000 500 El 1457 WKNOWN 21 Green J-KNOWN 2000 I | I 1000 E: l¢fifihll4fifi£ lfiiil m m m m WKNOWN 21 Yellow .Hotown L000 L400 L200 [I]!!! m 777 El! 0+040KMNN 21Ru1 .+KNOM01 900 00 300 83 14.3% Thesis date - Group 3 PR Licensed to Michigan 51019 Police. DNA 0011 GenotypetO 2.1 rr-rI—rrrrrrrf‘r r rfrrT—v—r‘F—T—r’rrrrr‘rtfirr'Y—r‘rrrr'rr F1"; r7 r7; r‘r‘rrr‘ ’80 100 120 140 160 180 200 220 240 260 280 300 320 340 11101.1111111111111101:105111111111111111;1vlgsrrgrr151 80 ‘00 120 140 160 180 200 220 240 260 280 300 820 340 CB-M-KNOWN 24 Blue Nil-KNOWN 0000 2000 1000 1 IE DE-M-KNOWN 24 Green 1.1-mama L4000 Laooo 52000 A 1 {31000 II] 10 SE Da-M-KNOWN 24 Yellow wmwn . E000 ‘faoo 4r300 at! 1079 m i 050 owmmoww 24 Bed M-KNOWN > 00 . 300 300 J ‘ 84 ASIA. PRlSM Thesis data - Group 2 Licensed to Michigan State Police. DNA Unit Genotypem 2. t rT—T fl T—FTI 1' r7—I I—III‘YVfI—rfrfifirthfirfirI’I—IV—Ir—TfiY—Yt—IY—TI—ll ii iii Jul 00 100 120 140 150 100 200 220 240 260 200 300 320 340 I‘III’IIIIIII’IIIIIII'III’III|III|III‘IlI‘III'III'IIIII so 100 120 140 160 100 200 220 240 260 200 300 320 340 010-O-KNOWN 25 Blue O-KNOWN 900 00 300 II; In 211 BE an IE £15 £213 EB DtO-O-KNOWN 25 Green O-KNOWN 1500 1000 E E] :0 E85]! {BIB Ell EB] BEE DtO—O-KNOWN 25Yellow o-KNOWN .5400 300 E200 E100 __ _ ‘ an GE an [E m: ~010-O-KNOWN 25Red O-KNOWN 000 000 400 200 85 ABIA Thesis data - group 3 PRISM Licensed to Michigan State Police. DNA Unit GenotyperO 2.1 I'IT—r—T [firrr‘trtlvrrerrII l'rrTI‘IIUm'rrWII'I‘TTYT 100 120 140 160 180 200 220 240 260 280 300 320 IIIIIIII’III'III’IIIIIII'III'DII'III'III'III‘IIIIIII 100 120 140 150 180 200 220 240 260 280 300 320 OG-U-KNOWN 23 80.10 U-KNOWN 1000 Ill 8! E 129 970 1037 D&U-KNOWN 1500 1000 500 IE EB 31-2 m m 1122] 770m [[3 1062 EH! 23Yellow U-KNOWN 600 00 200 E] [I] ,9!!! 700 518 1:0 DG-U-KNOWN Z) Hod U-KNOWN 900 00 300 86 ASIA. Thesis date 1 group 3 PRISM Licensed to Michigan State Police, DNA Unit Genotypero 2.1 YTTIUY'TYYI—IYIII'I'I’YII’VI[fit—I—rrrrYtfiTIIIII’YfiI’firIIUTrTI]I 80 100 120 140 160 180 200 220 240 260 280 300 320 340 I ' I l I ' I I I I I I I I I I I l I I I 1 I I c l | | 1 ' I‘ l 1 1 I l I I o I 1 I e I I 1 I l l 1 I l I ' I 80 100 120 140 160 180 200 220 240 260 280 300 320 340 C1 -O-KNOWN 1 4 Blue GKNOWN 800 600 400 200 IE Ill 3} ma EB Ell El Ell CPO-KNOWN 14 Green O—KNOWN 1500 1000 500 A . _ 1 -l . IRE rm: EH! EB Cl-Q-KNOWN 14 Yellow O-KNOWN 400 200 (a) 0 co CLO-KNOWN 14 Red O-KNOWN 87 AB'A Thesis data - group 3 FKQESBJI Licensed to Michigan State Police, DNA Unit Gonotypenb 2.1 Tvrrt—TIlitrlrrt[Tiflftir‘l‘r‘l'jTirltI‘I'I'w'IiIII"fi'I—IitFlt' 80 100 120 140 160 180 200 220 240 260 280 300 320 340 I 'II I‘I II III I'l III III |I II‘ III lII III II‘ III III III II' II l'l 80 100 120 140 150 180 200 220 240 250 280 300 320 340 C9BB-KNOWN 19 Blue BB-KNOWN F3000 -2ooo AAA L 11000 I13 II] III aflifl E3333 ‘065 26 EEII CS-BB-KNOWN 19 Gwen BB»KNOWN 1500 1000 A 500 E33 1317 C9~BB-KNOWN 19 Yellow ABS-KNOWN 1000 11 500 Ill 32% IE!!! 1103 900 00 300 88 ABIA Plots - c1809 results PRBM Licensed to Lynne Helton, Michignn State Police Genotypgro 2., TT'TWWI‘IYTTIT‘rUTTTT—fITWI1‘TfirT1l‘YrrhfimrTlerTVixtT YTUr‘I‘TII’II —— 80 100 120 140 160 180 200 220 240 250 280 300 320 340 360 380 400 IO'III'IIIIIII‘II. ICI‘I'I'IOI'COI'IIO‘III'III‘III‘OIO'IIIlIII‘III'S 80 100 12011$0 160 180 .200 220 240 260 280 300 320 340 360 380 400 ‘5 KNOWN F 5 5"“ KNOWN F 2000 1500 1000 500 A5 KNOWN F SGreen KNOWN F 000 2000 E m . am 0 A5- KNOWN F 5 Yellow KNOWN F 3 . 2000 -J 1000 II] III III 1523| m 1957 13 am As- KNOWN F 5 Rod KNOWN F 900 800 300 J 89 APPENDIX C Electropherograms generated from 5, 15, and 30 second samples. 90 Thesis date - Group 1 Licensed to Michigan State Police. DNA Unit 931%” Genotypero 2.1 '1 I I V] I1 I l T—jfijfiT—fiIT—lfitIYT I l'fiV—IrTYTT Ufi'UTT—r'ti UTIWTT 100 120 140 160 180 200 220 ‘ 240 260 280 300 320 340 IUI‘IOI’III'IIIIIII'IIIIIIIll!||1|I‘|II‘III‘II||‘\I'III 100 120 140 160 150 200 220 240 260 280 300 320 34a 012.0D-5 26 Blue DD-S ; 1500 1 1000 . l 500 IE 21 [EB [661 :23 012-00-5 zeemn DD-s 71500 -1000 II 4100' Eli 01200-5 26de DD—S 1500 1000 1 500 II] E] 497 IE 491 1312-00-55 269“ 00-5 800 600 00 200 91 ASIA Thesis data - Group 1 PRISM Licensed to Michigan State Police. DNA Unit Genotypero 2.1 U‘ijIVTYIVTFTII‘IVUY'I—FVTVIU‘TTUIlY—U’IIUU‘YUY'IITT'I'I' 100 120 140 160 180 200 220 240 260 280 300 320 340 II III 10' III III l.l III III .II I'l II' III .II Ill II] III 'II l'l II 100 120 ‘ 140 150 180 200 220 240 260 280 300 320 340 51-00-15 27 Blue 00-15 000 800 300 E1-DD-15 27 Green 00-15 ?eoo r800 -aoo 'V E1 -DD-15 800 800 00 ‘1 _200 A *A‘L E1-DD-15 27 Rod 00-15 00 00 200 1 *— L 92 ASIA Thesis date - Group 1 PR ISM Licensed to Michigan State Police, DNA Unit Genotypero 2.1 Tirlll’lrtT‘rrfi‘ll’UljfiYWVUUIIT'IT'IIIIIIIIIITIUri—IFI'I'IU 100 120 140 160 180 200 220 240 260 280 300 320 340 III'IIIIIII'III'III'lII'III'III.II‘I'IIIIIII'I'I'llllIII 100 120 140 160 180 200 220 240 260 280 300 320 340 5300-30 238100 00-30 -3oo L200 II L100 II] Ell HE! 135 6300-30 28 Green 00-30 53-09-30 23 Yellow oo-ao E30060 28 Red DD-m .}eoo r600 7400 r200 93 AB'A. Thesis data - group 1 PR 15M Licensed to Michigan State Police, DNA Unit GenotyperO 2.1 TrYIUIj—r‘I—TY—r Tl F—rYTT fir—VIIIYWY T711 ITTT'Y TW‘IITUY“ frl—IYYUI I TIT 10 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 ’IIIIIIIIIII’III'III'III’lll'III’IIIlIII'III'III’III,III’III| 10 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 BS-N-S “Blue N-S 400 300 200 100 86-N-5 11 Green NS 600 00 200 .0 .4 IE Ill! BS-N-S 11 Yellow NS 200 150 100 . 50 m 247 214 El stN-S 11Red N-5 900 600 300 94 Licensed to Michigan State Police, DNA Unit Genotypofg 2.1 f']'VVI'VVTVTYTIUfIYII'IIIIII—fIYYrIIIfifitfirriv[ItrlttrIrIIerf‘llv BO 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 APBEAM Thesis data - group 1 rli'UIIIOII|OIIlllll!|0|llll""I9I'III'IIOIIIIllIIIIII'OIIIIII'II 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 BB—N-15 12 Blue N-15 20 EB 88-N-15 12 Green N—15 30 20 1 0 88-14-15 12 Yellow N-15 20 10 12 Rod N-15 T 15L U1 J :22 95 ABIA Thesis date - group 1 PR 18M Licensed to Michigan State Police. DNA Unit Genotyperc 2.1 T TIT—lIjrrijfi‘f‘rrTT—T'ITT'r—VTfTIITjfiT—‘TFfiIY—IIri’f'lTfi’l’mYYUI—ITTII—I 80 100 120 140 160 160 200 220 240 260 280 300 320 340 360 380 tIIIlIIIlIII|IIIIIIIIIIIIIII‘IIIl1II|III|IIIIIII‘III|III'III'III'II 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 B10-N-30 13 Blue N30 80 60 20 B10—N-30 90 60 30 810-0140 13 Yellow N-SO 20 Ill B10—N-30 13 Rod N-SO M L i 2333 96 AB'A Thesis data ~ Group 1 PRW Licensed to Michigan State Police. DNA Unit Genotypem 2,1 YT—rIIIIIY—T FTYITTTfiT—Y'TTjilUlTfiTj rt'rirlfli‘riiirrt‘l 100 120 140 160 180 200 220 240 260 280 300 320 340 IIO.III'III’IIIIIII'III'II'IIIII'III'IIIIIII'IIIIIIIIIII 100 120 140 160 180 200 220 240 260 280 300 320 340 E11-X-5 32 Blue X~5 E1 1-X-5 32 Gmn X-5 300 200 100 354 E11-X—5 32 Yellow X-5 E11-X-5 900 00 300 97 gagsfi Thesis date - Group 1 . Licensed to Michigan State Police. DNA Unit GenotyperO 2.1 "I'YT'IYWII IjfiT—TITfiTfYr'rI—IIW—I’IIT‘FII Vivi—11W I? I'VI‘I 100 120 140 180 180 200 220 240 260 280 300 320 340 IIIIIII‘III'IIIIIII'III'III|III‘III|III‘IIIIIIIIIIIIIII 100 120 140 150 180 200 220 240 260 280 300 320 340 F2-X—15 33 3100 X45 200 150 100 50 a 10 18 EB 171 F2-X-1S 33 Green X-15 200 150 100 F2-X-15 33 Rod X-15 19500 r600 1-400 L200 L 98 AB'A. Thesis data - group 1 PRISM Licensed to Michigan State Police, DNA Unit Genotypem 2.1 Yl—TITr'fTIrVVITI'Y‘rrTUTTrT—IVrrTl’I’TTTTY—rlll‘IIUIIVTIrTT—rTiijtiIl'l 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 |II||II|III|III'lll'lll|lll|lll'IIIIIIIIIII'III'IIOIDIll|ll'|ld'.I 80 100 120 140 160 180 200 220 240 260 280 300 320 340 380 380 B12-X-30 14 Blue X-30 400 300 200 1 100 IE ‘ EH 1 812-X-30 14 Green x-eo 00 300 200 1 00 B12—X—30 14 Yellow X-GO 200 1 50 100 50 Biz-X430 14 Red x-eo 5900 3500 Leon 99 PRISM Licensed to Michigan State Police. DNA Unit Gonotypons 2.1 Ij r I W I I I r' I' 1 T r r I f—FW I T—’ 1' I ‘ f r r T ' F] V I ‘7 r r I I l 1'" U l r 100 120 140 160 180 200 220 240 260 280 300 320 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I ' I 100 120 140 160 180 200 220 240 260 280 300 320 C1-W-5 15 8100 W-S 100 50 [E [E El: 121 m m 18 IE EB m a; m CI-W-s 15 Green WS 150 100 50 frit“ L11. - 4-* ‘LvL‘Z‘: .2222- 28 IE 29 77 15 Yellow w-s 60 o 20 .51 IE E15 Ci-W-S 15 Red W-5 J- iii 1 i 353 100 ABIA_ Thesis data - Group 1 PRBM Licensed to Michigan State Police, DNA Unit Genotypero 2.1 FY'IIV—IIY—FIYYIIIV—UTT—YIIIIIIY—FUIVIII’(IlllYIT'Il‘l’IlIIIVU 100 120 140 160 180 200 220 240 260 280 300 320 340 plI'IIOlI..Illl|lll|lll'lll'lli|lllllIlllll'lll'IIOIIII 100 120 140 160 180 200 220 240 260 280 300 320 340 F8-W-15 36 Blue W-15 200 100 FEW-15 as Green W115 200 100 36 Yellow W-15 F8-W-1 5 36 Red W-1 5 l l M33 101 AB1A Thesis data - Group 1 PR W Licensed to Michigan State Police. DNA Unit GenetyperO 2.1 11'1I‘tl—er'I—IWYIIVV‘IT—TTIT‘I’ITITIT“I’TYUI'I’WI’IfiT‘T—IITUII 100 120 140 160 180 200 220 240 260 280 300 320 340 pl.IIII'III'III'lll'IlIlllI'111'III'III'IIIIIII'IIIIIII 100 120 140 180 180 200 220 240 250 200 300 320 340 F1 0-W-30 37 Blue W-ao 1500 1000 500 2000 1500 1000 500 37 Yellow W-SO soo 800 400 200 E] [E 11;: m HE cm In ‘ F10-W-30 37 Red W-SO 000 00 300 102 ABIA Thesis data - Group 1 PRISNT Licensedvto Michigan State Police, DNA Unit Genotypoie 2.1 r—rrfi I'T' 7T1 'Trj'? l rir11 1' YTUfir'r'VU—YTT—Y—Y—I—TT'j—rrfTrT‘UTrT—I 100 120 140 160 180 zoo 220 240 260 280 300 320 340 Ill'\I‘O|SI||00“I||’1I1i111'IIC'I1I|IIC|I|I,I1I'UCI'1II 100 120 140 160 180 200 220 240 260 280 300 320 340 F12-AA-5 38 Blue ANS 90 60 30 E1 F12-AA-5 38 Green AA-S 90 00 30 13 m m I!!! 0 IE F12-AA-5 38 Yellow AA-S 80 60 40 20 m F12-AA-5 38 Red M—S 800 00 00 200 103 ASIA PRISM Thesis data - Group 1 Licensed to Michigan State Police. DNA Unit Genotypero 2.1 YIITTYIIIUITITYTIj'TI'T'TWF—ITITUlrjIIrYTTIIIVI'YI'I‘UTITI‘fi 100 120 140 160 180 200 220 240 260 280 300 320 340 rlilllIlllllilll‘llllIli'IlI]llllllI’III|IIIIIII|III|III 100 120 140 160 180 200 220 240 260 280 300 320 340 Gt-AA-15 39 Blue AA-15 4O . 20 G1-AA-15 SDGreen AA‘15 100 50 121i Y Gi-AA-15 39Yeiiow AA-1s 40 30 20 10 G1-AA-15 399w AA-15 800 00 '00 200 104 ABIA Thesis data - Group 1 PRISM Licensed to Michigan State Police. DNA Unit Genotypem 2.1 III—ii—Tl—IUY—TVT'TIIYTTUIT][VIII—fillYTrlrrUlUTfltl’UITT—TITII 100 120 140 160 180 200 220 240 260 280 300 320 340 I l I ' I I I i I I I 1 I I l I I I I I I l I I I I I I I I I ' I I I I I I I I I I l l I I I I l 1 I I I I l 100 120 140 160 180 200 220 240 260 280 300 320 340 GS-AA-SO 40 Blue AA-30 300 200 100 200 100 E0 187 E0 IE 40 Yellow AA-SO 200 150 100 50 El! 0 [E 233 Ill EE 63-M-30 40 Rod AA-SO 000 00 300 105 ésgsfi Thesis data - Group 2 ' Licensed to Michlgan State Police. DNA Unit GenotypetO 2.1 'Y'i'rY—I'lrtil'rrl'l'1"I'rfififii‘rI—rITfi'lfi"Tj‘ltiilii 0 100 120 140 160 180 200 220 240 260 280 300 320 340 ' I I I ' I I I I I I I | I I I I I I I I I I I ' I I I I I I I I I I I | I I I I I I I I I I I | I I I l I I O 100 120 140 160 180 200 220 240 260 280 300 320 340 BS-FF-S 11 Blue FF-S 1500 1000 500 t1soo 1000 --500 b r600 r400 r200 BB-FFé 11 Rod FF-5 900 00 300 106 Thesis data - Group 2 Licensed to Michigan State Police, DNA Unit ASIA PRISM Genotypem 2.1 I' TTIIIlITITIITITTrj—r‘irjl’r‘l’r‘I—IY'YTr—tfi‘tY—l‘TIIWTrI‘U’I’rI o 100 120 140 150 150 200 220 240 260 280 300 320 340 blIl'III'III'III'III'III‘III'IIIIIIIIIIIIIIIIIII'IIIIII o 100 120 140 160 180 200 220 240 260 230 300 320 340 Bto—FF-ls 12 Blue FF-lS 800 600 on 200 @707“ Bio-FF-is 126mm FF-IS aoo .,eoo .400 9200 E in IE EE ES 888 EB 423 B10-FF-15 12 Yellow FF—15 300 200 100 m m EB IE B10-FF-15 12 Red FF—15 900 600 300 107 ABIA- Thesis data - Group 2 PRISM Licensed to Michigan State Police. DNA Unit Genotypero 2.1 TUIIUIYIY—rT‘I—r'III—I—TIII'rr"Irt'rl’"ljrrjt—‘rrTV—I‘ITT'T‘r o 100 120 140 160 180 200 220 240 260 230 300 320 340 'IIIIIII'III.IIIIIIIIIII'III'III'IIIIII‘IllII'III'IIIIII 0 100 120 140 160 180 200 220 240 260 280 300 320 340 B12-FF-30 13 Blue FF-ao 300 200 100 IE to En B12—FFo30 13 Green FF-SO L400 L300 L200 3100 13 Yellow FF—GO 90 60 80 EDIE 81 2-FF-30 13 Red FF-ao 800 00 00 200 108 ABIA. Thesis data - Group 2 PRISM Licensed to Michigan State Police, DNA Unit Genotypeno 2.1 'T' ‘I—Yj—Y'V'ITTTI' T—‘T'T' T I I t T Y I VT. rlfirrt I I Y Y'IT' t ' I I ‘I—fI I '—' 100 120 140 160 180 200 220 240 250 230 300 320 340 I I I ' I I I ' I I I | I I C I I I I ' I I I I I I . I ' . I i I I I | ' I I ‘ | I I ‘ ‘ I I l I I . I I I I 100 120 140 160 180 200 220 240 260 280 300 320 340 CQ‘J-5 13 Elm J-S 150 100 50 69-4-5 18 Yellow J-5 094-5 10 Rod J-5 9000 9000 5300 b- 109 ABIA_ Thesis data - Group 2 PRISM Licensed to Michigan State Police. DNA Unit acnotypcro 2.1 7W,r'I'T—‘T't'r'TTYT—YTVTYr'r"I'Vr‘Trfi—YT" VIIII‘leIrTUU 100 120 140 160 no 200 220 240 260 230 300 32o 340 II!‘III'III‘III‘III'III'IIIIIIl‘lll'lll'llI‘IOI'III'III 100 120 140 160 180 200 220 240 260 280 300 320 340 GHQ-15 19 Blue J45 1500 1000 500 k L k _ + m m [E m C11+15 19W J45 2000 1500 P‘OOO . A -500 IE m 795 C11415 19 YOIIOW $15 00 00 200 m 745 0114-15 19 Rod J-15 in: J J 35‘? 110 ABIA Thesis data - Group 2 PRISM Licensed to Michigan State P0lico.-DNA Unit Genotyporo 2.1 lIler'rrtlIITr'rfij’r—YII'UI‘VY'I'I’V‘UIUI’IIYVU'UY'Ir‘TrI'I 100 120 140 160 130 200 220 240 260 280 300 320 340 100 120 140 100 130 200 220 240 250 280 300 320 340 024-30 ZOBluo J-ao 1500' 1000 I I 500 Iii EEIJ II] 024-30 20 Gwen +30 1500 1000 00 490 txa+eo zowwflmu.400 #000 L400 E200 024-30 20 Rod J-m 800 00 00 200 111 ABIA, m. m . em 2 PRISM Licensed to Michigan State Poison. DNA Unit Gonotyporo 2.1 rIVTItIIrrT'IIVrTYYTIYTTI'T’TY—V'TTYtY—IYI’TIUW"TTI'I'IITI 0 100 120 140 160 180 200 220 240 260 280 300 320 34'!) IIIO|III'IIIIIIIIIIO'III'Ullllll'lll'lll'lllIIIO'IIOIII 0 100 120 140 160 180 200 220 240 260 280 300 320 340 AT-M-s SBiuo NM) 30 20 10 .H l ‘ , , i ‘ A l I . I ' A7-M-5 5 Gmn M-S 20 AHA-S 5 Yellow 01.5 30 20 10 A7-M-5 5 Rod M-5 112 AB'A Thesis data ~ group 2 PRISM Licensed to Michigan State Police. DNA Unit GanotyparO 2.1 YrYIY'TfI'I{Uri—T'VfirYrr'UrVTVTTr'YVIYIT—II’TIIIIVIIYTIYTII'IVII 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 PIIIIIIIIIIIIIIIIIIIIIl.l0|'lIlIltllllt'lll|IOIIlll'llIllllll 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 A7-M-15 5 Blue M45 400 300 200 ‘1 L ‘°° ’ EB IE HE A7-M-15 SGmon M-15 }000 7800 740° 200 A7-M-15 s Youow M-15 00 0 30 m n; [E13 115 IE Ill [IE EB A7-M-15 5 Rod M-15 900 600 300 113 Ucensed to Michigan State Police. DNA Unit GonotyperO 2.1 fY‘YVTIIIIrTrIIITTI‘YrT—rTUIliiflVrI‘TIUITI—TITT‘r' '1'“. UI'VUTI' $00 120 140 160 180 200 220 240 260 200 300 320 340 350 380 Ang%- Thesis dais - group 2 I.IIIOI.OlllIII.OII'III'III'lll'lllllll'lllllll'IOI|III|IOIII 100 120 140 150 180 200 220 240 260 280 300 320 340 350 380 AQ-M‘m BBIUO M-30 O 80 40 20 II} E? IE ADM-30 8 Gwen M-ao 900 600 300 114 ABIA Thesis w. . Group 2 PRW Licensed to Michigan State Police. DNA Unit Genotypeio 2.1 fivtltT—‘I—IYVTIYIT]Ir111rfritrIITf'IVTIYY‘TIfIVfiYIIIYY'rIV 0 100 120 140 160 160 200 220 240 260 280 300 320 340 llll'lilIIII'lll'lll'll‘l'il'llllI'I'lillllll'llfi'lillll O 100 120 140 160 180 200 220 240 260 280 300 320 340 82-0-5 88iue 0-5 30 20 82-0-5 8 Green 05 20 82-0-5 8 Yeliow 0-5 30 20 1 O 32-0-5 8906 0-5 900 00 115 ABIA_ Thesis data - Group 2 PRISM Licensed to Michigan State Police. DNA Unit Genotypem 2.1 TIIII'IT—rrT—ITIrIITIIIIIIITI'IIIIYYIIVIIrT—YIITTV['Irrtr O 100 120 140 160 180 200 220 240 260 280 300 320 340 I I I I I I I I | I I I I I I I I l I I l I I I ' I I I I I I I I I I I I I I I ' I I I I I I I I I I I | I I O 100 120 140 160 180 200 220 240 260 280 300 320 340 34-0-15 9 Blue (>15 40 .2 20 a! :3 84-0-15 QGreen 0-15 0 0 0 20 EB E 84-0-15 9Yeiiow 0-15 0 20 :2 84-0-15 980d 0-15 800 600 400 200 116 Thesis data - group 2 Licensed to Michigan State Police. DNA Unit GenotyperO 2.1 TU'IIYITIIIIUVI‘IITTIIYIYVIIIII‘IrT'fiITTIIII]TUW'IIII'1' 100 120 140 160 180 200 220 240 260 280 300 320 340 ABIA PRISM 100 120 140 160 180 200 220 240 260 280 300 320 340 560-30 10 Blue 0-30 0 20 IE m 380-30 10 Red 030 000 00 300 117 ABIA Thesis data ~ Group a PRW Licensed to Michigan State Police, DNA Unit Genotypero 2.1 I‘YIYI‘III'IIrII’rIII’IIIrl‘IIITTT—YIrIYIrV'I—vTrVYIIII'UII 100 120 140 160 180 200 220 240 260 280 300 320 III.III|III|III|III'III[III'III'III'III'III'III'III 100 120 140 160 180 200 220 240 260 280 300 320 Ct t-U-5 20 Blue U-5 2 0 15 C11-U-5 20 Green ch 4o 30 20 1 0 Ct 1-U—5 20 Yellow U-S C1 1-U-5 20 Red U-S 118 ABIA, PRISM Thesis data - Group 3 0241-15 D2-U-15 Licensed to Michigan State Police, DNA Unit Genotypeto 2.1 I'IITII‘VIIY'T'IUI'UT'I'Tr'l'tlTII'IUrTII—UIIiI'IIr 100 120 140 160 180 200 220 240 260 280 300 320 III'III'III'III'III'III’III'III'III'III'III'III'III 100 120 140 160 180 200 220 240 260 280 300 320 D2-U-15 21 Blue U-15 BO 60 0 20 m E! E m '02-U-15 21 Gwen U-15 21 Yellow U-15 40 20 m 21 Red U-15 _U L A y SEE 119 ASIA. Thesis date - Group 3 PRISM Licensed to Michigan State Police. DNA Unit Genotypero 2.1 'UIj—TfitiTFIITIIVrTjtTI—rVIIIUIIIIIIrV‘Ufi‘ITT'Tir'Yrr 100 120 140 160 180 200 220 240 260 280 300 320 I I I I I I I I I I I | I I I ' I I I ' I I I I I I I I I I I I l I I ' I I I I I I I I I I I | I I I 100 120 140 160 180 200 220 240 260 280 300 320 04-an0 228iue U430 0 ‘. 20 ‘ i , .' i 51-: Hi El 04-an0 226mn U-30 80 0 0 20 B [E El! [E El D4~U~3o 22 Yellow U-ao 0 30 20 10 22 Red U-3O 04-11-30 900 00 300 120 APEIA Thesis data - Group 3 ISM Licensed to Michigan State Police. DNA Unit GenotyperO 2.1 fi'rvtrTIfTTrfi‘erTrl’ITI—YwI—TI—‘IIIfi’IYrTIITT—rtjfjiriIr 100 120 140 160 180 200 220 240 260 280 300 320 III'III'III'III'III'IIIIIIIIIII'III'III'IIIIIIIIIII 100 120 140 160 180 200 220 240 260 280 300 320 88-0-5 118iue 0-5 mm m 32 M 88—0-5 11 Green 0-5 IE [E] [D me an a; 88—0-5 11 Yellow 0-5 m a m (I: m IE m ma w “Red 05 “i 1111 J 200 100 100 50 121 ABIA_ Thesis data-Group 3 PRISM Licensed to Michigan State Police. DNA Unit Genotypes-O 2.1 rr'r'ItTl[IYTIF'rr'T—T‘TYfiTITIIrl—IilrIrTj‘II'llIII’VU' 1 DO 120 140 160 180 200 220 240 260 280 300 320 III'III'IIIIIIIIIIIIIII'III'III'IIIIIIIIIII'IIIIIII 100 120 140 160 180 200 220 240 260 280 300 320 810-045 12 Blue 0-15 150 100 so IIEIIEEI IIEIIIi [IE] III! II! 810-045 12Green 0-15 00 300 200 1 100 ill IE 3 IE m Hill :53 144 £21 I!!! Ell III] 810-0-15 12 Yeliow 0-15 150 100 50 Ill .13 I12 Ilia III -li§ Ila Ill 910-015 12 Red 0-15 P900 . }600 E300 L22 ABIA Thesis data . group 3 PR ISM Licensed to Michigan suns Police, DNA Unit GenotyperO u 1'1Itt'ftl[III]IT!'IITTrUVITijUIYTW—UTII—T—TTTFU'III 100 120 140 160 180 200 220 240 260 280 300 320 I I I I I I I | I I I II I I I I I I ' I I I i I I III I I I I I I l I I I | I I I | I I I I I I I 100 120 140 130 180 200 220 240 260 280 300 320 812-030 138iue 0-30 }400 ~300 ll 9200 7100 32 E!!! E15 E15] £113 B12-O-m 13 Green 0-30 E‘ m Elli 1113 £123 Ell EEK} 812-030 13 Yellow 030 . 300 200 _#uh“ 100 [I] [3 I15 EIEJ E2!) 171 IE} EIEJ 8120-30 13 Red 030 . P900 ' }s00 5300 123 ASIA Thesis data - group 3 PRISM Licensed to Michigan State Police, DNA Unit Genotypene 2.1 I 1 I T 1’1 r I T I I I "l 0 I I I ‘l' I” T l U I T 1 Y 1' ‘ I T f I I r I I W r Y I I ' ‘ ‘ I I j 100 120 140 160 180 200 220 240 260 280 300 320 IIIIIII'IIIIIII'III'Ilt'lllllII'IIIIIIIIIIOlIIl'III 100 120 140 160 180 200 220 240 260 280 300 320 (Xyaafi HIBWO 336 .L300 1200 -1oo I13 II] E!) 1358 I!!! III E13 1! 03-885 16 Green 88—5 200 150 100 (X¥886 16th! 8845 ~1000 #500 12Ai ArggsAM Thesis data - group 3 . Licensed to Michigan State Police. DNA Unit Genotypem 2.1 ll'I‘l"[Uif‘lfilvifiITIIIY'I’TIYY—[T'VthIIrYTr!IIITYT 100 120 140 160 180 200 220 240 280 280 300 320 lll'lll'lllIIII‘III'OII'IIC'III.IOI'III'lll'llllIt! 100 120 140 160 180 200 220 240 260 230 300 320 05-8845 17 3'00 35-15 L400 L200 J__ _ __ __ ‘U . m * 517 a [m E Hi 0586-15 17 Green 88-15 05-88-15 17 Rod 88-15 900 00 00 125 ABIA Thesis data - Group 3 PRISM Licensed to Michigan State Ponce, DNA Unit Genotypcie 2,1 '7 I I I I I r I r I r I I I r 1 I I I I I I U I I I l I I I I I I I I I I I I I I j I I I I I I I I 100 120 140 160 180 200 220 240 280 280 300 320 III'III'III'lIO'IUI|III'IIIIIDD'DII'OIO'IIIIOIIIIIO 100 120 140 160 180 200 220 240 260 280 300 320 (”-38.30 18 Blue 88-30 oo 00 200 I. A JLLJI ._ - ., c__ IE ID ED 736 716 [[33 [EB 07-88-30 18 Green 38-30 00 r400 haoo a i 559 Em (27-88-33 200 I 100 IE [IE 07-88-30 18 Red 83-30 - Leon E-eoo gaoo 126 ABIA PRISM Thesis data - Group 2 Licensed to Michigan State Police. DNA Unit GenotyperO 2.1 rIr'IIIUTfIIFYITT'TT'IYI'WTI'— T'IUU'YIYIII'II'YI’III’YIIIT 100 120 140 160 180 200 220 240 260 280 300 320 340 llI'lll'lllIlll'lll'lltlllI'llI'III'IIO'OIIIIII'III|Ill 100 120 140 160 180 200 220 240 260 280 300 320 340 DS—F-S 23 Blue F-S 300 200 I I I 100 IE 18 E 114 “E [E 2:: EH] [IE rm DS—F-S zaGreen F-5 Leoo H00 I 200 J A; M L 4 E III [E] 739 E IE 06-F-5 23Yeiiow F6 150 190 50 800 00 00 200 127 AB'A Thesis data - Group 2 PRW Licensed to Michigan State Police, DNA Unit GenotypeiO 2.1 IVIUTITYVYIIiinII’T‘I—TIIIIIII'ITTTYITI—‘Vr—II'T'IIY'IIT'III 100 120 140 160 180 200 220 240 260 280 800 320 840 pll'Ill'lll'llIlIll'lll'llI'Oll'lllllll'III'IIIIIIIIIOI '00 120 ‘40 150 180 200 220 2‘0 250 280 300 320 340 03-F'15 24 Blue F-15 MOO L l I ~200 Eli EM 034-15 24 Gwen F15 19000 igeoo I I 9300 E in EE an em 03-315 24 Yellow F-ts ' zoo 200 I 100 m [EB ma EB! IE rm oe-Hs 24 Red F-15 .Esoo r800 .400 5200 128 Thesis data - Group 2 Licensed to Michigan State Police, DNA Unit ABIA PRISM Genotyper® 2.1 F0 100 120 140 160 180 200 220 240 260 280 300 1 6 0 F-SO 100 120 140 9 Blue 30 180 200 220 240 260 280 300 B44230 B4-F-3O 9 Green F-30 In! M 34-53-30 9 Yeliow F-30 'Ill'illllll'llll'llllllllll'lll'lll'lllII'O'III' 320 340 320 340 .- A“ _.__ ‘rK—Il'Vfi—TTTUVTTTVIFIVvaiiljIle‘YTTV'IIrrTIIIITjV‘UIlfl‘IVUT—I 360 380 360 380 300 200 100 600 00 200 ~200 ~150 ~100 r-SO —— v 344-30 800 600 400 200 129 AB'A Thesis data - Group 2 PRISM Licensed to Michigan State Police. DNA Unit Genotype“ 2.1 I'IIVTIYY ‘IIU—[fi—YTYUIli'r'rlr1W117TYTWrV'W YT'rTYIYIVIITT' l0 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 'III'III’III'llI’llI'lll‘llI'Ill'lll'lllllll'lll'lll'III'III' 0 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 BZ-L-S 8 Blue L-5 BZ-L-S 8 Green L-5 200 100 x a 262 m E E m £19 an BZ—L-s a Yellow L-5 0 40 20 EE Bz-L-S 900 00 300 130 ASIA- Thesis date . Group 2 PRISM Licensed to Michigan State Police, DNA Unit GenotypeiQ 2.1 I' ' T [fir T r IfT T l I I t Ij—T’fi I I I 1' hr 'j 1' I ' lfi 1 Ij I ' T—t I' ‘7 r F‘ V r r I f‘ I 1' 0 100 120 140 160 180 200 220 240 260 280 300 320 340 ' I I I l I I I ' I I I I I I I I I I I I I I I l I I I ' I I I I I I I I I I I I I I I I I I I | I I I I I I O 100 120 140 160 180 200 220 240 260 280 300 320 340 CS-L-lS 16 Blue L-tS 00 200 8% El 595 iii] “:1 a ‘1 D CS-L-ts 16 Green L-15 .300 E-BOO MOO r200 C5-L-15 16 Yellow [:15 C5-L-15 18 Red L45 9900 .—500 -aoo 131 ABIA Thesis data - Group 2 PR ISM Licensed to Michigan State Police, DNA Unit GenotyperO 2.1 VITIIUIITT'Y—f I IrrIIIIIVY‘fiIY'rIIIIIIITWITI’TT' TUT'fIfi'I O 100 120 140 160 180 200 220 240 260 280 300 320 340 p I I I | I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 0 100 120 I40 160 180 200 220 240 260 280 300 320 340 C7-L-30 17 Blue L-ao 800 600 00 Ml . I I 200 + A) AA AA A AAAAA A AA A 7 IE 18 m «0 an IE m em C7-L-30 17 Green L-30 1000 500 A. L A 12 144 13 187 200 . I 100 95 IE [EB C7-L-30 900 000 300 132 ABIA PRISM Thesis date - Group 3 Licensed to Michigan State Police. DNA Unit Genotype“ 2.1 IIIUT'III'IIT'TYIT'IrIrj'IIIYlT'IlIT,IIIW'TjI'I'I 100 120 1 40 160 180 200 220 240 260 280 300 320 IIIIIII'III'IIIIIII'IIIIIII'IIIIIIIIIIIIIIIIIII'III 100 120 1‘0 160 180 200 220 240 260 280 300 320 8244-5 BBiue HS 150 100 50 8241-5 300 200 l 100 III? IE [IE aa-u-s 8Yellow 1+5 80 0 0 20 8244-5 900 00 300 133 ASIA Thesis data - Group 3 PRISNT Licensed to Michigan State Police, DNA Unit Genotypero 2.1 Tl'fV'ITrII'Tt'W‘IrrTViI’I'IU'fIfiIIIrTrUIITYII'I’II’Y' 100 120 140 160 180 200 220 240 260 280 300 320 I II I' II I' II II I a. II a. II n .0 II II II II II II II I‘ I u. II r. II I 100 120 I40 150 180 200 220 240 250 280 300 320 M15 9 Blu. H45 800 800 400 200 -1000 $500 i2 M15 9 Yellow H45 ‘r400 ~300 #200 r100 8441-15 , 134 ASIA PRISM Thesis data - Group 3 Licensed to Michigan State Police. DNA Unit Genotypero 2.1 86-H-30 864-1-30 10 Blue H~3O ‘10 Yellow H-30 10 Red H-30 90 60 30 100 $0 80 4O 20 900 600 300 135 APPENDIX D Electropherograms generated from samples containing 3ul of PCR product. 136 ABIA Thesis data - Group 1 PRISM Licensed to Michigan State Police, DNA Unit Genotyperfl 2.1 rrIIIrTrTI—TIIITTITTIII—rrrI‘f‘I'IfTIITTTITrhrr'YT1"I’rllI 60 80 100 120 140 160 180 200 220 240 260 280 300 320 IIIIIIIIIIIIIIIIIIIIIIIIIIIIII'III'III'IIIIIIIIIIIIIIIII 60 80 100 120 140 160 180 200 220 240 250 280 300 320 8344.15 5 BIUO N-15 200 100 IE IE 88-N-15 5 Green N-15 150 I00 50 EB 31 ea-N-is 5 Yellow N-15 _ 150 100 50 III III EB BB-Nds 5 Red N-lS 600 00 200 137 ABIA Thesis data - changed amp. volume PRISM . Licensed to Michigan State Police. DNA Unit Genotypero 2.1 VIII!IIIITTII[IIrIIIITTrTITI'YIIT—T'YII'IITrfiI[IIII 100 120 140 160 180 200 220 240 260 280 300 320 340 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I l 100 120 140 160 180 200 220 240 260 250 300 320 340 A5-X-5(3UL) 4 Blue X—5(3UL) 00 200 ‘J‘A ‘ A A5-X-5(3UL) 4 Green X-5(3UL) IE [E E 699 Km HE 30.2 cm. A5-X-5(3UL) 4 Yellow x-5(3u1.) 200 150 100 1 o m. E El! HE [E In IE3 IE A5-X-5(3UL) 4 Red X~5(3UL) 00 300 200 100 138 ABIA. Thesis date - changed amp. volume PRISM Licensed to Michigan State Police, DNA Unit GenotyperO 2.1 jji—YT—rY—rTITIt—rtliifillffifiIfI’IIIrIYI'TI'T‘ftlUfiTIII’IT 80 100 120 140 160 180 200 220 240 260 280 300 320 3‘0 I I I I I I I I I I ' I I I I I I I I I l I I I I I I I I I I I I I | I I I ' I I I '~I I I I I I I I I I I I so 100 120 140 160 180 200 220 240 260 260 300 320 34c A7—X-15(3UL) 5 Blue x-zswuu 00 300 200 100 A7-X-15(3UL) 5 Queen X—15(3UL) ‘9400 IrSOO 200 E100 A7-x-15(3UL) 5 Yellow X—15(3UL) #‘ ‘ LAL ___ AA A A7-X-15(3UL) 5 Rod X-15(SUL) E400 ~3oo L200 P100 L- “‘ ‘A‘.‘ A L ‘- A 139 9R§ISIAW Thesis data - changed amp. volume Licensed to Michigan State Police. DNA Unit Genotypeto 2.1 ‘I I l I I I I I T rT T’j’l I—r f I l I I l I 1" I Y T '1 fr I I I I I I I I T I T T r I 100 120 140 160 180 200 220 240 260 280 300 I I I I I | I I I . I I I ' I I I | I I I ' I I I ' I I I I I I I | I I I I I I I | I I I 100 120 140 160 180 200 220 240 260 280 300 BSoW-15(3UL) 12 Blue W-15(3UL) 900 l 00 “I 00 W 788 IE an A; m 18 ea-w-wwuu 12 Green w-vswuu Egon on 400 L {-200 IE Ba-W-15(3UL) 12 Yellow W-15(3UL) A no ‘ zoo “2 an 451 EB £33 In sew-mam.) 12 Red W-15(3UL) {-400 r300 200 L“00 140 ABIA Thesis data - Group 1 (re-run) PRISM Licensed to Lynne Helton, Michigan State Police Genotypero 2.1 rIIY—‘rTjrlIrTTT'IIIjr‘FTII’TIjjIII—IITI‘ITT'IIIII 100 120 140 160 180 200 220 240 260 280 300 320 IIIIIIlIIIIIII'III'III'III|IIIIIIIIIIIIIII'IIIII 100 120 140 160 180 200 220 240 260 280 300 320 DG—AA-S 16 Blue AA—S .200 100 A AA- - L LT; DB-AA-S 16 Gwen AA-S 200 150 I00 50 E IQ m E *3 IE I] IE E [E E1! [IE DO—AA-S 16 Yellow M-S 200 150 100 n - - g; A» LJL- 5° III E III IE 237 me E! DB-AA-S 16 Rod AA-S on 200 141 7 ABIA Thesis dale - changed amp. volume PRISM Licensed lo Michigan State Police, DNA Unit GenotypetO 2.1 U V f T I 1' I r I I I ‘ T t I I I I 1 r r—' I l I I 'j I I 1 I 1 r I I j IV r [T ‘ Ir—i 100 120 140 160 180 200 220 240 260 280 300 o | o I o | v I I | n I I I I 1 I ' 1 I I I I 1 I I I I 1 1 o I n I I I 1 I o 1 a ' 1 1 1 100 120 140 150 180 200 220 2‘0 260 280 300 BB-M-30(3UL) 11 ENG AA-30(3UL) 200 150 100 50 gagla Bis-“mum 11 Gwen AA-30(3UL) 150 100 50 150 100 BB-AA—30(30L) 11 Red M-30(3UL) 00 300 200 100 142 ASIA Thesis data - changed amp. volume. PRW Licensed to Michigan State Police. DNA Unit Genotypeco 2.1 I I I r I l I I 1 T T I I I I I I I—I' f I I I I I I I T 1 I I I I l' I I T I I I—j' r I I I 100 120 140 180 180 200 220 240 260 280 300 100 120 140 160 180 200 220 240 260 280 300 B12-FF-30(3UL) 14 Blue FF-30(3UL) 00 00 200 RE 513 5E1 EB B12-FF-30(3UL) 14 Green FF-30(3UL) 00 00 00 200 um B12-FF~30(3UL) 14 Yellow FF-30(3UL) 200 150 100 50 Elm B12-FF-30(3UL) 14 Red FF-30(3UL) 7400 7300 7200 r100 143 ASIA. PRISM Thesis data - changed amp. volume ANSBU L) ANSQUL) w HE 3E a Yellow J-5(3UL) mm Im- a Red J-5(SUL) @BQB Licensed to Michigan State Police, DNA Unit Genotypeifi 2.1 TIfiIIIII‘T'TT‘I’IIII’jfiI’I—l—I—IIITITIIffir'fi'jIIII T—‘II' 100 120 140 160 180 200 220 240 260 280 300 I'IIIIIII'III'III'III'III'IIIIIII'IIIIIIIIIII 100 120 140 160 180 200 220 240 260 280 300 A9-J-5(3UL) 6 Blue J-5(3UL) 300 200 100 ‘ ll! El 257 E12 IE IE Eil 3E A9-J-5(3UL) 6 Green J-5(3UL) 00 400 200 150 100 ?‘°° >300 fizoo E100 144 ABIA thesis data . changed amp. voiume PRISM Licensed to Michigan State Police. DNA Unit Genotypem 2.1 IIIIrffilTTTIrIIIIIIITTIIITYIU'I—TIIIl—TITT‘IIII'T"'rlI—rIT 100 120 140 160 180 200 220 240 260 280 300 320 340 360 I I I I I I I I I I ' I I I I I I I ' I I I l I I I I I I I ' I I I I I I I ' I I I I I I I I I I I I I I I I l I 100 120 140 150 130 zoo 220 240 260 280 300 320 340 360 Ci-M-5(3UL) 15 Biue M-5(3UL) 60 4O 20 C1-M-S(3UL) 15 Green M.5(3UL) 1 00 50 E El 30 C1-M—5(3UL) 15 Yeliow M-5(3UL) ' 0 20 CI-M-5(3UL) 15 Rad M-5(3UL) 400 200 145 ABIA- Thesis data - changed amp. volume , PRISM Licensed to Michigan State Police. DNA Unit Genotypam 2.1 r I I ' t I l7 I I I I f I T I T I '1 I T- I I I I I l ' I T I T t I I 1 I ' T r I I l I I t I I I U r I T I ‘ I r 100 120 140 160 180 200 220 240 260 280 300 320 340 360 I I ' I I I ' I I I | I I I ' I I I ' I I I l I I I ' I I I I I I I I I I I | I I I | I I I ' I I I i I I I I I I 100 120 140 160 180 200 220 240 260 280 300 320 340 360 CS-O-S(3UL) 16 Blue 05mm.) 40 30 . , 20 . l 1 0 ' , I i. A . ‘ v 03-05(3UL) 16 Green O-5(3UL) 60 - 0 20 03-05(3UL) 16 Yeflow O-5(3UL) 20 1 0 ' l _ I I i I '1 ca-o-suauu 18 Red O-5(SUL) 00 200 A ‘ A- M _ _- A - 146 Thesis data - changed amp. volume Licensed to Michigan State Police. DNA Unit ABIA PRISM Gonotyperfl 2.1 r I I Y 160 I' I j 180 [YTTIITIIIYI'I—V‘I'I 200 220 240 260 280 V I I T I I I 100 120 I I' I I I 140 an' 280 I 260 220 240 140 160 180 13 BIUO U15(3UL) 200 ' I ' I 100 120 B10-U-15(3UL) HEM [EB [E 13 Green U-15(3UL) B10-U-15(3UL) B10—U-15(3UL) 13 Yellow U-15(3UL) 53 UI B10-U-15(3UL) 1 3 Rod U-15(3U L) I' ‘ I’ I' I 300 300 200 150 100 50 00 300 200 100 30 00 300 200 100 147 ABIA_ PRISM thesis data - changed amp. volume Licensed to Michigan State Police, DNA Unit Gonotypom 2.1 100 120 140 160 180 200 220 240 260 280 300 II'OII'lII.IUU'IDI'O|I'Illtllt'lII|lll' 100 120 140 160 180 200 220 240 260 280 300 CS-U-30(3UL) 17 Blue U-30(3UL) m m m [:5 ER (in CS-U-30(3UL) 17 Gwen U-30(3UL) X E 161 m 63 cs-U.30(3UL) 17 Youow U-30(3UL) m cs-u-aomuu 17 Rod U—30(3UL) 320 320 340 340 II'VW'UrIfi'IF‘IIjTW'IIIIIT‘IIWIYrYYYI'UIIU'I'I'UT‘I'T'UIII 330 360 80 40 20 150 100 50 60 00 200 148 APBIA Thesis data - changed amp. volume Licensed to Michigan State Police. DNA Unit GenotypetO 2.1 I I I V I I ‘l T I l I T r I Tfi‘ T_' fil 1 l l i I I T I I I r I I I T 1' Ti Y I I T—I' I I 100 120 1 40 160 1 80 200 220 240 260 280 300 I I I I I I I I I I I I I I I I I I I I I I I l I l I I I ' I I I I I .I I I I I I | I I l 100 120 140 160 180 200 220 240 260 280 300 Att-L—15(3UL) 7 Biue L-15(3UL) 900 600 L 300 @- IIIIE Ilia II] E113 Att-L-15(3UL) 7 Green L-15(3UL) ~1500 ~1000 1-500 A A _ LA 32 EIIE E33 317 Att-L-15(3UL) 7 Yeliow L-15(3UL) L300 .I E200 ‘ I 7100 - m. Ei33 I13 E113 A11-L-15(3UL) 7 Red L-15(3UL) L400 L300 5200 rtoo 1119 ABIL PRISM Thesis data - changed amp. volume Licensed to Michigan State Police, DNA Unit GenotyperO 2.1 V I ‘l l’ l I I l I 1' I I I I I 1' I I I I T I I V ‘I I I I I I I I T I I T‘I lfi' T I I 1 I’ r 100 120 140 160 t80 200 220 240 260 280 300 I'III'III'III'III IIIlIII'IIIIIIIIIII'IIIIIII 100 120 140 160 180 200 220 240 260 280 300 82-88-5(3UL) 9 Blue BB-SlSUL) r800 L400 L200 *4 M ‘ - M L - ‘ ‘A ’ EB IE! 82-88-5(3UL) 9 Green BB-S(SUL) 300 200 100 “.11 EB IE an E1] IE EE IE [E III] a: Bz-BB-S(3UL) 9 Yellow BB-S(3UL) 200 150 100 50 2in E13 82—88-5(3UL) 9 Red BB-S(3UL) -r400 7300 7200 100 150 Thesis data - changed amp. volume Licensed to Michigan State Police, DNA Unit ABIA, PRiSM Genotypero 2. t 1 ‘l I I I I I I I I I I I I I I I I r I r I fIIIrfiIIrIIIIII'IfiI—Iert 100 120 140 150 180 200 220 240 250 230 300 I'lII'III'III’III'III lll‘lll'lIlllll'lllllll 100 120 140 160 180 200 220 240 260 280 300 mmmu to Blue 015(301.) 400 200 52 BE EB GE :25 E? m 54415900 10 Green 015(301.) 1500 1000 500 L - in - 44L [2 IE E13 IE [E E!!! am 470 E]! m: 34-0-15(30L) 10 Yellow 0.15901.) 00 200 am m an an an: an elm-1590:.) to Red 0-15(3UL) 400 300, 200 100 151 APPENDIX E Electropherograms generated from samples containing 3ul of concentrated PCR product. 152 ASIA. Thesis data - concentrated amp. product PRISM Licensed to Michigan State Police, DNA Unit GenotypeiQ 2.1 Ill‘IIIIIIIII'TI'IIII'TTIT‘IIIIIIIIYY'ITIIIII‘II'IIIIYVIIIIIII’IIIIII'III'I 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 ll'lll'llDIIII'IOIIIII'III'Ill'lID'lll'lll'llI'tlllIIO'IIIIIIOIIII'OIIIO 80 100 120 140 150 180 200 220 240 260 280 300 320 340 360 380 400 420 A9—N—15(3UL) 4 Blue N-15(3UL) 600 00 200 A9—N-15(3UL) 4 Green N-15(3UL) 00 200 A9-N-15(3UL) 4 Yoiiow N-15(3UL) 200 100 A9—N-15(3UL) 4 Red . N«15(3UL) 111 111 111L133 153 ABIA Thesis data - concentrated amp. product PRISM Licensed to Michigan State Police. DNA Unit Genotyparo 2.1 TYIYTI’TIII[YYfIlI—‘FFITIIttfifrIifiIiiilfttI'IITTTVVI'IIY'FII 80 100 120 140 160 180 200 220 240 260 280 300 320 340' 360 l'o ll. Iii 'III 'II III ii'i II' III 'lIl Iii O'Il I'I il| llll iii '0! l'l ii 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 C1 ~X-5(3UL) 15 BIUO X-5(3UL) 1500 1000 500 C1-X-5(3UL) 15 Green X-5(3UL) -2000 ~1500 -1 000 I l -500 cr-x-slaul.) 15 Yellow x-slam.) been r800 r400 7200 9. C1 ~X-5(3UL) 1 5 Rod X-5(3UL) 11 - 1 111SEE 154 AB“; Thesis data - concentrated amp. product PR1SM Licensed to Michigan State Police, DNA Unit Genotyperw 2.1 IIITr'rrIIIITTIIIIrT—rTII—rrTIIIIIIIIIITIIIIII'll'lll'l'jI'IrI 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 'III...IIIIO'Ili'IIIlIII'|IO|III!I."IIIIIIC'III"IIIIIO|OII 80 100 120 140 150 180 200 220 240 260 280 300 320 340 360 C5-X-15(3UL) 17 Blue X-15(3UL) 2000 1500 1000 l 500 I I - J A , m 1 6 2127' 1255] 13 1a 21 5 3 cs-x-15(3UL) 17 Green x-15(3UL) 2000 1500 . 1000 l ‘ .1 500 ii! IEJF IE! 1!! 1595: 1953 I!!! 704 ll!!! v EEEII cs-x-15(3UL) 17 Yellow Xo15(3UL) 900 500 300 “.1 IE (I!) cs-x-15(3UL) 17 Red x-15(3UL) 800 00 00 200 155 ABIA_ thesis data - concentrated amp. product _ Licensed to Michigan State Police. DNA Unit Genotypero 2.1 T—rIIIYfiIT‘II'IIIIIIIIIIIrIrTITW’TrrTI'IT 200 220 240 260 280 300 320 340 360 TTTYT'IIIITIIIT 100 120 140 160 100 100 120 140 160 180 200 220 240 260 280 300 320 340 360 E5-W-15(3UL) 30 Blue W-15(3UL) 3000 2000 1000 3209 2201 18 23 1232 E5-W-15(3UL) 30 Green W-15(3UL) 2000 n 1000 [15 1209| 766 E5—W-15(3UL) 30 Yeflow W~15(3UL) 1500 1000 ‘ j 600 11] 1322 ES-W-15(aUL) 30 Red w-15(3UL) 600 00 200 156 ABIA Thesis data - concentrated amp. product Licensed to Michigan State Police, DNA Unit Genotypero 2.1 IIII'IIIjiffII’IIlrf"II—T'IIITWTIj—I‘IIIIjTrTWTIIrT'ITIIYTI'III 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 ll.ll'lll'lllllll'Illllll'lil'lll'Illllllllll'ltllIUIIIII'III 80 100 120 140 160 180 200 220 240 250 280 300 320 340 360 A5-AA-5(3UL) 4 Blue M-5(3UL) 600 600 400 200 A5.M»5(3UL) 4 Green AA-slauL) 400 L400 L ~200 RE] 30 A5oAA~5(3UL) 4 Yellow AA-5(3UL) 00 . 00 L 200 [I] :3 IE EB BEE mm 111! IE [IE A5-AA-5(3UL) 4 Red AA-5(3UL) 11111 111 157 AB'A Thesis data - concentrated amp. product PRISM Licensed to Michigan State Police, DNA Unit Genotyper® 2.1 TTIIIfi'I‘I—rTIII1YI—TIIII—[TTI]II‘V'ITTIYITlfiTrIIIIIIYrIII—rfT 100 120 140 160 180 200 220 240 260 280 300 320 340 380 1 III III III III III ill '1! 1'. II. III Ill 1'. III iii '1! ll. ll. lit III 100 120 140 160 180 200 220 240 260 280 300 320 340 360 E1oM-30(3UL) 28 Blue AA-30(3UL) 2000 1 500 1 000 500 1500 1000 500 E1-AA-30(3UL) E1~AAo30(3UL) 28 Yellow AA-30(3UL) 1000 500 l l- 11 _ , E1-AA-30(3UL) 28 Rod AA~30(3UL) 600 00 200 158 AB'A Thesis data-concentrated amp. product PRISM Licensed to Michigan State Police, DNA Unit Genotypem 2.1 TlrIlI'rTIll ll—rWIIIIII[lrf’UYTlIII'rfT—IIIIITII‘IIIIIIIIT‘I 100 120 140 160 180 200 220 240 260 280 300 320 340 360 IIIIlllllil‘lllilll'lll‘lllllII'IDIIIII'IIIIIII'III'IOI'II. 100 120 140 160 180 200 220 240 260 280 300 320 340 360 F2-FF-30(3UL) 34 Blue FF-30(3UL) 3000 2000 . l 1000 1 _ e - ---A _ IE IE I18 m 3176 F2-FF-30l3UL) 34 Green FF-30(3UL) r4000 -3000 LA! 92000 A] 71°°° x 27| 4261‘ - 1779' Y] 1 28 3597 1566 F2-FF-30(3UL) 34 Yellow FF-30(3UL) l l J @233 %300 Ilfl iii 11 1087 1049' m 12 700 F2-FF-30(3UL) emed FF-30(3UL) E600 -400 ~200 F 15S) AB'A Thesis data - concentrated amp. product PRISM Licensed to Michigan State Police, DNA Unit Genotypenv 2.1 IvIrTIITrerI—FTTI'ITIII'TTIIIIf‘rrTI‘TTTiYTIIT—rlrleITTTIIVIVI 100 120 140 160 180 200 220 240 260 280 300 320 340 360 OIIIIII'IIIIIII'III'III'IIIIIII'IIIIIIO'IIIIIIIIIII'III'II 100 120 140 160 180 200 220 240 260 280 300 320 340 360 CQ-J-5(3UL) 19 Blue J-5(3UL) 000 600 400 200 m a! 719 Gee-slam) 19 Green J-5(3UL) 1500 1000 500 {394] m 339 ce-aoslauu 19 Yellow J.5(3UL) 400 200 m 519 IE 551 E IE EH CNSGSUL) 19 Red J-S(3UL) 000 00 400 200 160 ABIA_ Thesis data- concentrated amp. product . Licensed to Michigan State Pollce, DNA Unit Genotypero 2.1 1V,IIV'l"I‘ITTTTYYrVTTIVTTI'YUI'VTI'IIIIfiIjrrrI"IIYII[III‘IIIIYI 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 so 100 120 140 160 130 200 220 240 260 230 300 320 340 360 380 82-M-5(3UL) 6 Blue M-5(3UL) 32146901.) 5 Green M-5(3UL) 00 300 200 100 E‘ m in E]?! [E HE m [E BE EB IE [it emu-slam.) 3 Yellow M-5(3UL) 200 150 100 so 82-M-5(3UL) a Red M-5(3UL) 000 00 200 161 thesis data - concentrated amp. product ABIA PRISM Licensed to Michigan State Police. DNA Unit Genotypenb 2.1 lTjIr‘iI‘ijTIIT'IIIIIIIITII'IIIl‘TrlII'IIIIIYU IIIIIIVIIr 100 120 140 160 180 200 220 240 260 200 300 320 340 360 380 IIIOI|III'IIII IIIIIII'II'I1|..1II'UIIIIIIIIII'II I'III'IIII 100 120 140 160 100 200 220 240 260 280 300 320 340 360 380 BS-O—S(3UL) a Blue O-SlSUL) 400 300 200 100 .AA A !— ‘L. Be-o-slauL) 400 200 a I Bs-o-slaul.) a Yellow O~5(3UL) 200 150 100 50 BS—O-5(3UL) a Red 05901; 600 400 200 162 AB'A Thesis data - concentrated amp. product Licensed to Michigan State Police. DNA Unit Genotypero 2.1 IIIIIfirIIYTTrIII'TTI’I‘I’IrI—I—II]III'IITIITrlI—VIrfiTIIIrI O 100 120 140 160 180 200 220 240 260 200 300 320 340 360 1|II'III'IIIIIIIlIIIIIII'Ill'lll'llI'llllIllllII'IIlIIII' O 100 120 140 160 180 200 220 240 260 280 300 320 340 360 E9-U-15(3UL) 13 Blue U-15(3UL) 600 400 200 ‘d '_A_ __ ‘ . LL- 3W _ ‘ ‘ A- L-.- “A . ' mil-I leg - a: m 739 E] gm [E HE a3 [Hi IE m m IE 17 m 167 288 an 444 m EO-U-15(3UL) 13 Green U-15(3UL) 1500 1000 500 14 m E 230 [E 247 m E9-U-15(3UL) 13 Yellow U-15(3UL) 300 200 100 Es—u-lslauL) 13 Flea U-15(3Ul.) #— 163 ABIA Thesis data - concentrated amp. product PRISM Licensed to Michigan State Police, DNA Unit Genotypr 2.1 GIVIWIIIII'ITT'IIYIIr‘I'ITIT‘ITII—I—IIIII‘IIIITIIII‘III'VIIYI’IIII‘ 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 Illll'lllllll'III'III'IIIIIIllilI'lll'Ill‘tltlIIIIOIIIOII‘ 100 120 1‘0 160 180 200 220 240 250 280 300 320 340 360 380 B 1 0°U-30(3U L) 1 1 Blue U‘30(3U L) 600 400 ; 1 200 LA. ‘ ._ - A__ 4...“ u u. m an [$25 [D Em rm IE 54 IE rm EB ma 161 200 676 BtO-U-30(3UL) 11 Green U—30(3UL) 900 600 300 B10-U-30(3UL) 11 Yellow U-30(3UL) 400 300 200 100 B10-U~30(3UL) 11 Red . U-30(3UL) 600 400 200 164 AB'A. Thesis data - concentrated amp. product PR‘SM Licensed to Michigan State Police. DNA Unit Genotypero 2.1 Y1‘rIITIII—III'II'Ir‘Ij—IUII'TTTI—TllllIIIIIjIIII'iTV—TVIfrIII'II 100 120 140 160 180 200 220 240 260 280 300 320 340 360 Illllllllllllll‘lllllll'llllll‘lltt'ttlllll'lll’Ill'llt'lI 100 120 140 150 180 200 220 240 260 280 300 320 340 360 D10-Q-15l3UL) 26 Blue Q15(3UL) 1500 1000 J 500 [E m an E141 1079 IE3] at: - E . EB DtO-Q-tS(3UL) 20 Green o-lslaul.) 000 000 2000 1000 : [E m D10—Q-15(3UL) 26 Yellow 0-15(3UL) 1000 500 El 1389 13 1116 Dto-G-15(3UL) 20 Bee o-lsalUL) 00 400 200 165 ABIA Thesis data - concentrated amp. product PRISM Licensed to Michigan State Police, DNA Unit GenotyperQ 2.1 fijw'IIIIII—IIITrTTI'I[III'IIIIIIIITI—TITIIIIIT—rI—I—TirtrrtIT'AYI' 100 120 140 160 180 200 220 240 260 280 300 320 340 360 I I I I I I I‘| I I I ' I l I ' I I l i I I I I I I I ' I I I I I I I I I I I I I I I I I I I l I I I I I I I l l I 100 120 140 150 130 200 220 240 250 230 300 320 340 350 Bees-slam) 23 Blue BB»5(3UL) 1 500 1000 500 J 4 . IE IE E11 m an DS—BB-5(GUI..) 23 Green BB—5(3UL) 900 00 300 1 51 256 IE IE DS-BB-S(3UL) 23 Yellow BB-S(3UL) 600 00 200 m an W IE IE 33 DG-BBFSGSUL) 23 Red BB-S(3UL) 600 00 200 166 AB'A_ Thesis data — concentrated amp. product PRISM Licensed to Michigan State Police, DNA Unit Genotyper® 2.1 I'II‘III'IIII‘TIIIIIIY—IITITI'rIIIrI—rmrmIIIITIIIIl'T—T'Y—Ift 100 120 140 160 180 200 220 240 260 280 300 320 340 360 III'IIIIIII'III'III'III'IIIIIIl'III'III'IIIIIII'III'III'II 100 120 140 160 180 200 220 240 260 280 300 320 340 360 D4-L-15 22 Blue L-15' 1500 1000 500 D4-L-t'5l 22 Green L-15 2000 1000 A A AA 29] IE 479 423 30 Ilfl 534 415 D4-L-15 22 Yellow L-15 400 200 ll_l 3 IE Ella-178 D4~L~1S 900 600 300 1(57 APPENDIX F Electropherograms generated from ladders and quality control samples. 168 ABIA Thesis data PRISM Licensed to Michigan State Police. DNA Unit Genotypem 2.1 IIl[TII'TIIIT‘IIIFTrTIIIIr‘IYTTIT‘rfiI‘rIlIrIfIITWTIjTVIIFD 100 120 140 160 180 200 220 240 260 280 300 320 340 360 IIII‘IIIIIIIIIIIllll'lll'Ill'III'III'III'II||III'III'III' 0 100 120 140 150 180 200 220 240 260 280 300 320 340 360 A3310 BLANK 3 BIUB 310 BLANK 80 60 A3—310 BLANK 3 Green 310 BLANK A3—310 BLANK 3 Yellow 310 BLANK 3O 20 10 A3—310 BLANK 3 Bed 310 BLANK 1000 500 169 PRISM Licensed to Michigan State Police. DNA Unit GenotyperQ 2.1 ITWTIIT—r—rIfiI—rrllIIIIrTIW—rIITII1IrIrrI—III—III‘III—TIrrTTITr'IrI—fYTI'II IO 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 IIIIIIII'IIIIIIIIIII‘III‘IIIlIII'III'III'III'IIIIIII'III'III'III'll IO 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 A3—310 BLANK 381m 310 BLANK 100 50 A3—310 BLANK 3 Green 310 BLANK 40 20 l A3310 BLANK 3 Yellow 310 BLANK A3-310 BLANK 3 Red 310 BLANK 1 1111 - 111 1‘3?" 01 170 ASIA Thesis data PRISM Licensed to Michigan State Police, DNA Unit Genotypero 2.1 IIITr—IYjI'IlIl—r'IIIrITTTrrI[IT—rrIIYIIIIIIrrIIjII 100 120 140 160 180 200 220 240 260 280 300 320 340 III I I | I I I | I I I I I I I | I I I ' I I I | I I I I I I I ' I I I I l I I | I l I I I I I I I I I I 100 120 140 160 180 200 220 240 260 280 300 320 340 A3—310 BLANK 3 Blue 310 BLANK 15 10 AIS-310 BLANK 3 Green 310 BLANK 20 I 'l I - ' . . p I : l ‘.I I." ' l . .l I ‘l I ". t I A3—310 BLANK 3 Yellow 310 BLANK 20 10 A3—310 BLANK 3 Red 310 BLANK 900 600 300 171 ABIA Thesis data PRISM Licensed to Michigan State Police. DNA Unit Genotypero 2.1 IIIIITfirfiITIIrIfIIUTIlll’I'IIIlf—ITII I—FIYII‘TT‘YIIII'IVIITI 100 120 140 160 180 200 220 240 260 280 300 320 340 360 III'IIIlIII'III'III'III'III'III'III'III'III'IIIlIIIIIII'l 100 120 140 160 180 200 220 240 260 280 300 320 340 360 A3-310 BLANK SBIue 310 BLANK 30 i 20 a 10 i i 1 1 1 1 A3310 BLANK SGreen 310 BLANK 80 60 0 20 A3-310 BLANK a Yellow 310 BLANK O 20 A3—310 BLANK 3 Red 310 BLANK 900 600 300 172 APR§IS%A Thesis data Licensed to Michigan State Police, DNA Unit Genotyperfi 2.1 T‘I—IITTI’I'II] TTI’rITIITV I‘ITI I'lij—I—IT—erU‘I—I—I [ITI] UTIIIT—I—[rlj'I 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 llIIIII'III'III'III'III'III'IIIIIII'III'III'IIIIIIIIIII'III'I 100 120 I40 160 180 200 220 240 260 280 300 320 340 350 380 A3310 BLANK 381UB 310 BLANK No Size Data A3-31O BLANK 3 Green 310 BLANK No Size Data A3—310 BLANK 3 Yellow 310 BLANK 20 15 10 . ,‘ l ‘, ‘ a g I - g . , , ‘ i. -. .. 1.. ‘ “ i 1 s . - . . - i A3—310 BLANK 3 Red 310 BLANK 1000 500 173 ASIA Thesls data PRISM Licensed to Michigan State Police, DNA Unit Genotypem 2.1 ti‘jrjr'II[TITIYT—rri’l'lritj'f'rrTTT—I'Y—TIIVIlrleIfTrIr O 100 120 140 160 180 200 220 240 260 280 300 320 340 ’III'IIIIIII'IIIIIII'III[Ill'IlI|llI|lIIllIIlIII|III|II O 100 120 140 160 180 200 220 240 260 280 300 320 340 A3-310 BLANK 3 Blue 310 BLANK 20 10 A3—310 BLANK 3 Green 310 BLANK fl‘I :- ‘ ' 1 III I | HI ~. '1" 5 A3-310 BLANK 3 Yellow 310 BLANK 1 5 i . 1 0 A3310 BLANK 3 Red 310 BLANK 600 1 IIILI+1-J1‘233 174 ASIA Thesis data PRISM Licensed to Michigan State Police, DNA Unit GenotyperO 2.1 YIfTitrIrl’IIvr'rrt—tfiitttjIV—rrfittlVIVITUTIlti’ltifi' 100 120 140 160 180 200 220 240 260 280 300 320 340 I.III'IIIIIIIIIII'IIIIIII'III.IIIIIIIIIII|IIIIIII' 100 120 140 160 180 200 220 240 260 280 300 320 340 A3-310 BLANK (3) 3 Blue 310 BLANK (3) 20 . 10 I l I ' . u i_| A3—310 BLANK (3) 3 Green 310 BLANK (3) 20 15 ' ' . I I r , 10 M3 ' -" I ' ._ . ' K ' ‘ .' :tL'; '. _ .l 9' ~.‘ 5 g .. "r A3-310 BLANK (a) 3 Yellow 310 BLANK (a) A3—310 BLANK (3) 3 Red 310 BLANK (3) 1000 500 175 . Licensed to Lynne Helton, Michigan State Police Genotyper® 2_1 ABIA Thesis data~contamination check PRISM IIIfiITITIY1I|VITIIl’T'I’rITTTU'rTj‘I’TTTY—IIIIIll'rrTIlIIUIIII'II'II‘I'I'IV I) 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 III.IIIIIIIIIIIIIII'III'IIIIIII'III'III'III‘IIIlIII‘III'IIIIIIIIIII'II D 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 A7'AMP BLANK (C) 5 Blue AMP BLANK (C) 40 20 A7-AMP BLANK (C) S Green AMP BLANK (C) No Size Data A7-AMP BLANK (C) 5 Yellow AMP BLANK (C) No Size Data A7-AMP BLANK (C) 5 Red AMP BLANK (C) M11111 333 176 ASIA Thesis data - Group 1 PRISM Licensed to Michigan State Police, DNA Unit Genotypem 2.1 L'lII'III'III'III’IIIIIII'III'III|III'III'IIIIIIIIIII'III'I 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 A5-AMP BLANK 4 Blue AMP BLANK 30 20 10 I v | i I . I . | A5-AMP BLANK 4 Green AMP BLANK 30 20 1 I , ,- , I 1' H} . .. .' I .Y I ‘ ‘lilj' i I 'i i ' AS-AMP BLANK 4 Yellow AMP BLANK 1 5 10 AS-AMP BLANK 4 Red AMP BLANK 600 7 400 200 177 ABIA Thesis data - Group 2 PRISM Licensed to Michigan State Police, DNA Unit Genotypem 2.1 Y‘ITTTWII1IITIII’TjFI—I'YI‘II'Irr'I'II'IIUI'TII‘rIT‘IjIIITIf 100 120 140 160 180 200 220 240 260 280 300 320 340 DIIIIIIIIII'III'IIIIlll’IlI'Ill'IlI'IlI'IIIIIII'IIIIIII 100 120 140 160 180 200 220 240 260 280 300 320 340 AS-AMP BLANK (2) 4 Blue AMP BLANK (2) 4O 20 l "I I ' l I AS‘AMP BLANK (2) 4Green AMP BLANK (2) 20 . 15 . _ i , t 10 , '1 I I- ," ‘1‘ ._._i 'l I . .l' 'l .7" ‘-. ‘ . ,5.“ ‘ ffl 1‘ ‘(r ‘ .l l'. ' .. "lll" 5 A5-AMP BLANK (2) 4 Yellow AMP BLANK (2) 15 10 . " '. l . I ‘ . . ', I i l . I- , ,It | . ,' f , , .: ‘ I , ,: 5 . I ".‘I '. ‘ l . It ,- -; | l 1' [till ; ‘ ’ AS-AMP BLANK (2) 4 Red AMP BLANK (2) 800 600 400 200 178 éBISfi thesis data - Group 3 ' Licensed to Michigan State Police. DNA Unit GenotyperQ 2,1 TlIT'IYUUIrf'IrrjIUII—ITB']'ffi'frI'Ii'Vr'lIrV‘UU'l 100 120 I40 160 180 200 220 240 260 280 300 320 340 u I I u I I o a I I I n . I I I I | 0 I I I I I I I I I I I I n l I l I I I I o I I o I I ' I i I I 100 120 140 150 180 200 220 240 260 280 300 320 340 AS-AMP. BLANK (3) 4 Blue AMP. BLANK (3) 4o 30‘ 20 l t ’ i 10 ' '.l , l . - -_ , . ' A5-AMP. BLANK (a) 4Green AMP. BLANK (3) 20 p 15 . I. p I; ‘ . ‘ . lo .‘ “3| ' l l I :III ‘1." ' ' 1' As-AMP. BLANK (a) 4Yellow AMP. BLANK (a) . l I I ' II .. . - l ' I' I " A5-AMP. BLANK (3) 4 Bed AMP. BLANK (3) 800 00 400 200 179 AB'A Thesis data - Group 1 . PR'SM Licensed to Michigan State Police, DNA Uni! Gonotyporo 2.1 II'7'!lililrrlllltf'lr—rllVIIIUUU‘IIIIIrTjUUrr‘TTIl’IUYIVVt'I 00 120 140 160 180 200 220 240 260 280 300 320 340 360 380 iIIIIIIIIII.IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 100 120 140 150 180 200 220 240 260 280 300 320 340 350 380 AWMKXIJOONNNJ. 58MB “KKXJCONTROL A7—WOOD CONTROL 5 Gwen WOOD CDNTROL .'| '._‘ i 1| . . 4‘ , I . | , .I - 200 150 100 50 800 800 00 200 180 A 1%:ng Thesis date - Group 1 Licensed to Michigan State Police. DNA Unit GenotyperO 2.1 I I 'U‘II‘l—T'UIV'YIYITI‘YTITWYIl'fI‘IfiIUijjjfrliiltlrj 100 120 140 160 180 200 220 240 260 280 300 320 340 IIIIIIIIIII'III'OOUII.I"IIIIUII'IIII'IIlIlI'I'I'IU'I 100 120 140 160 180 200 220 240 260 280 300 320 340 E1-MB-ILC 273MB MB-ILC 1000 I l 500 Ill 1464 an “E EIII Ei-MB-iLC 27 Gwen MB—lLC 1500 1000 500 E! [[3 I15 HE} ([1 112! ifiiiiifli 1333 [2:1 EI-MB-ILC 27 Yellow MBv-ILC ' 400 300 200 100 Ei-MB-iLC 278M MB'ILC 800 00 on 200 181 ABIA PRISM Thesis data - contamination study Licensed to Michigan State Police, DNA Unit Genotypem 2.1 l’ I’ I f'ffil I If I Y I Yrj’j’fi Y "rj rT—l' l' l' T I r I I rfijfi I T—rrl I I I I too 120 140 160 180 200 220 240 260 280 300 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I too 120 140 160 180 200 220 240 260 280 300 012—M8 (C) 26 Blue M8 (C) 400 I I M 200 IE 33 237 257 IE E 813 232 th-MB(C) 266mn MB (C) 600 00 200 IE1 IE1 in HE IE ID 734 292 a 240 um Die-Ma (C) 26 Yellow MB (C) 200 150 100 50 Ill IE 11 204| [m 129] 12 m 117 me we D12-M8 (C) 26 Red M8 (C) 900 600 300 182 ABIA Thesis data - contamination check PRISM Licensed to Michigan State Police, DNA Unit Genotypero 2,1 Il‘l’firlTT—‘IljlrlI[jIfirili[UTTITIW1TVI‘Till1U—I 100 120 140 160 180 200 220 240 260 280 300 IIIIIIIIIIIIIIIIIIIIIIIIIIIII'IIIIIIIIIIIIIII 100 120 140 160 180 200 220 240 260 280 300 E3-+CONTROL (C) 28 Blue +OONTROL (C) 1000 A I 500 32 E3»+CONTROL (C) 28 GNOfl +OONTROL (C) 1500 1000 500 E [E E IE [E m 1407 EE (ES E3+OON1ROL (C) 28 Yellow +CONTFiOL (C) @900 9600 I I 9300 m m ng m 749 II] E Es-KJONTROL (C) 28 Red +OONTHOL (C) . Leon 54500 {-400 7200 183 ABIA_ Thesis data - Group 1 PRISM Licensed to Michigan State Police. DNA Unit Genotypem 2.1 IFIrrtt'l’lTTIlrrr‘IVIIIIIYTTIIITrTIIrY'fiTfirVIIITT‘IT'II’I’IIII’f'III 0 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIiIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 50 30 100 120 140 150 180 200 220 240 260 280 300 320 340 360 E3-+ CONTROL 28 Blue +OONTROL —1ooo —soo E3—+ CONTROL 28 Green + CONTROL 2000 1500 1000 AI I47 500 E3 IE3 Ell (E! II] 1 5 5 6 EH 7 3 3 53+ CONTROL 28 Yeliow + CONTROL 1000 500 II] III II; flfiii 1014 E!!! H“ III] E3-+ CONTROL 28 Red + CONTROL 800 00 400 200 #4 A _ 184 QBIA Thesis data - Group 2 Licensed to Michigan State Police, DNA Unit Genotypens 2.1 I I I r‘T I I r r't t t T r I I I 1 l’ I I r‘rfi r I I I rtfi I I I I I r I I r I I 1 U 100 120 140 160 180 200 220 240 260 280 300 I I I I I I I I I I I l I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 100 120 I40 160 180 200 220 240 260 280 300 E5-+CONTROL (2) 29 Blue «CONTROL (2) E5-+CONTROL (2) 29 Green +CONTROL (2) 1000 see IKE mm [EB EH3 £5-+CONTROL (2) 29 Yellow +CONTROL (2) L800 9400 M P200 P III II] [£1 2:! 507 £12! [I] ES-+OONTROL (2) 29 Fled +CONTROL (2) ' I900 i800 r300 185 ABIA Thesis data - gmup 3 IDFEESIVT Licensed to Michigan State Police. DNA Unit Genotypero 2.1 T “F I’ l' I I T I i I T T r r r I I'— l I I I I I I ‘ ‘I’ Y 1"1' 1 I I I I Y I l I l’ T I I I I 100 120 140 160 180 200 220 240 260 280 300 I ' I I I l I I I I I I I I I I I I I I I ' I I I ' I I I | I I I | I I I | I I I I I I I 100 120 140 160 180 200 220 240 260 280 300 E1-+CONTHOL (3) 27 Blue +CONTROL (3) 2000 ‘AJ[ 1000 ' E!!! E1-+CONTROL(3) 27 Green +CONTROL'(3) 4000 fl 2000 E3 ‘13 El] II] EEEEI [£521 lain! 155‘ fliflfi E1o+OONTROL (a) 27 YoIIow +CONTROL (3) 2000 h I! 1000 III III I]! fiflifll 2371 {155 III EEII E1—+CONTROL (3) 27 Red «tmNTROL (3) 900 600 300 1863 Thesis data Licensed to Michigan State Police. DNA Unit ABIA PRISM 100 120 140 160 180 200 220 240 260 280 300 320 340 GenotyperO 2.1 360 100 120 140 160 180 200 A1-LADDEH-3 15 8103 LADDER 220 240 260 280 300 320 340 1 21 24 2 3 21 33 2 2 2 1 2 2 2 391 33 19 25 29 21 27 41 23 2 23 AIcLADDER-a 15 Green LADDER 24. 2 31 3 462 28. 3 3 2 77 32. 3 1 51 73 2 40 47 54 301351 2 2 29. 3 34 10. 1 2 34 21 53 3 33. 35 33 24 36 2 30. 61 11 14. 19 29 678 40 38 46 19 27. 31. 46 73 31 76 353 35. 1 107 501 27 831 62 46 32 42 34. 46 67 187 360 ABIA Thesis data PRISM Licensed to Michigan Siale Police, DNA Unit A1-LADDER-3 9 Yellow LADDER 300 200 100 A1-LADDER-3 9 Red lADDER Lilli 11-11.33? 188 ABIA. Thesis data PRISM Licensed to Michigan State Police. DNA Unit Genotypem 2.1 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 IIII‘III|III|III'III|IIIIIII'IIIIIIIIIII‘III'III’III‘III' 100 120 140 180 180 200 220 240 260 280 300 320 340 350 380 A1-LADDER‘3 9 8100 LADDER A1-LADDEH-3 189 ABI;- Thesis data PRISM Licensed to Michigan State Police. DNA Unit GonotyporO 2.1 A1-LADDER-3 15 Yolow LADDER 300 200 100 A1-LADDER-3 15 Rod LADDER LDUL A i- i- i 333 190 QEISIAM' Thesis date Licensed to Michigan State Police, DNA Unit Genotypeto 2.1 100 120 140 160 180 200 220 240 260 260 300 320 340 360 380 100 120 140 160 180 200 220 240 260 260 300 '320 340 360 380 Ai-LADDER-a 10 Blue LADDER At -LADDER-3 10 Green LADDER 24. 2 3 454 2 31 2 768 38 73 81 3 2 45 6 3 3 3 3 3 11 O 35 54 3 32 2 45 65 391 27. 34 6 31. 62 7 45 47 104 49 28. 33. 29. 34 42 30. 65 32. 191 ABIA Thesis date PRISM Licensed to Michigan State Police, DNA Unit Genotypero 2.1 A1-LADDER-3 10 Yellow LADDER 300 200 100 7 9 12 15 a 11 14 s 3 11 14 341 11 14 312 12 15 253 9 12 1s a 317 258 9 221 7 1 13 374 ‘27s 16 254 13 214 166 194 332, 13 10 217 233 191 10 31s 21s 1239 243 214 333 231 277 227 211 fifiifl 232 A1-LADDERo3 10 Rod LADDER 111 1 - 1- 1L1??? 1192 $394” Thesis date Licensed to Michigan State Police. DNA Unit Genotypem 2.1 ‘I’II'IIIIIIrIIIYIIII'I IIIIVTrIIIT‘TW—U[I—YVTIIF—rrIUTUV—I—IlVIII 100 120 140 160 180 200 220 240 260 280 300 320 340 360 III‘III'III'IIIIIII'III.III'III.III|III|IIIIIII'IIIIIII‘I 100 120 140 160 180 200 220 240 260 280 300 320 340 350 A1-LADDEH 1311.10 LADDER 000 00 . 1 ‘ . 200 J ' ' w I ‘ ‘ ' 1 . ‘ A“ .. AA A £35191” :0 I0 3.1 n; $15211- in W “film 831111211 Em 33153133] IE “:3 ENE] '13 E13113} E113 390 I!!! EIEIJEIH E115 Elligzzil 8!! 5E!) II? liifl;flil E3; £333|21a an IE] em EB EE|242 797 M Em @ 254 260 740 El] E1] 20. 200 mucosa 1 Green LADDER @900 9300 €300 |e4§ 642 193 ABIA Thesis data PRiSM Licensed to Michigan State Police, DNA Unit Genotypem 2.1 A1-LADDEFi-4 29 Yeiiow LADDER A1-LADDER-4 29 Red LADDER 194 ABIA_ Thesis data PRISM Licensed to Michigan State Police. DNA Unit Genotypene 2.1 rt r'ITfiT‘T—IIITIjITjYITT'lf'r‘r'rl'I—Vl'lI'TII'WI U—I 'Fr‘j‘T'I'r“ —‘ i0 100 120 140 160 180 200 220 240 260 280 300 320 340 360 IIIIIII'IIIIIIIIIIIIIII'III'IIIIIIIIIII'III|III'III'III| O 100 120 140 150 180 200 220 240 260 280 300 320 340 360 A1-LADDER-4 29 8100 LADDER 2 301 21 28 4 241 23 24 24 26. 19 A! -LADDER4 29 Green LADDER 195 ABIA Thesis data PRISM . Licensed to Lynne Helton, Michigan State Police At—LADDER tYeitow LADDER 300 200 i l 2 , 3 100 ’ LL ‘ —4-+ A I'éJtIIIm e in II: 3mm] Ina 5m [23 re a: ism] tie: a 223 m EB 10] 231 m 240 EB HE ma EB 286 A1-LADDEF1 1Fied LADDER 400 200 196 ABIA Thesis data ' PRISM Licensed to Lynne Hench, Michigan State Poiice Genotypeio 2.1 100 120 140 160 180 200 220 240 260 280 300 320 340 360 illlutlItl'nncltnillnI'ItojfluutuclionoluII|I0I|0|IIII||u 100 '20 ‘40 160 150 200 220' 240 260 280 300 320 340 360 A1-LADDER 1 Blu. LADDER I’d-LADDER 1 Green LADDER 88 50 63 44 34 481 401 31. 35. 37 49 197 ABIA Thesis data PRISM' Licensed to Michigan State Police. DNA Unit Ai-lADDER 1Yeiiow LADDER . 300 200 i 100 ! -.-- . - ' L U - EWH4 9mm 13mm 34010 13 16 BE [BEE EQIIEIIE a 329 is @321 mlml 372 12 266 RE [B EB @1200 322 297 [E E 241 [E2] 337 259 E25 E3} 333 213 274 1289] HE E El] HE M-LADDER med LADDER on 300 200 100 198 ABIA. Thesis data PRISM Licensed to Michigan Stats Police. DNA Unit Genotypow 2.1 pulvn—rrr.,..y,rn—,..v,.nrr..r...,...,...VH7. fl. 0 100 120 140 160 150 200 220 240 260 280 300 320 340 IiII'Illllllllllllll'llllll III'III'II D l I I l | I I I ' I I I ' I l I l 0 100 120 140 160 180 200 220 240 260 280 300 320 340 AI-LADDER 1 Blue LADDER 800 600 400 200 A1-LADDER IGmn LADDER 21.115223” 51.125523. Imam In: III -! Ilia Ell IEE EB :23 mi IE3 ER! EH SE Em 199 ABI; Thesis data PRISM Licensed to Michigan State Police. DNA Unit GenotyperO 2.1 At-LADDER 1 Yellow LADDER A1 MDDER 1 Red LADDER 200