Generating forensic DNA profiles from;contact;DNA on cartridge cases and gun grips

1、GENERATING FORENSIC DNA PROFILES FROM CONTACT DNA ON CARTRIDGE CASES AND GUN GRIPS Lisa Branch B.A., University of California at Santa Barbara, 2002 THESIS Submitted in partial satisfaction of the requirements for the degree of MASTER OF SCIENCE in BIOLOGICAL SCIENCES (Molecular and Cellular Biology

2、) at CALIFORNIA STATE UNIVERSITY, SACRAMENTO SPRING 2010 ii 2010 Lisa Branch ALL RIGHTS RESERVED iii GENERATING FORENSIC DNA PROFILES FROM CONTACT DNA ON CARTRIDGE CASES AND GUN GRIPS A Thesis by Lisa Branch Approved by: _, Committee Chair Dr. Ruth Ballard _, Second Reader Dr. Brett Holland _, Third

3、 Reader Dr. Nicholas Ewing Date:_ iv Student: Lisa Branch I certify that this student has met the requirements for format contained in the University format manual, and that this thesis is suitable for shelving in the Library and credit is to be awarded for the thesis. _, Graduate Coordinator _ Dr.

4、James W. Baxter Date Department of Biological Sciences v Abstract of GENERATING FORENSIC DNA PROFILES FROM CONTACT DNA ON CARTRIDGE CASES AND GUN GRIPS by Lisa Branch Advances in DNA typing methods have enabled forensic scientists to obtain genetic profiles from cells shed onto objects that have bee

5、n touched or handled. This type of DNA is called “contact DNA,” and crime labs are currently receiving many requests from attorneys and investigators to try to find it on crime scene evidence and analyze it. Cartridge cases and firearms are common types of evidence recovered from crime scenes and ar

6、e potential sources of contact DNA because criminals may handle cartridges or gun grips directly, without gloves. As a result, analysts are often asked to swab cartridge cases and gun grips for contact DNA when examining evidence from crime scenes where firearms were used. DNA typing is expensive an

7、d time consuming, and many crime labs are backlogged with unsolved cases. Moreover, many analysts report that, in their experience, fired cartridge cases are a very poor source of DNA and that it is probably a vi waste of time and money to test them. Therefore, a comprehensive study of the factors t

8、hat affect the deposition of contact DNA on ammunition and guns, and the types of cartridges and gun grips that are most likely to generate useful genetic information, is badly needed. This project examined whether it is possible to generate human DNA profiles from fired and unfired cartridge cases,

9、 and from gun grips, under varying conditions relevant to forensic casework Seven common cartridge cases were examined, including 0.22 caliber brass cases, 0.38 caliber brass, aluminum and nickel cases; and 9mm brass, steel, aluminum and nickel cases. Gun grips made of wood, plastic and rubber, with

10、 either textured or smooth surfaces, were also sampled. In addition, two other variables were studied: (1) whether the length of time between hand washing and handling affects the amount of DNA shed onto a case or grip, and (2) whether the amount of time the person handles a case or grip affects DNA

11、 shedding. The Applied Biosystems AmpFSTR Minifiler system was used to genotype all of the samples. While it is less informative than the more widely used AmpFSTR Identifiler system, it is more sensitive. However, samples producing Minifiler profiles with 6 or more alleles and containing at least 0.

12、1 ng of DNA were also genotyped with Identifiler to see if more genetic information could be obtained. A total of 690 swabs were collected and analyzed, 590 for the cartridge case studies and 150 for the gun grips studies. Of these, 51 samples were contaminated with DNA from the examiner or another

13、source and were excluded from downstream analyses. Therefore, results are reported for only 501 cartridge cases and 138 gun grips. vii The DNA from the samples was extracted on the BioRobot EZ1 using the trace protocol, amplified with ABI AmpFSTR Minifiler kits, and genotyped on a 3130 Prism xl gene

14、tic analyzer. Only profiles containing alleles above a minimum threshold of 75 RFUs were included in the evaluation of a profile. Samples with alleles present at all loci were designated as “full profiles” since, during casework involving an unknown donor, they could be interpreted by an analyst as

15、containing all of the donors alleles. Samples with at least one allele observed at any of the loci were designated as having “partial profiles,” and samples with no alleles were designated as having “no profile.” Overall, 135 of the 639 non-contaminated samples yielded “full” or “partial” profiles w

16、ith Minifiler, 63 from the cartridge cases and 72 from the gun grips. Of the samples that also underwent additional AmpFSTR Identifiler typing, 24 produced profiles. One “full profile” was produced from a cartridge case and six “full profiles” were produced from gun grips. In addition, four “partial

17、 profiles” were produced from the cartridge cases and 14 were produced from the gun grips. The caliber of the cartridge, the material from which it was made, and the length of time it was handled did not affect DNA recovery. Similarly, the material and texture of the gun grip and the length of time

18、it was handled did not alter DNA yields. However, cartridge cases and grips that were handled one hour after donors washed their hands yielded significantly more DNA than cases and grips handled immediately after hand washing. In addition, unfired cases yielded significantly more DNA than fired case

19、s, and gun grips yielded significantly more DNA than cartridge cases. viii After the genotyping was complete, all “full profiles” and “partial profiles” were compared to the donors known profile to detect allelic drop out. While partial allelic drop out is common in contact DNA profiles analyzed by

20、Identifiler, the second allele can usually be detected on the electropherogram below the stochastic threshold but above background. Therefore, the second allele can be used for inclusionary or exclusionary purposes. However, 58 of the 135 Minifiler profiles exhibited the complete drop out of an alle

21、le at one or more heterozygous loci. This presents a challenge because, if a contact DNA sample is from an unknown source, its Minifiler profile could be interpreted incorrectly and potentially mislead an investigation. The fact that 7.2% of the Minifiler profiles in this study exhibited contaminati

22、on only serves to exacerbate the problem. Therefore, although Minifiler has the advantage of being more sensitive than Identifiler, analysts should carefully weigh the need for sensitivity against the possibility of increased contamination and the complete drop out of some alleles. In some instances

23、, Minifiler may be the best choice for obtaining genetic information from a contact DNA sample. However, it is critical that forensic DNA analysts understand the limitations of the system and interpret their results with caution. This study provides a wealth of information about the factors that may

24、 influence DNA recovery from cartridge cases and gun grips and should provide the forensics community with empirical support for accepting or denying requests for contact DNA analyses. In particular, this study indicates that trying to obtain genetic information from fired cartridge cases is probabl

25、y not worth the time and expense involved. Investigations ix would be better served by developing new methods for generating such profiles or by focusing on evidence that is likely to be more probative. _, Committee Chair Dr. Ruth Ballard _ Date x ACKNOWLEDGEMENTS I would like to thank all the membe

26、rs of my committee (Dr. Brett Holland and Dr, Nick Ewing), and specifically Dr. Ruth Ballard for helping me through the ebbs and flows of this project. Also Dr. Jamie Kneitel for helping out with the statistics performed during this research. I would also like to thank Mary Hansen for giving me the

27、opportunity to be part of the Sacramento County District Attorneys Office Laboratory of Forensic Services and helping me through out this project, she has been a rock. Also all the members of the DNA/Serology section of the laboratory (Jeff Herbert, Michelle Chao, Angel Shaw, Ryan Nickel, Nikki Sewe

28、ll, Ann Murphy , Dolores Dallosta, Kristie Abbot, Kristin Bejarano, Gerald Arase and Joy Viray, they taught me everything I needed to know in order to complete this project and a number of friendships have been formed from this experience. Also members of the Firearms section (Phil Hess, Cara Gomes

29、and Mike Saggs) of the laboratory, without them it would have been difficult. Also, Dr. Anglim, who, provided tremendous help throughout the project. Last, I would like to thank my family and friends for putting up with me during these last two years, especially my Mom for always supporting me and b

30、elieving in me. xi TABLE OF CONTENTS Page Acknowledgements.x List of Tables.xii List of Figures.xiii Introduction.1 Materials/Methods .26 Validation of the BioRobotEZ1.26 Recovery of Contact DNA from Cartridge Cases and Gun Grips.34 Results.43 Discussion.79 Appendix A: Protocols from the Sacramento

31、District Attorneys Office, Laboratory.88 Of Forensic Services for DNA extraction, quantitation and amplification Appendix B: Permission to use figures from Applied Biosystems and Elvesier.93 Literature Cited.95 xii LIST OF TABLES Page Table 1. Fabrics for inhibition study.33 Table 2. Substrates cont

32、aining PCR inhibitors.53 Table 3. Mean DNA yields Montpetit, Fitch and ODonnell, 2005). Automated systems for DNA extraction have proven to be extremely useful, especially when processing reference samples (samples of known origin that contain ample amounts of DNA). Automated extraction is much fast

33、er - approximately thirty minutes for completion compared to the longer time required for organic extraction (one to one half days). With automated systems there are less manipulations of the sample and no organic solvents are used. In the context of the laboratory, the absence of organic solvents m

34、akes the process safer for the person performing the extraction and the automation allows for faster extraction. Automated extraction methods use solid-phase extraction, compared to liquid-phase extraction, which is used in the organic extraction. Liquid-phase extraction requires multiple manipulati

35、ons, requires organic solvents and is harder to automate, whereas solid-phase extraction is much easier to automate and does not require organic solvents. 9 The Qiagen Corporation developed an automated DNA extraction method, the BioRobot EZ1 workstation Figure 4. This system can purify high quality

36、 DNA from up to six samples in approximately thirty minutes. The BioRobot EZ1 workstation extracts DNA by magnetic bead particle technology. The DNA sample is isolated in one step by binding to silica beads. The process involves lysis of the sample, addition of magnetic beads to the sample, and bind

37、ing of the DNA to the particles. The DNA Figure 4. The Qiagen BioRobot EZ1 (photo courtesy of Lisa Branch). 10 purification is done using a chaotropic extraction with paramagnetic silica beads. The chaotropic agents, guanidine thiocyanate (GuSCN)/guanidine hydrochloride (GuHCl), denature proteins, i

38、nhibit nucleases and promote the binding of the DNA to the magnetic silica beads by breaking the weak hydrogen bonds, in water, which destroys the hydration shell around the DNA and exposes the negatively charged backbone. The exposed backbone can then adhere tightly to the positively charged magnet

39、ic silica beads. A magnet is used to separate the particles from the lysate. The DNA is then washed and eluted (J. M. Butler, 2002; Montpetit, Fitch and ODonnell, 2005; (Anslinger et al., 2005; Kishore et al., 2006). One single barrier pipette tip is used for the entire DNA extraction and all washes

40、, which minimizes contamination via multiple tube switches. Contact DNA Study The History of DNA Typing In the mid 1980s, human DNA identification typing was first described by English geneticist, Alec Jeffreys. He identified certain regions of the human genome that 11 contained sequences that are t

41、andemly repeated and found that the number of repeats differs from one individual to another (Jeffreys, Wilson and Thein 1985). These repetitive units, variable number tandem repeats (VNTRs), have repeat units that vary in size from 10-100 base pairs. It is estimated that there are thousands of VNTR

42、 loci located in the human genome, providing a rich source of genetic variability (Siegal, Saukko, however, it had several important limitations. It required at least 50 nanograms of DNA (1 ug was optimal), it could not be used on 12 samples compromised by age or enviromental exposure and it was tim

43、e consuming and laborious (Siegal, Saukko, while 3% of these regions consist of STRs (Schneider, 1997, Fan and Chu, 2007). STRs, or microsatellites, are smaller version of VNTRs that can be amplified by PCR and maintain their high level of discriminatory power. STRs are short repeated sequences, wit

44、h a repeat unit of only 3-5 bps and a total amplicon length of less than 500 base pairs. These polymorphic markers are commonly found throughout the non-coding regions of the DNA and do not reveal information about genetic traits or predisposition for genetic diseases (Butler, 2002, Schneider, 1997)

45、. STRs are highly variable for two reasons. First, these regions are non-coding and are not subjected to selective pressures during evolution. Coding portions of the human genome, i.e. expressed genes, are subject to selection pressures during evolution in order to maintain their specific function,

46、so variation, as a result of mutations, between 14 individuals is rather limited (Schneider 1997). In contrast, mutations in these non-coding regions are usually retained and passed to offspring creating high genetic variability. Second, they are mutation hotspots because they mutate more quickly th

47、an other regions of the genome. Mutations in STRs can occur via strand slippage during DNA replication (Fan and Chu 2007, Levinson and Gutman 1997). This mechanism involves alteration of repeat sequences or slippage on the two complementary strands of the double helix, leading to an incorrect pairin

48、g of repeats, causing deletions or insertions in the DNA. These occur by the formation of an unpaired hairpin loop. The hairpin loops are often more stable than the original structure, increasing the probability of expansion and deletion (Petruska, Hartenstine and Goodman 1998). When a mutational ch

49、ange occurs, a new repeat unit is created from the nascent (new) strand which can potentially expand these further. A deletion is created when slippage occurs on the template strand (Levinson and Gutman 1997) Figure 5. Most of these hairpin loops are recognized and repaired by the mismatch repair sy

50、stem, but for those that do not get repaired, new STR alleles are created (Fan and Chu 2007). The difference in the number of repeats present at any given locus on the chromosome is what makes STRs unique. STRs are classified by the different repeat units, such as mono-, di-, tri-, tetra-, penta- an

51、d hexanucleotides (Fan and Chu 2007). As tetranucleotide repeats, such as gata (the STR locus D7S820 located on chromosome 7), are less likely to undergo mutations during PCR and contain less PCR stutter products (minor fragments (artifacts of PCR) that differ in size than the major allele usually b

52、y one 15 repeat unit) compared to shorter repeat units, they are more popular for forensic uses (Montpetit, Fitch and ODonnell 2005). One key advantage of STRs over VNTRs is they can be amplified by PCR. STRs also require only 1 ng of DNA as opposed to the 50 ng required by RFLP analysis (Wickenheis

53、er 2002). These new STR loci offer high discriminatory power and the STR markers allow for identification of individuals. 16 Figure 5-Schematic illustration of the strand slippage mechanism (Fan and Chu 2007, copyright permission from Elvesier, Appendix B) Identifiler and Minifiler-STR Systems The A

54、pplied Biosystems AmpFSTR Identifiler kit, released in 2001, amplifies 15 human specific STR markers plus the gender marker amelogenin in a single reaction. Commercial kits like Identifiler have amplicon sizes that usually range from 100 to 500 base pairs. In these kits, each primer is fluorescently

55、 labeled with a dye which is attached to the 5 end of the PCR primer and is detected by passing through a light source scanner that detects the spectrum of light from the different fluorophores. This is done via gel capillary electrophoresis, which separates the DNA fragments by size. Results obtain

56、ed from kits are seen on an electropherogram. An electropherogram contains a y-axis, which measures the relative fluorescent units (RFUs, a measurement of the amount of emitted light that has passed through a laser) and an x-axis, which measures time or size of the DNA. 17 While Identifiler works we

57、ll on a majority of samples encountered in criminal cases, it may not produce full human genetic profiles on compromised samples. Such samples occur if DNA is exposed to destructive elements, such as UV or heat, or if the samples have been exposed to environmental contaminants, such as soils. These

58、elements have the possibility of inhibiting the PCR process (Butler, Shen and McCord 2003) (Grubwieser, et al. 2006). In these samples there is a potential loss of signal in the larger-sized STR products, due to fragmented DNA molecules or PCR inhibitors present in the sample (Chung et al., 2004; Gr

59、ubwieser, et al. 2006; Hill et al., 2007). This loss of signal is referred to as allelic dropout and can result in a failure to amplify one or both of the individuals alleles at a locus, which in turn can result in the generation of a partial genetic profile or potentially no DNA profile at all. This creates a decay curve in commercial kits, like Identifiler, where the peak height is inversely proportional to t