Comparison of Four Differential DNA Extraction Methods for Casework Analysis of Sexual Assault Kit Swabs

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1 University of North Texas Health Science Center UNTHSC Scholarly Repository Theses and Dissertations Comparison of Four Differential DNA Extraction Methods for Casework Analysis of Sexual Assault Kit Swabs Francine J. Brignac University of North Texas Health Science Center at Fort Worth, Follow this and additional works at: Part of the Medical Sciences Commons Recommended Citation Brignac, F. J., "Comparison of Four Differential DNA Extraction Methods for Casework Analysis of Sexual Assault Kit Swabs" Fort Worth, Tx: University of North Texas Health Science Center; (2016). This Thesis is brought to you for free and open access by UNTHSC Scholarly Repository. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UNTHSC Scholarly Repository. For more information, please contact

2 Brignac, Francine. Comparison of Four Differential DNA Extraction Methods for Casework Analysis of Sexual Assault Kit Swabs. Master of Science (Biomedical Sciences, Forensic Genetics). May pp, 11 Tables, 13 Figures, 36 References. Sexual assault kits make up 40-50% of a typical Forensic Laboratory caseload. The traditional method to process these samples is time-consuming and requires the use of hazardous chemicals such as Phenol:Chloroform:Isoamyl Alcohol (PCIA). This study compares another manual differential extraction method and two automated methods to the traditional standard differential extraction. Results indicate that as sperm sample concentration decreases, automated methods produce superior results both in DNA quantity obtained and in quality of STR profiles produced. Automated methods reduce hands-on time, facilitate higher through-put of samples, and reduce analyst contact with hazardous chemicals such as PCIA, making it an excellent choice for labs.

3 COMPARISON OF FOUR DIFFERENTIAL DNA EXTRACTION METHODS FOR CASEWORK ANALYSIS OF SEXUAL ASSAULT KIT SWABS Francine J. Brignac, B.S.C.L.S. APPROVED: Major Professor Committee Member Committee Member Committee Member Committee Member University Member Chair, Department of Molecular and Medical Genetics Dean, Graduate School of Biomedical Sciences

4 COMPARISON OF FOUR DIFFERENTIAL DNA EXTRACTION METHODS FOR CASEWORK ANALYSIS OF SEXAUAL ASSAULT KIT SWABS THESIS Presented to the Graduate Council of the Graduate School of Biomedical Sciences University of North Texas Health Science Center at Fort Worth in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE By Francine Brignac, B.S.C.L.S., Fort Worth, Texas May 2016

5 ACKNOWLEDGMENTS I would first like to thank my thesis advisor Dr. Joseph Warren of the University of North Texas Health Science Center for the useful comments and remarks through the process of engaging in this master thesis. He consistently allowed this paper to be my own work, but steered me in the right the direction whenever he thought I needed it. Furthermore, I would like to thank Jie Sun of the University of North Texas Health Science Center for introducing me to this topic as well as all the support she has provided to me on the way. Christina Capt, M.S. and Darice Yoshishige, B.S. of the University of North Texas Center for Human Identification for helping me to refine the methodology for this thesis. Thank you Jonathan King, M.S. and Dr. Bruce Budowle from UNTHSC for use of the Applied Biosystems 3500xL Genetic Analyzer and the Qiagen EZ1 Advanced XL; Deborah Burch, M. Bryan Davis, Mary Jones Dukes, Lesley Eschinger, Dr. Mark Guilliano, and Dr. Meredith Turnbough from Qiagen for reagents and technical expertise. I would also like to acknowledge my good friend and fellow student F. Nicole Proctor of the University of North Texas Health Science Center, as I am gratefully indebted to her for her very valuable input on this thesis. Finally, I must express my very profound gratitude to my parents and to my husband, Blaise, for providing me with unfailing support and continuous encouragement throughout the process of researching and writing this thesis and the years of study leading up to it. This accomplishment would not have been possible without them. Thank you. Francine Brignac ii

6 TABLE OF CONTENTS LIST OF TABLES... iv LIST OF FIGURES... v I. INTRODUCTION AND BACKGROUND... 1 II. MATERIALS AND METHODS... 9 A. REAGENT PREPARATION... 9 B. SAMPLE PREPARATION C. DNA EXTRACTION AND PURIFICATION D. QUANTIFICATION, AMPLIFICATION AND CAPILLARY ELECTROPHORESIS III. RESULTS IV. DISCUSSION V. CONCLUSION VI. REFERENCES VII. APPENDIX VIII. DISCLOSURE iii

7 LIST OF TABLES Table 1 Equivalent amounts of Saliva and Semen in each Mixture Table 2 Two-Way ANOVA P Values Using α=0.05 to Assess Whether a Significant Difference Existed Between Method and Mixture by IPC, C T and Quantity Estimate for the Non-Sperm Fraction Table 3 Dropout in the Separate Fractions Table 4 P Values for Peak Height (PH) and Peak Height Ratio (PHR) of Unaged Samples by Mixture, Separated by Method Table 5 P Values for PH and PHR of Unaged Samples by Method, Separated by Mixture Table 6 P values for PH and PHR of Aged Samples by Mixture, Separated by Method Table 7 P Values for PH and PHR of Aged Samples by Method, Separated by Mixture Table 8 P value for Peak Height and Peak Height Ratios of Unaged samples by Mixture, separated by Method and donor peaks Table 9 P value for Peak Height and Peak Height Ratios of Unaged samples by Method, separated by Mixture and by donor peaks Table 10 P Value for PH and PHR of Aged Samples by Mixture, Separated by Method and by Donor Peaks Table 11 P Value for PH and PHR of Aged Samples by Method, Separated by Mixture and by Donor Peaks iv

8 LIST OF FIGURES Figure 1 Total DNA (ng) Extracted, Non-Sperm Fraction, Unaged Samples, Mixtures 1, 2, and Figure 2 Total DNA (ng) Extracted, Non-Sperm Fraction, Unaged Samples, Mixtures 2, and Figure 3 Total DNA (ng) Extracted, Sperm Fraction, Unaged Samples, Mixtures 1, 2, and Figure 4 Total DNA (ng) Extracted, Sperm Fraction, Unaged Samples, Mixture Figure 5 Total DNA (ng) Extracted, Sperm Fraction, Unaged Samples, Mixture Figure 6 Total DNA (ng) Extracted, Non-Sperm Fraction, Aged Samples, Mixtures, 1, 2, and Figure 7 Total DNA (ng) Extracted, Sperm Fraction, Aged Samples, Mixtures 1, 2, and Figure 8 Total DNA (ng) Extracted, Sperm Fraction, Aged Samples, Mixture Figure 9 Total DNA (ng) Extracted, Sperm Fraction, Aged Samples, Mixture Figure 10 Overall Peak Height, Non-Sperm Fraction, Unaged Figure 11 Overall Peak Height, Non-Sperm Fraction, Aged Figure 12 Overall Peak Height, Sperm Fraction, Unaged Figure 13 Overall Peak height, Sperm Fraction, Aged v

9 I. INTRODUCTION AND BACKGROUND On average, someone in the United States is sexually assaulted every two minutes [31]. DNA evidence has been haled as the greatest crime-fighting breakthrough since crossexamination [28], and its value cannot be underestimated in cases such as these. DNA evidence can determine the source of a biological sample with a high degree of certainty, which can provide invaluable support to an investigation. Though DNA cannot prove guilt or innocence, the clues provided by the genetic data recovered from this intimate sample type are highly impactful. The Attorney General issued a report in 2004 that estimated nearly 543,000 criminal cases with biological evidence were waiting DNA testing, and 169,000 of those were sexual assault cases [15]. This backlog can cause delays in the trial or sentencing of cases where the prosecution relies on DNA evidence to either acquit or sentence the suspect. According to The Innocence Project, over 300 people have been exonerated based on DNA evidence, and over 300 other people are waiting for their aid in proving their alleged innocence [33]. Many labs struggle to keep up with the high volumes of sexual assault cases due to the increasing demand for DNA testing across the board [17]. One year a lab will receive a certain quantity of cases; if the lab can only process 90% of the cases, then 10% carries over to the next year. The following year the lab must attempt to complete the new cases submitted in addition to the excess from the previous year. By the end of the second year an even greater backlog has developed. It becomes a cycle of increasing demands and the inability of labs to maintain a balanced case-in to case-out ratio. 1

10 The cycle is made worse by the CSI effect. Due to the multitude of criminal justice television shows, the common perception of a juror is that DNA evidence is required to convict a person in a criminal case. If no DNA evidence is presented, jurors may wrongfully acquit a defendant [30]. One district attorney said, Jurors now expect us to have a DNA test for just about every case. They expect us to have the most advanced technology possible, and they expect it to look like it does on television."[30] Even though labs have increased their capacity to run the lab tests, many still cannot keep up with the demands that increase at a faster rate than their own growing work capacity will allow. In 2003 New York City launched an initiative to end the sexual assault backlog. After the backlog was ended, the 18% conviction rate rose an additional 11% [19]. When looking at cases involving rape by strangers, the conviction rate jump was even more profound [19]. According to the National Institute of Justice (NIJ) the definition of a backlogged specimen is one that has not been tested for greater than 30 days after it is received in the laboratory [3]. There are several important implications of a laboratory s backlog. The most important implication is the statute of limitations. The Federal Statute of Limitations for sexual offenses is five years, and state statutes of limitations for rape range from six to fifteen years depending on the state [18]. If a significant backlog accumulates, the results of any testing performed on a sexual assault kit may no longer have probative value, and the perpetrator could not be prosecuted if a case is simply too old. DNA casework labs often have to extract DNA from aged samples. This is especially true for backlogged samples. The older a sample gets, the greater the likelihood the sample will remain bound to the substrate. This study will perform four separate differential extraction methods to determine the best method for DNA extraction of epithelial/sperm mixtures on cotton 2

11 swabs. There were two manual methods and two automated methods. Automated methods most often utilize magnetic beads as the beads lend themselves more easily to automation for DNA extraction [10] as opposed to manual methods which use Phenol:Chloroform:Isoamyl Alcohol. Yoshida et al. pioneered the first differential extraction technique to separate male and female DNA for sexual assault cases [35]. This method became the gold standard for differential DNA extraction. It was successful due to the particular nature of the sperm cell. Dithiothreitol (DTT) breaks down the disulfide bonds present in high quantities in a sperm cell s acrosome [4]. Without DTT, all non-sperm cells can be preferentially lysed separately from the sperm cells. Before differential extraction was conceived, obtaining a suspect profile from a sexual assault case was difficult, as a large quantity of female DNA can prevent detection of the male DNA present in much smaller quantities. After the traditional organic differential extraction was presented, separating the male sperm DNA from the epithelial DNA became simpler. The sample is digested in a mild detergent, leaving the sperm cells intact while lysing the more fragile epithelial cells. Afterward, the sample is centrifuged so the sperm can be pelleted at the bottom, leaving the epithelial lysate portion in the liquid supernatant. The supernatant can then be pipetted off into a new tube resulting in two separate fractions for the original sample: the epithelial lysate and a sperm fraction belonging to the male perpetrator. This technique enables independent analysis of the two fractions thereby reducing the need for mixture deconvolution. The traditional Differential Extraction, while an important breakthrough for the processing of sexual assault cases, has many drawbacks. It is time-intensive and has many hands on steps for the analyst making it labor intensive and time-consuming [27]. It uses harsh and dangerous chemicals in the form of PCIA for organic extraction. Past efforts to automate the 3

12 process have produced lackluster results. Additionally, not only is PCIA a hazardous organic chemical, but also DNA extraction success is dependent on the skill of the technician performing the extraction. To improve efficiency of processing, DNA extraction technology should be easy to use, require as minimal amount of hands-on time as possible to assist analysts to do more and maximize efficiency in the lab by enabling the ability to process multiple samples simultaneously [13]. An easy way to fulfill these requirements is an automated method. Automated methods are increasingly investigated to process DNA samples in an attempt to keep up with the laboratory workload. However, an automated method may not fulfill the needs of the lab if the process loses too much DNA. Ultimately, the purpose of this study is to identify the extraction method with the greatest DNA extraction efficiency for sexual assault samples, and which also supplies quality DNA for analysis. Identifying and selecting the best method of DNA extraction, and thereby increasing extraction efficiency, will have many anticipated benefits. Among these benefits would initially be increasing the percentage of cellular material extracted from cotton swabs. Larger amounts of DNA of superior quality would provide more useful profiles. The most effective DNA extraction and purification method would allow analysts to perform tasks with greater efficiency, which would ultimately free up analyst time for other tasks. One way to assess extraction efficiency is to measure DNA quantity and assess which method produces the greatest amount of DNA from the swabs. This will be especially essential in casework samples that contain smaller quantities of DNA to extract. However, since quantity of DNA is not sufficient, STR profile quality must be assessed. 4

13 PCIA has been used since 1956 in the purification of nucleic acids when K.S. Kirby published a paper that described a new method to isolate nucleic acids from mammalian tissues [12]. When mixtures are extracted with PCIA proteins are denatured and collected in the organic phase, nucleic acids remain in the aqueous phase, and the location where the two phases touch is known as the interphase. This new method utilized phenol to separate the organic solids. Eventually scientists realized that while phenol worked as an excellent organic solvent and is effective at denaturing and precipitating most proteins, it does not inhibit DNase activity. A combination of Phenol:Chloroform:Isoamyl Alcohol (25:24:1) resolves this problem [20, 32, 36]. Chloroform sharpens the interface between aqueous and nonaqueous phases, and mixed with phenol together denature proteins more efficiently than they each do alone [36]. The addition of isoamyl alcohol helps in avoiding foaming, which sometimes occurs with pure phenol chloroform [36]. PCIA is hazardous to work with. Phenol can easily be absorbed through the skin and causes severe burns to any biological surface it touches. It also can act as a local anesthetic, so if any contact is suspected the area should be washed thoroughly and immediately [21]. Chloroform is also a skin irritant, but it has the added hazard of being carcinogenic [29]. Adding the chloroform to phenol enhances their ability to be absorbed by the skin [21]. Another serious risk of chloroform is inhalation. The primary hazard of the last portion, the isoamyl alcohol, is its extreme flammability. Due to the risk factors listed here and others not listed, PCIA must be carefully disposed of as a hazardous waste. Early DNA testing involved cutting DNA with restriction enzymes that cut at particular sequences, separating the fragment pieces by gel electrophoresis, and then analyzing the different fragment lengths. This was called Restriction Fragment Length Polymorphism (RFLP). 5

14 Unfortunately, the method needed a great deal of DNA to run and not all crime scenes provided an adequate amount of DNA. Modern DNA analysts use Short Tandem Repeats (STRs) to individualize DNA. A breakthrough in the Polymerase Chain Reaction (PCR) process allowed for amplifying specific portions of DNA with fewer template strands [5]. PCR is the process used to make numerous copies of specific portions of DNA. An additional benefit of PCR is the ability to add markers to the strands during the replication process so the segments of DNA can be visualized with specialized instruments during capillary electrophoresis (CE). The CE analyzer captures the fluorescent signals, which can then be analyzed using software such as GeneMapper ID-X (Thermo Fisher Scientific). GeneMapper ID-X (Thermo Fisher Scientific) translates the fluorescent activity recorded in the CE analyzer into understandable STR peaks. STRs are polymorphic regions on a chromosome. Each STR location on the chromosome has a specific name, and a range of lengths based on the number of repeats present for that person. Each location, called a locus, will have a variant of the gene, called an allele, inherited from each parent. If the alleles are the same length when the STR is run, only one peak will appear; however, if the alleles are differing lengths, then two peaks will appear at the locus when the electrophoresis is run. A problem arises when DNA is mixed so that instead of a maximum of two peaks at each locus, three or more can appear. If the profile is a mixture, first the analyst must identify the minimum number of contributors [1]. Next, the analyst must identify if there is a major or minor contributor, or if the relative contributions are approximately equal [1]. If the mixture has a major and minor contributor, the peak sizes will be imbalanced. The major contributor will have taller peaks, and the minor contributor will have smaller peaks. Third the analyst must consider the possible genotype combinations of the contributors [1]. If the mixture has a major and a 6

15 minor contributor, identifying the combination of the major contributor would be facilitated by the larger peaks, however the minor contributor may have some peaks obscured by the major contributor if the two contributors share an allele call at a particular locus [1]. Once major and minor peaks have been determined, statistical analysis must be performed. Sexual assault samples are collected on cotton swabs, allowed to dry and sent to lab. The typical cell types found in sexual assault samples are epithelial cells and sperm cells. Epithelial cells of all types may be present based on the type of sexual assault and penetration. Ultimately, there is little difference beyond histological between the different epithelial cell types and are treated identically [16, 8]. The original struggle labs had was to separate profiles from the two differing contributors. With the breakthrough to lyse sperm cells separately from the non-spermcells, perpetrator DNA can be isolated from victim DNA, which would produce a single-source STR profiles of the perpetrator separate from the victim. Often, when extracting sample from the cotton substrate, a percentage of cellular material remains bound to the cotton swab and ultimately becomes discarded when the substrate is removed from the supernatant. This problem becomes exacerbated with aged samples. Current extraction methods for DNA recovery of spermatozoa in sexual assault cases typically leave behind a significant portion of the cellular material on the cotton swab substrate. In cases with small amounts of DNA present, it is especially important to extract as much of the material as possible for analysis. Older samples are more difficult to separate from their substrate [11]. The standard differential extraction pioneered in 1995 using PCIA is the gold standard for any kind of differential extraction. In this study, three different methods were compared against the standard differential extraction method. 7

16 The first alternate method used was similar to the standard differential extraction save for the first step. A different detergent was used to separate the cellular material from the cotton substrate and lyse the epithelial cells. Additionally, the solution was incubated at a lower temperature more suited to the activity of Proteinase K (ProK) before the non-sperm fraction was separated from the sperm fraction. These changes were made to optimize the lysis buffer and incubation conditions. Afterwards, the steps were identical to the standard differential extraction technique. The second and third methods that were used were automated purification methods. The AutoMate Express TM DNA Extraction System (ThermoFisher Scientific, Carlsbad, CA) did not have a specific procedure for differential extractions, but was easily modified for differential extraction purification of DNA. The other automated method, the QIAcube and the Qiagen EZ1 Advanced XL (Hilden, Germany), had a protocol that was used for purification purposes. Both methods used magnetized silica beads to isolate the DNA and rinse the rest of the cellular material out. Both methods use proprietary formulae for their DNA extractions. Both methods were chosen for their automation and to also compare the silica bead purification performance methods to the organic purification methods used. DNA binds to silica beads in the presence of chaotropic salts and low ph solution. The chaotropic salts formed a salt-bridge between the negatively charged DNA backbone and silica particles. The beads are then attracted to one side of the tube with a magnet to allow the DNA and beads to be washed so contaminants would be removed. A low salt-high ph solution was used to release the DNA from the silica beads [26]. 8

17 One reason these comparisons were made was to test the efficacy of the traditional differential extraction method that was pioneered in 1995 by Kanako Yoshida against other methods that were developed later and had more research to draw upon to adjust the process for lab work. Developing the differential extraction was important, but it was just the first step. As two of the methods tested were automated, another reason to compare the methods was to see if analyst time could be freed from the time-intensive standard differential extraction with a comparable or superior method. Also, the comparison of manual method results to automated method results was determined to be beneficial, as manual methods require a skilled analyst to extract the DNA efficiently with minimal loss of DNA. An unskilled analyst could negatively affect the amount of DNA ultimately extracted and purified from a sample. One of the concerns of this study is to investigate whether there is an automated method that is capable of extracting and purifying DNA as well as or better than the manual method. Simply increasing the quantity of DNA extracted is not sufficient, as DNA of appropriate quality is necessary to give a complete STR profile. Greater efficiency of sample processing would help reduce sample backlog. With less backlog, it is more likely that criminals will be brought to justice when caught instead of being released due to lack of evidence. On the other hand, more innocent accused will be exonerated with DNA evidence to corroborate innocence of that crime. The hypothesis of this study was that the automated methods would perform the process of purification more efficiently than the manual methods, and of the two automated methods, the Qiagen method would be superior to the AutoMate method in its consistent results due to the minimized hands-on time for the analyst. II. MATERIALS AND METHODS A. Reagent Preparation 9

18 Buffers and supplies were made to the following specifications. For TE -4 buffer, 10mM Tris-HCL (Invitrogen, Life Technologies) and 1mM EDTA (Invitrogen, Life Technologies) solution was made with molecular biology grade water (Genemate, BioExpress) with a final ph of 8.0. The Lounsbury buffer was made with 10mM Tris (Invitrogen, Life Technologies), 10mM MES [2-(N- morpholino)ethanesulfonic acid] (Sigma-Aldrich), and 1% SDS (Gibco, Thermo Fisher Scientific) and brought to volume with water to have a final ph of 8.7 after adjustment with NaOH (Thermo Fisher Scientific). The Stain Extraction Buffer (SEB) was made with 10mM Tris-HCl (Invitrogen, Life Technologies), 0.1M NaCl (Sigma-Aldrich), 2% Sodium Dodecyl Sulfate (SDS) (Gibco, Thermo Fisher Scientific), and 10mM EDTA (Invitrogen, Life Technologies) and brought to volume with MBGW (Gibco, Thermo Fisher Scientific) for a final ph of 7.1. B. Sample Preparation The swabs were made with three different mixtures with each mixture run in triplicate for each method for aged and unaged samples. To simulate aged and unaged samples the same mixture was applied to swabs at two different times. Each method had a total of 18 initial swab samples run. Each method produced 18 non-sperm fractions, and 18 sperm fractions. The automated methods each had 2 reagent blanks, while the manual methods each had 1 reagent blank. One analyst ran the study samples to ensure the swabs were as similar age as possible when processed. To decrease variation, there was one male sperm donor and one female donor for the non-sperm fraction. To simulate female DNA from a sexual assault sample, saliva was collected and used due to its similarity to vaginal epithelial cells. Before sample was added to swab heads, the cotton swabs were removed from the applicator stick. After application of the 10

19 sample, the swabs that were to mimic aged swabs were allowed to dry for 2 weeks in a 36 C (±1 C) heat oven, then allowed to remain at room temperature for an additional 6 weeks. This allowed the swabs to dry further and mimic the behavior of an aged sample [11]. The unaged swabs were created with the same protocol and mixtures created for use in the aged swabs and allowed to dry at room temperature for 3 days before processing. For all differential mixtures, the dilutions were vortexed for an initial 20 seconds, and an additional 10 seconds after each third swab head. The first differential extraction mixture required 5000μL saliva to be diluted with 5000μL TE -4 (1/2 dilution); and 500μL semen to be diluted with 500μL TE -4 (1/2 dilution). Next, 1800μL diluted saliva was mixed with 600μL of the diluted semen. For a final quantity of 10μL of the 1/2 diluted semen and 30μL of the 1/2 diluted saliva, 40μL of the mixture was applied to each swab. The equivalent amount of undiluted semen and saliva were 5μL and 15μL respectively. The second differential extraction mixture required that 100μL of the 1/2 dilution of semen was further diluted with 400μL TE -4 for a total 1/10 dilution. Next, 1,800μL of the 1/2 diluted saliva was mixed with 300μL of the 1/10 diluted semen. For a final quantity of 5μL of the 1/10 diluted semen and 30μL of the 1/2 diluted saliva, 35μL of the mixture was applied to each swab. The equivalent amount of undiluted semen and saliva were 0.5μL and 15μL respectively. For the third differential extraction mixture 100μL of the 1/10 dilution of semen was diluted with 900μL TE -4 for a total 1/100 dilution. Next, 3000μL of the 1/2 diluted saliva was mixed with 500μL of the 1/100 diluted semen. For a final quantity of 5μL of the 1/100 diluted semen and 30μL of the 1/2 diluted saliva, 35μL of the mixture was applied to each swab. The equivalent amount of undiluted semen and saliva was 0.05μL and 15μL respectively. 11

20 Table 1: Equivalent Amounts of Saliva and Semen in each Mixture Total Volume Equivalent Saliva Equivalent Semen Mixture 1 40μL 15μL 5μL Mixture 2 35μL 15μL 0.5μL Mixture 3 35μL 15μL 0.05μL C. DNA Extraction and Purification Method 1: Standard Differential Extraction [35] Each cotton swab head was digested in 450μL Stain Extraction Buffer (SEB), and 10μL of 20 mg/ml ProK in a 1.5mL conical tube. Each tube was vortexed for 20 seconds, pulse spun to remove liquid from lid and incubated at 56 C (± 1 C) for 2 hours. After incubation, the substrate was moved to a spin basket and spun down at 5,000 x g for 2 minutes. After disposal of the spin basket and substrate, the tube was centrifuged at 10,000 x g for 5 minutes. What was to become the non-sperm fraction of the sample was found in the supernatant and was carefully removed from the original tube and transferred to another appropriately labeled tube. The non-sperm fraction was set-aside until the PCIA extraction step. The remaining fraction in the original tube was the sperm fraction. The sperm fraction was washed three times by adding 500μL SEB, briefly vortexing and centrifuging at 10,000 x g for 5 minutes each wash step, discarding the supernatant at the end of each cycle. After the third wash, 500μL molecular biology grade water (MBGW) was added and the tube was briefly vortexed and centrifuged at 10,000 x g for 10 minutes. All but 50μL of the liquid was discarded. 12

21 The sperm pellet was digested with 450μL SEB, 10μL ProK, 20μL DTT (1.0M). The tubes were briefly vortexed and pulse spun to mix thoroughly and remove liquid from the cap, then incubated at 56 C (± 1 C) for 2 hours. The tubes were pulse spun to force condensation to the bottom of each tube. 500μL of PCIA was added to each sample of both fractions to purify the DNA. The samples were vortexed for 30 seconds to attain a milk emulsion, then centrifuged for 3 minutes at 16,300 x g. The aqueous layer was removed from each tube and transferred to a new appropriately labeled tube for ethanol precipitation. 1.0 ml of cold absolute ethanol was added to each sample tube and vortexed for 5 seconds. The samples were placed in a freezer at -20 C for 30 minutes. After the cold incubation, the samples were centrifuged at 16,300 x g for 20 minutes and afterwards the alcohol was decanted. Next, 1.0 ml of 70% ethanol was added to each tube and spun at maximum speed 16,300 x g for 10 minutes. The 70% ethanol was decanted and allowed to evaporate in the 56 C (± 1 C) heat block. Once dry, each tube had 100μL TE -4 added, was vortexed briefly, then incubated at 56 C (± 1 C) for 30 minutes to resolubilize the DNA. Method 2: Lounsbury s Modified Differential Extraction with Optimized Incubation Buffer and Incubation Times [14] Each swab had 500μL of the Lounsbury buffer, and 5μL of 20mg/mL ProK added. Each tube was incubated at 42 C (± 1 C) for 30 minutes. The tubes were then vortexed briefly and centrifuged to remove condensation from the lid. The substrate was transferred to a spin basket and centrifuged at 5,000 x g for 2 minutes to remove as much cellular material as possible from the substrate. 13

22 Each tube was then centrifuged 10,000 x g for 5 minutes. The supernatant was transferred to a new appropriately labeled tube as the non-sperm fraction. The non-sperm fraction was put aside until PCIA purification. The remaining fraction in the original tube was the sperm fraction. At this point the two fractions were treated identically to the standard differential procedure as listed in Method 1. Method 3: Modified AutoMate Express Forensic DNA Extraction System (Thermo Fisher Scientific). [2] Just as in the standard differential extraction method, each cotton swab head was digested in 450μL SEB lysis solution, and 10μL of 20 mg/ml Pro K. The tubes were vortexed for 20 seconds and pulse spun to remove liquid from the lid, then incubated on a thermal shaker at 56 C (± 1 C) for 2 hours. After incubation, the substrate was moved to a spin basket and spun down at 5,000 x g for 2 minutes. The spin basket was removed and discarded, and the tube centrifuged at 10,000 x g for 5 minutes. The supernatant was carefully removed and transferred to an appropriately labeled AutoMate PrepFiler Sample Tube as the non-sperm fraction. The non-sperm fraction tubes were loaded onto the AutoMate Express Forensic DNA Extraction System and run with the PrepFiler Express method to purify the non-sperm fraction. The sperm fraction was washed three times by adding 500μL SEB, briefly vortexing then centrifuging at 10,000 x g for 5 minutes and discarding all but 50μL of the supernatant at the end of each cycle. After the third wash, 500μL MBGW was added to each tube, then briefly vortexed and centrifuged at 10,000 x g for 10 minutes. All but 50μL of the supernatant was discarded and 14

23 150μl SEB was added to each sample (volume of sperm cell fraction at this point was 200μL). Each sample had 10μL 20mg/ml ProK and 20μL 1.0M DTT added. The samples were briefly vortexed and pulse spun, then incubated on a thermal shaker at 56ºC (± 1 C) for 2 hours. After incubation the sperm cell fraction lysate was transferred to a new, appropriately labeled PrepFiler LySep Column/hingeless PrepFiler Sample Tube and centrifuged. After centrifugation 300μL PrepFiler Lysis Buffer was added and centrifuged again to wash any lingering DNA into the column. This brought the total volume to 530μL. After briefly vortexing to ensure the contents were well mixed the column/tube was centrifuged for 2 minutes at 10,000 x g remove any liquid from the filter column. The sperm fraction tubes were loaded onto the AutoMate Express Forensic DNA Extraction System and run with the PrepFiler Express Method. Method 4: Qiagen EZ1 Advanced XL purification method (Hilden, Germany) [24, 25, 26] Each cotton swab was digested with 480μL Buffer G2 and 20μL ProK provided with the kit. The tubes were vortexed for 10 seconds then incubated at 56 C (± 1 C) for 2 hours in a thermal mixer at 900rpm. The tubes were then centrifuged briefly to remove liquid from the lid and the substrate was placed in a spin basket and spun down to dislodge cells from the substrate. The spin basket and substrate were discarded. The samples were loaded in the rotor adaptor along with the appropriate reagents and supplies in the QIAcube as indicated in the protocol. The protocol Separate and Lyse 12A Mod was selected to separate the non-sperm fraction. Once the run completed, the non-sperm fraction was removed for DNA extraction and the reagents and supplies were reloaded. The non-sperm fraction was ready for purification after adding 400μL warm Buffer MTL to each tube. After 15

24 placing the sample tube in row 4 of the Qiagen EZ1 Advanced XL and loading the appropriate supplies and reagents the DNA Purification (Large-Volume Protocol) was selected and run for the epithelial cells. The procedure Separation and Lysis 12B Mod was then selected to complete sperm fraction washing and to prepare for lysis. 160μL Buffer G2, 10μL ProK and 40μL 1.0M DTT was added to each sperm fraction tube and vortexed then incubated at 70 C (± 1 C) for 10 minutes. The sample tubes were vortexed for 10 seconds and briefly centrifuged before being loaded onto the Qiagen EZ1 Advanced XL and run with the protocol DNA Purification (Trace Protocol). D. Quantification, Amplification and Capillary Electrophoresis After extraction, all samples were quantified using the Quantifiler Trio DNA Quantification Kit (Thermo Fisher Scientific, Carlsbad, CA). Quantifiler Trio (Thermo Fisher Scientific) calculates the amount of diluent and source DNA needed for normalization based on the kit used for amplification. The DNA was normalized according to the Quantifiler Trio DNA Quantification Kit (Thermo Fisher Scientific) instructions for the GlobalFiler PCR Amplification Kit (Thermo Fisher Scientific). Two-way ANOVA testing using RStudio (RStudio Inc., Boston, MA) was performed on the Internal PCR Control (IPC), C T, and Quantity Estimate by method and by mixture to assess if a significant difference in total DNA quantity existed between the extraction methods and between the three mixtures. The normalized samples were amplified on a 9700 GeneAmp PCR System 96 well Thermal Cycler (Thermo Fisher Scientific). The STR profiles were generated on a 3500xL Genetic Analyzer (Thermo Fisher Scientific). It was supplied with POP-4 polymer (Thermo 16

25 Fisher Scientific), and a 36cm capillary, and calibrated using the DS-36 Matrix Standard (J6 Dye Set) (Thermo Fisher Scientific). The STR results were assessed with GeneMapper ID-X version 1.4 (Thermo Fisher Scientific) set to the kit manufacturer s analytical settings. Artifacts were manually removed from all profiles, and the resulting profile information was exported to Microsoft Excel (Microsoft Corp., Redmond, WA) for further analysis. Average peak heights were calculated by profile. Two-way ANOVA testing using RStudio (RStudio Inc., Boston, MA) was performed on the peak heights by mixture and by method if a significant difference in peak heights existed between the four fractions (S and non-s, aged and unaged), and if significant difference was found it was followed by a TukeyHSD to see where the difference was. Peak height ratios for the saliva donor and the semen donor were calculated after subtracting shared peaks. Two-way ANOVA testing using RStudio (RStudio Inc., Boston, MA) was performed on the peak height ratio by mixture and by method if a significant difference in peak height ratios existed between the fractions and donors. If significant difference was found it was followed by a TukeyHSD to see where the difference was. III. RESULTS Quantification and Analysis DNA extraction and purification was completed in three days to minimize the effects of aging samples. Due to limited space on the automated analyzers, the first two replicates of each sample was placed on the first run with the third replicate being placed on the second run for each fraction. All samples waited at room temperature to be loaded on the machines. After adding the 300μL PrepFiler TM Lysis Buffer to the samples before being placed on the AutoMate 17

26 Express Forensic DNA Extraction System, a white precipitate formed in the samples tubes and remained present in original samples tubes after DNA purification. Also, the second replicate for the unaged non-sperm fraction did not purify. After purification protocol was run, the reagent tray displayed bubbles in the reagent wells. The original sample tube had been discarded and DNA could not be recovered. The standard differential and Lounsbury methods were run on the same day. The standard differential samples were placed on the incubator before the Lounsbury method was started. After the initial incubation both methods were identical, so the methods were batched together. Sperm and non-sperm fractions separated the quantification runs. Both runs yielded an R 2 regression value of or greater without deletion of standard points. Two-way ANOVA testing using α=0.05 was performed on the quantification values on the IPC, C T, and Quantity Estimate between each method and mixture in each fraction. In the non-sperm fraction the IPC C T values ranged from 25.4 to 26.3, and for the sperm fraction the IPC C T ranged from 25.5 to As the C T measures the threshold where DNA can be measured above background noise, none of the C T values indicated the samples were inhibited. The sample C T values in the non-sperm fraction ranged from 26.6 to 32.6, and ranged from 24.2 to 37.0 for the sperm fraction. For both fractions, the higher C T values belonged to samples that had a higher dilution value and required more cycles for DNA to be detected. A possible reason for Table 2: Two-Way ANOVA P Values Using α=0.05 to Assess Whether a Significant Difference Existed Between Method and Mixture by IPC, C T and Quantity Estimate for the Non-Sperm Fraction. The results indicated that each group had a significant difference in the fractions except Quantity Estimate (QE) by Method. All values were then subjected to a TukeyHSD test to assess where the difference lay. Significant values are represented in gray. Table A represents the 18

27 adjusted p values for IPC by method. Table B represents the adjusted p values for C T by method. Table C represents the adjusted p values for QE by method. Table D represents the adjusted p values for IPC by mixture. Table E represents the adjusted p values for C T by mixture. Table F represents the adjusted p values for QE by mixture. IPC by Method C T by Method 19 Quantity Estimate by Method IPC by Mixture C T by Mixture Quantity Estimate by Mixture Non-S fraction Unaged E E-13 S fraction Unaged E E-10 Non-S fraction Aged E-05 S fraction Aged E E-14 A. IPC by Method Non-S fraction Unaged S fraction Unaged Non-S fraction Aged S fraction Aged Lounsbury-AutoMate Qiagen-AutoMate Standard Differential-AutoMate QIAgen-Lounsbury Standard Differential-Lounsbury Standard Differential-QIAgen B. C T by Method Non-S fraction Unaged S fraction Unaged Non-S fraction Aged S fraction Aged Lounsbury-AutoMate Qiagen-AutoMate Standard Differential-AutoMate QIAgen-Lounsbury Standard Differential-Lounsbury Standard Differential-QIAgen C. QE by Method Non-S fraction Unaged S fraction Unaged Non-S fraction Aged S fraction Aged Lounsbury-AutoMate Qiagen-AutoMate Standard Differential-AutoMate QIAgen-Lounsbury Standard Differential-Lounsbury Standard Differential-QIAgen D. IPC by Mixture Non-S fraction Unaged S fraction Unaged Non-S fraction Aged S fraction Aged Mixture 2-Mixture Mixture 3-Mixture Mixture 3-Mixture E. C T by Mixture Non-S fraction Unaged S fraction Unaged Non-S fraction Aged S fraction Aged Mixture 2-Mixture Mixture 3-Mixture Mixture 3-Mixture F. QE by Mixture

28 Non-S fraction Unaged S fraction Unaged Non-S fraction Aged S fraction Aged Mixture 2-Mixture Mixture 3-Mixture Mixture 3-Mixture this in the non-sperm fraction could be the addition of greater amounts of diluent when creating the dilutions for the mixtures or settling of the sample despite care taken to preserve homogeneity when creating the dilutions. Also, while attempts were made to keep the approximate amount of saliva consistent from sample to sample in all mixtures, semen also carries epithelial cells as well as sperm cells that could contribute to the overall DNA present in a sample. Due to the presence of the semen epithelial cells, the overall amount of DNA in the nonsperm fraction could easily drop from one mixture to the next mixture. Results of two-way ANOVA analysis of the IPC, C T and DNA Quantity Estimate can be found in Table 2. All results were subsequently analyzed with a TukeyHSD (honest significance difference) test in RStudio (RStudio Inc., Boston, MA). The analyses from Table 2 that indicated a significant difference can be found in sub-tables A-F. Only the pairwise comparisons from the TukeyHSD that indicated significant difference were highlighted in gray. Even though the Quantity Estimate (QE) by Method did not indicate a significant difference was present, the TukeyHSD results were also included. A significant difference in the quantification results was found mostly in C T by mixture and in QE by mixture, which would be expected as C T rises as the DNA quantity decreases, and the quantity estimate would decrease as the actual amount of DNA decreases as well. The lack of a significant difference in the IPC by method and mixture would also be expected as none of the methods should have inhibitory factors remaining after extraction and purification. 20

29 The values for the unaged non-sperm fraction can be found in Figures 1-2. Due to the dramatic difference between Mixture 1 and the remaining two mixtures, the Mixtures 2 and 3 are represented on a graph without Mixture 1. For the non-sperm unaged fraction, the variability of the specimens is highest in Mixture 1, with the amount of variation decreasing as the mixtures become more dilute. The total amount of DNA present in the non-sperm fraction drops severely from mixture 1 to mixture 2, but less so from mixture 2 to mixture 3. A reason behind this is the presence of epithelial cells in semen. These epithelial cells would still be lysed in the first lysis of the sample leaving DNA from the semen donor in the non-sperm fraction. The values for the unaged sperm fraction in can be found in Figures 3-5. Due to the dramatic difference between the three mixtures Figure 3 holds the three mixtures for comparison, but Figures 4-5 hold Mixture 2 and Mixture 3 respectively. This fraction has greater variability than the non-sperm fraction. This is where the elution of sample from the cotton swab is most important, as it is difficult to elute the sperm from the cotton substrate and becomes more difficult as time progresses [6]. Unlike the non-sperm fraction, the sperm fraction variability remains high. The values for the aged non-sperm fraction can be found in Figure 6. The variability of the mixtures in the aged non-sperm fraction specimens is lower when compared to the aged sperm fraction samples. 21

30 DNA Quantity (ng) DNA Quantity (ng) Mixture 1 Mixture 2 Mixture 3 Standard Differential Lounsbury AutoMate Qiagen Figure 1: Total DNA (ng) Extracted, Non-Sperm Fraction, Unaged Samples, Mixtures 1, 2, and 3. The bars represent the Standard Error present in each set of triplicate. Qiagen Mixture 2 does not have a Standard Error bar due to the failure to purify of one of the replicates Mixture 2 Mixture 3 Standard Differential Lounsbury AutoMate Qiagen Figure 2: Total DNA (ng) Extracted, Non-Sperm Fraction, Unaged Samples, Mixtures 2, and 3. The bars represent the Standard Error present in each set of triplicate. 22

31 DNA Quantity (ng) DNA Quantity (ng) Mixture 1 Mixture 2 Mixture 3 Standard Differential Lounsbury AutoMate Qiagen Figure 3: Total DNA (ng) Extracted, Sperm Fraction, Unaged Samples, Mixtures 1, 2, and 3. The Bars represent the Standard Error present in each set of triplicate Mixture 2 Standard Differential Lounsbury AutoMate Qiagen Figure 4: Total DNA (ng) Extracted, Sperm Fraction, Unaged Samples, Mixture 2. The Bars represent the Standard Error present in each set of triplicate. 23

32 DNA Quantity (ng) DNA Quantity (ng) Mixture 3 Standard Differential Lounsbury AutoMate Qiagen Figure 5: Total DNA (ng) Extracted, Sperm Fraction, Unaged Samples, Mixture 3. The Bars represent the Standard Error present in each set of triplicate Mixture 1 Mixture 2 Mixture 3 Standard Differential Lounsbury AutoMate Qiagen Figure 6: Total DNA (ng) Extracted, Non-Sperm Fraction, Aged Samples, Mixtures, 1, 2, and 3. The Bars represent the Standard Error present in each set of triplicate. 24

33 DNA Quantity (ng) The values for the aged sperm fraction can be found in Figures 7-9. Due to the dramatic difference between the three mixtures Figure 7 holds the three mixtures for comparison, but Figures 8-9 hold Mixture 2 and Mixture 3 respectively. The visual representation in the amount of DNA (ng) returned from these samples is important due to the fact that the automated methods returned 2-5 times the amount of DNA than did the manual methods in Mixtures 2 and Mixture 1 Mixture 2 Mixture 3 Standard Differential Lounsbury AutoMate Qiagen Figure 7: Total DNA (ng) Extracted, Sperm Fraction, Aged Samples, Mixtures 1, 2, and 3. The Bars represent the Standard Error present in each set of triplicate. 25

34 DNA Quantity (ng) DNA Quantity (ng) Mixture 2 Standard Differential Lounsbury AutoMate Qiagen Figure 8: Total DNA (ng) Extracted, Sperm Fraction, Aged Samples, Mixture 2. The Bars represent the Standard Error present in each set of triplicate Mixture 3 Standard Differential Lounsbury AutoMate Qiagen Figure 9: Total DNA (ng) Extracted, Sperm Fraction, Aged Samples, Mixture 3. The Bars represent the Standard Error present in each set of triplicate. 26