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)
at
CALIFORNIA STATE UNIVERSITY, SACRAMENTO
SPRING
2010
© 2010
Lisa Branch
ALL RIGHTS RESERVED
ii
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 Reader
Dr. Nicholas Ewing
Date:____________________
iii
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. James W. Baxter
Department of Biological Sciences
iv
____________
Date
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 been 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 are 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 and 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
v
waste of time and money to test them. Therefore, a comprehensive study of the factors
that 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, 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
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 shedding.
The Applied Biosystems AmpFℓSTR Minifiler™ system was used to genotype all
of the samples. While it is less informative than the more widely used AmpFℓSTR
Identifiler™ system, it is more sensitive. However, samples producing Minifiler™
profiles with 6 or more alleles and containing at least 0.1 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 source and were excluded from downstream analyses.
Therefore, results are reported for only 501 cartridge cases and 138 gun grips.
vi
The DNA from the samples was extracted on the BioRobot® EZ1 using the trace
protocol, amplified with ABI AmpFℓSTR Minifiler™ kits, and genotyped on a 3130
Prism® xl genetic 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 containing all of the donor’s 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
with Minifiler™, 63 from the cartridge cases and 72 from the gun grips.
Of the samples that also underwent additional AmpFℓSTR 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 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 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 cases, and
gun grips yielded significantly more DNA than cartridge cases.
vii
After the genotyping was complete, all “full profiles” and “partial profiles” were
compared to the donor’s known profile to detect allelic drop out. While partial allelic
drop out is common in contact DNA profiles analyzed by 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 allele 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 contamination 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, 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 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 probably not worth the time and expense involved. Investigations
viii
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
ix
ACKNOWLEDGEMENTS
I would like to thank all the members 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
opportunity to be part of the Sacramento County District Attorney’s 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 Sewell, 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 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 believing in me.
x
TABLE OF CONTENTS
Page
Acknowledgements ..............................................................................................................x
List of Tables .................................................................................................................... xii
List of Figures .................................................................................................................. xiii
Introduction ..........................................................................................................................1
Materials/Methods .............................................................................................................26
Validation of the BioRobot®EZ1 ..........................................................................26
Recovery of Contact DNA from Cartridge Cases and Gun Grips .........................34
Results ................................................................................................................................43
Discussion ..........................................................................................................................79
Appendix A: Protocols from the Sacramento District Attorney’s 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
xi
LIST OF TABLES
Page
Table 1. Fabrics for inhibition study .................................................................................33
Table 2. Substrates containing PCR inhibitors .................................................................53
Table 3. Mean DNA yields & genotyping success of saliva stains on cigar
paper ...................................................................................................................55
Table 4. DNA shed onto .22 caliber brass cases by 10 volunteers. ..................................57
xii
LIST OF FIGURES
Page
Figure 1. Revolver and semi automatic pistol .....................................................................2
Figure 2. Firing pin impressions and ejector marks ............................................................4
Figure 3. Photos of common grip types ..............................................................................5
Figure 4. Qiagen BioRobot®EZ1 .......................................................................................9
Figure 5. Schematic illustration of the strand slippage mechanism..................................15
Figure 6. Shortening of miniSTR primers ........................................................................18
Figure 7. Comparison of Identifiler™ and Minifiler™ ....................................................19
Figure 8. Electropherogram of Minifiler allelic ladder .....................................................20
Figure 9. Arrangement for samples in the contamination study .......................................32
Figure 10. Flow chart for collection of samples for Phase 1 of Part 2 .............................38
Figure 11. Histogram of DNA yields for saliva samples ..................................................44
Figure 12. Histogram of DNA yields for blood samples ..................................................45
Figure 13. Histogram of mean DNA yields for 50 µL of semen samples ........................47
Figure 14. Histogram of DNA yields for hair samples .....................................................48
Figure 15. Histogram of DNA yields for 50 µL of urine ..................................................50
Figure 16. Histogram of DNA yields for 200 µL of urine ................................................51
Figure 17. Comparison of organic and EZ1 DNA extraction methods ............................56
Figure 18. Histogram of percent of samples for caliber type ..........................................60
Figure 19. Histogram of percent of fired vs. unfired cases ...............................................61
xiii
Figure 20. Histogram of percent of samples for handling time-cartridge
cases................................................................................................................63
Figure 21. Histogram for percent of samples for case material ........................................65
Figure 22. Histogram for percent of samples for hand washing timecartridge cases ................................................................................................67
Figure 23. Effect of gun grip material on % of full, partial and no profile.......................70
Figure 24. Effect of handling time on the % of full, partial and no profiles
from gun grips ................................................................................................71
Figure 25. Percent of gun grips for which no profile, a partial profile or
full profile for hand washing ..........................................................................74
Figure 26. Flow chart comparing the results obtained from the gun grip
and cartridge case studies ...............................................................................77
Figure 27. Full profile at 75 RFUs or greater no allelic dropout ......................................86
Figure 28. Profile with allelic drop-out.............................................................................87
xiv
xv
1
INTRODUCTION
In 2006, almost fifteen thousand homicides occurred in the United States and over
seven thousand of these homicides involved handguns (Bureau of Justice Statistics 2007).
Since the 1970’s, gunshot wounds have been the leading cause of death by homicide in
the United States and despite the best efforts of law enforcement agencies and
prosecutors, thirty five percent of these homicides remain unsolved (Federal Bureau of
Investigation, 2006). Therefore, it is of great importance to develop new methods and
techniques to analyze evidence recovered from crime scenes in which firearms were
used. Handguns are especially of interest because they are the most common firearms
involved in homicides. Potential evidence in these cases includes unfired cases, cartridge
cases that are ejected after the gun is fired, and the firearms themselves.
A cartridge contains a projectile or bullet, the primer (a small explosive charge in
the base of the case which is used to set off the propellant powder when hit by the firing
pin) and the case, which holds these elements together (Fisher, 2000). When a subject
loads a firearm, their hand(s) and finger(s) can come into contact with the case. The
cartridge is then either loaded directly into the firearm, where the case remains after the
bullet is fired (revolvers) [Figure 1A] or into a detachable magazine, from which the
empty case is ejected after firing (pistols) [Figure 1B]. When fired from a pistol,
cartridge cases are often left behind at crime scenes and are retrieved by investigators to
determine critical information about the firearm and, in some instances, the shooter.
2
A
B
Figure 1—A: Revolver. B: Semi automatic pistol. (Photos courtesy of the Sacramento County
District Attorney’s Office Laboratory of Forensic Services, Firearms unit)
3
Sources of information include rifling marks, firing pin impressions, ejector marks
[Figure 2A], breech face marks [Figure 2B], chamber marks, and latent fingerprints
(Schehl 2000, Given, 1976, Migron, et al. 1998). The most common types of cartridge
cases found at crime scenes are .22, .38, .45 and 9 mm in caliber and are made of brass,
aluminum, nickel and steel (only made for 9 mm). Gun grips can be a potential source of
evidence if the firearm is left behind at a crime scene. Gun grips vary in composition and
can be made of wood, plastic and rubber and have either textured or smooth surfaces
[Figure 3].
DNA analysis of firearm-related evidence appeals to law enforcement agencies
and investigators because it is a highly sensitive and informative analysis that has the
potential to link the suspect to the firearm more successfully than fingerprint analysis
alone. Cells (e.g. epithelial cells from the mouth and skin) contain DNA that can be
retrieved from almost any surface, whereas fingerprints must be deposited on relatively
hard, smooth surfaces in order to be useful in an investigation. Recent studies have
demonstrated that DNA profiles can be generated from objects that have simply been
touched by an individual (known as ‘contact’ DNA).
Cartridge cases and firearms are common types of evidence found at crime scenes
and are potential sources of ‘contact’ DNA. It may be possible to retrieve sufficient
quantities of DNA from these surfaces for analysis.
4
A
B
Figure 2—A: Firing pin impression and ejector marks. B: Breechface impression. (Photos
courtesy of Phil Hess, Sacramento County District Attorney’s Office Laboratory of Forensic
Services, Firearms unit).
5
T
Figure 3- Photos of some of the most common types of gun grips. A. Smooth-plastic. B.
Smooth wood. C. Rubber. D. Plastic checkered. (Photos courtesy of Lisa Branch).
6
The goal of this research was to test gun grips and fired and unfired cartridge
cases to determine how DNA can be retrieved from them and if the amount is sufficient
to provide probative information in a crime scene investigation. This type of evidence is
routinely submitted to forensic laboratories by law enforcement agencies.
Validation of the BioRobot®EZ1
Prior to beginning the cartridge case and gun grip study, I assisted the laboratory
in the validation of the BioRobot®EZ1 extraction system, an automated extraction
platform that reduces DNA extraction times from many hours to 30 minutes. Crime
laboratories are required to conduct internal validation studies on each method employed
in DNA analysis prior to implementation of the method in casework. The validation was
conducted in accordance with the Quality Assurance standards for forensic DNA Testing
Laboratories, issued by the F.B.I. director, issue date 07/04, Rev. 6 (42 Code of Federal
Regulations n.d., American National Standards Institute 1990, Technical Working Group
on DNA Analysis Methods 1995, Federal Bureau of Investigation DNA Advisory Board
C
1998 Federal Bureau of Investigation DNA Advisory Board. 1999, (American Society of
Crime Laboratory Directors/Laboratory Accrediation Board 2003). These studies allow
laboratories to check the reliability, reproducibility, sensitivity and limitations of the
methods. Since the information obtained from my project was going to be used by the
Sacramento County District Attorney’s Crime Laboratory to aid in their evaluation of
performing DNA analysis on samples collected from unfired and fired cartridge cases and
7
gun grips, validation of the BioRobot®EZ1 extraction system was required.
My portion
of the validation involved optimization of analytical procedures using standard samples
(blood, saliva, hair and urine) to determine the efficiency of DNA recovery using the
BioRobot®EZ1, comparing DNA yields obtained from the BioRobot®EZ1 to those
obtained from the organic extraction method utilized by the laboratory, evaluating the
capability of the BioRobot®EZ1 in removing PCR inhibitors (dyes) from samples, and
verifying that sample-to-sample contamination did not occur. Extraction of ‘contact’
DNA samples using the BioRobot®EZ1 were performed separately as part of the
validation of the Minifiler™ system.
The most critical step of the analysis is the extraction of the DNA from forensic
evidence samples: blood, saliva, urine, hair and semen (Montpetit, Fitch and O'Donnell
2005). The cells of interest to forensic scientists (nucleated cells) contain other
substances besides DNA such as, proteins, carbohydrates, lipids, etc. Therefore, the
DNA molecules must be separated from this cellular material before it can be further
examined (J. M. Butler 2002). The most common technique employed is the phenolchloroform method (the organic method). This method involves a detergent-mediated
cell lysis (rupturing the cell and exposing the DNA) and proteinase K treatment (to break
down the proteins). Once the cell has been lysed, phenol-chloroform is used in multiple
washes to separate proteins from DNA by creating an upper aqueous phase (containing
the DNA) and a lower organic phase (containing the proteins and lipids). The organic
extraction method is well suited for forensic samples because of the high recovery rate
8
and the purity of the DNA recovered. However, it requires multiple steps which can lead
to contamination and/or loss of sample. It is also time consuming and does not remove
all PCR inhibitors (J. M. Butler, 2002; Montpetit, Fitch and O'Donnell, 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 faster - 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 makes 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 manipulations, requires organic solvents and is
harder to automate, whereas solid-phase extraction is much easier to automate and does
not require organic solvents.
The Qiagen Corporation developed an automated DNA extraction method, the
BioRobot® EZ1 workstation [Figure 4]. This system can purify high quality 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 binding of the DNA to the particles. The DNA
9
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, inhibit 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 magnetic 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
O'Donnell, 2005; (Anslinger et al., 2005; Kishore et al., 2006). One single barrier
pipette tip is used for the entire DNA extraction and all washes, which minimizes
contamination via multiple tube switches.
Contact DNA Study
The History of DNA Typing
In the mid 1980’s, human DNA identification typing was first described by
English geneticist, Alec Jeffreys. He identified certain regions of the human genome that
contained sequences that are tandemly 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 loci
located in the human genome, providing a rich source of genetic variability (Siegal,
Saukko, & Knupfer, 2000)
11
Restriction fragment length polymorphism (RFLP) analysis technology utilized
restriction enzymes that cleave the DNA around these repeated sequences. Once the
DNA fragments were cut, they were separated by size using polyacrylamide gel
electrophoresis and denatured in an alkali solution in order to separate the DNA strands.
Probes (specific DNA sequences) that hybridize with one or more of the digested DNA
samples were labeled with a radioactive isotope and, after hybridization, were viewed
using autoradiography. RFLP testing began as multi-locus probes that recognized
multiple nucleotide sequences on multiple chromosomes. However, these probes were
difficult to interpret if samples contained more than one source of DNA, such as a male
and a female source, as occurs in most rape evidence samples (Siegal, Saukko, &
Knupfer, 2000). These samples also required a large amount of sample DNA. To
overcome these limitations, Jeffrey’s created single locus probes, which bind to a single
location on a single chromosome. Single locus probes required less DNA than the multilocus probes method. The RFLP technology was the first accepted forensic DNA
analysis method in the United States; however, it had several important limitations. It
required at least 50 nanograms of DNA (1 ug was optimal), it could not be used on
samples compromised by age or enviromental exposure and it was time consuming and
laborious (Siegal, Saukko, & Knupfer, 2000). Therefore, it was rarely used for forensic
casework because very few pieces of crime scene evidence contained enough good
quality DNA for a robust VNTR analysis.
While Jeffreys was developing the RFLP technology, the polymerase chain
reaction (PCR) was developed in the United States. PCR makes millions of copies of
12
short, targeted regions of DNA. At the end of one PCR cycle, two copies of the original
template have been made and as the number of PCR cycles increases, exponential copies
of template DNA are generated. PCR is better suited for forensic analysis because it
requires a much smaller amount of sample for amplification (~1 ng), and is an easier
process to automate. It was quickly adopted by the forensic community as an alternative
to RFLP typing.
The first PCR test used in forensics was HLA-DQα (DQA1), a human leukocyte
antigen locus. This system included eight alleles and single-stranded, labeled probes for
PCR, which were used to detect each of these different alleles (Siegal, Saukko and
Knupfer, 2000). It was an informative test and could be used on trace and/or degraded
samples, but it lacked the discriminatory power of RFLP typing. In approximately 16%
of cases, the victim and suspect could have the same DQA1 genotype. Commerical kits
were developed and, eventually, expanded to included an additional set of five markers
(low density lipoprotein receptor (LDLR), glyphorin A (GYPA), hemoglobin G
gammaglobin (HBGG), DS78, and group specific component (Gc)) collectively called
Polymarker (PM). The DQA1-PM kit, marketed by Applied Biosystems, was the first
example of a multiplex PCR kit, in which all six marker systems were amplified in a
single PCR reaction. However, even with the additional markers, it was still far less
discrimnatory afford than RFLP technology (Siegal, Saukko and Knupfer 2000).
Therefore, there was a need for the development of a more informative PCR-based
system, with more genetic markers with a higher discrimination potential.
13
Short Tandem Repeats
The human genome is comprised of three billion basepairs of DNA, 90% of
which are non-coding regions that do not contain genes that code for proteins; 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, with 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).
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, so variation, as a result of mutations, between
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 than 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 pairing of repeats, causing deletions or insertions in
the DNA. These occur by the formation of an unpaired hairpin loop. The hairpin loops
14
are often more stable than the original structure, increasing the probability of expansion
and deletion (Petruska, Hartenstine and Goodman 1998). When a mutational change
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 system, 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- and 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 by one
repeat unit) compared to shorter repeat units, they are more popular for forensic uses
(Montpetit, Fitch and O'Donnell 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
(Wickenheiser 2002). These new STR loci offer high discriminatory power and the STR
markers allow for identification of individuals.
15
Figure 5--Schematic illustration of the strand slippage mechanism (Fan and Chu 2007, copyright
permission from Elvesier, Appendix B)
16
Identifiler™ and Minifiler™-STR Systems
The Applied Biosystems AmpFℓSTR® 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 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 obtained 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.
While Identifiler™ works well 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
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;
Grubwieser, 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 individual’s alleles at a
17
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 the amplicon length (Chung et al.,
2004). If there is a low level of DNA, it is likely that one allele may, by chance, be
amplified more than the other. In samples with low DNA quantities, allelic drop out can
be greater than 50% (Miller et al., 2002). Therefore, Identifiler™ may not work on
degraded DNA samples. A solution to this problem is to reduce the size of the amplicons
targeted for PCR, by moving the primers as close to the STR repeat as possible (Chung et
al., 2004; Opel et al., 2006) [Figure 6.]. These smaller STRs, using reduced primers to
create smaller amplicons, are called miniSTRs (Wiegand and Kleiber, 2004) [Figure 7].
These miniSTRs allow for a reduction in product size down to < 299 base pairs, with
most amplicon reductions ranging from 60-200 base pairs (Chung, et al. 2004). Applied
Biosystems employed this mechanism for the PCR system, AmpFℓSTR® Minifiler™
using nine STR loci. Minifier™ reduces the amplicon size of only the largest eight STR
loci in the Identifiler™ PCR Amplification kit, as the remainder of the STR loci in
Identifiler™ contain smaller amplicons (D7S820, D13S317, D16S539, D21S11,
D2S1338, D18S51, CSF1PO, FGA and amelogenin) [Figure 8]. Wiegnad and Kleiber
(2004) reported that using the miniSTRs in PCR amplification can produce full genetic
profiles at those loci in degraded DNA samples.
18
Figure 6. Picture depicting the shortening of the miniSTR primers compared to the
conventional PCR primer system (Applied Biosystems, 2008, copyright permission
granted see Appendix B).
19
Figure 7- A comparison of Applied Biosystems Identifiler™ and Minifiler™
amplicon sizes in their corresponding fluorescent colors along with a corresponding
size standard (shown in orange) (J. Butler 1997).
20
Figure 8—An electropherogram of the allelic ladder of AmpFℓSTR® Minifiler™.
(http://www3.appliedbiosystems.com/cms/groups/applied_markets_marketing/documents/gen
eraldocuments/cms_039944.pdf, accessed November 11, 2007).
21
The MiniFiler™ primer sets have been shown to fully provide highly concordant
results to the commercial kits, like Identifiler™, and can also provide improved detection
of degraded DNA (Wiegand and Kleiber 2004, Butler, Shen and McCord 2003). For
example, in one study Opel et al. (2006) took naturally degraded DNA from human
skeletal remains and performed PCR amplification. The miniSTRs had a greater chance
of producing full genetic profiles from degraded DNA (64%) when compared to other
commercial kits (16%).
Minifiler™ provides a more sensitive technology that can be utilized for the
determination of DNA profiles when only partial or no genetic profiles are generated
from standard amplification kits because of the effects of DNA degradation.
Cartridge cases and gun grips contain low quantities of DNA and amplifying with
Minifiler™ might make it possible to produce a human genetic DNA profile from these
samples or improve detection of DNA in these samples
‘Contact’ DNA
‘Contact’ DNA, also known as trace DNA or ‘touch’ DNA, is minute amounts of
DNA transferred through skin contact to an object or surface (Wickenheiser 2002).
Contact DNA occurs in relatively low amounts, usually less than 100 picograms (Findlay
et al., 1997, Van Hoofstat et al., 1998, Migron et al., 1998). Contact DNA typing and the
ability to yield DNA profiles from less than 100 picograms may be possible due to the
improved sensitivity of existing techniques (Minifiler™), as well as to the development
of new technologies, such as automated extraction processes (Lowe et al., 2002).
22
Skin is the largest organ in the body and humans shed approximately 400,000
skins cells daily. Epithelial cells, as found in skin, are some of the fastest regenerating
cells in the human body. When skin cells have reached the outermost layer, the stratum
corneum, the dead skin (non-nucleated) cells are sloughed off. The skin matrix contains
sebaceous oil glands and sweat glands. Secretions and nucleated cells from these glands
are brought to the surface of the skin via the ducts and pores and create a potential source
of DNA (Schiffer et al., 2005, Wickenheiser, 2002). The fingers or hands, acting as a
vector, have the capability of transferring the cells/DNA as well as transmission of
nucleated cells from the eyes and mouth, to objects that have been handled, such as
knives, cartridge cases, rope, etc. (Wickenheiser 2002).
Given the improved detection technology, and the fact that researchers have been
able to generate human DNA profiles from the mere contact between an object and an
individual, it has recently been suggested that contact DNA might be present on gun grips
and cartridge cases, as a result of the individual handling the evidence prior to firing.
While it is likely that DNA which has been deposited onto the cartridge case before firing
has been destroyed via the heat and pressure inside the chamber of the gun, several
studies still report the use of contact DNA to generate human identification DNA
profiles. Studies have shown that it is possible to recover DNA from items such as
exploded pipe bombs, hammers and chisels (Van Hoofstat, et al. 1998, Esslinger, et al.
2004). Esslinger, et al. (2004) were able to report one full profile and three partial
profiles from twenty-seven, 1-inch diameter, exloded pipe bombs. A full reportable
genetic profile for this study was declared if all ten loci examined had alleles with RFU
23
values between 150 and 4500, while a partial profile was declared when at least three
STR loci had alleles with RFU values between 150 and 4500. This study demonstrates
that it is possible to obtain usable genetic profiles from materials that have been exposed
to extreme heat (Esslinger, et al. 2004). Polley, et al., (2006) conducted a study
examining the recovery of DNA from various locations on the firearm as well as recovery
from fired and unfired cartridge cases. They reported one fired cartridge case from a .45
pistol, when examined using Identifiler™, yielded peaks at the amelogenin locus only.
However they did recover DNA from gun grips that generated sufficient quantities of
DNA for further DNA analysis using Identifiler™. These researchers determined that
there is less contact DNA present on unfired cases and fired cases when compared to gun
grips. It is more probable to recover DNA from items that have a larger, textured surface
area, such as gun grips, knife handles, etc. (Polley, et al. 2006).
At this time, no reports describing experiments in which full DNA profiles have
been recovered from fired cartridge cases using traditional methods exist. Nevertheless,
labs may still be required to perform the analysis of such evidence at the request of
attorneys or law enforcement agencies. Also no extensive studies using Minifiler™ for
contact DNA exist, but this technology could potentially be far superior to Identifiler™
for performing such examinations.
Another consideration when examining contact DNA is whether hand washing
affects the ability to collect DNA. Hand washing is classified as scrubbing the skin by
mechanical agitation caused by the movement and pressure of both hands in liquid. The
dirt and dead skin cells are separated from the skin by a combination of the movement of
24
the hands as well as the wet chemical action of the soap. When hand washing occurs, an
individual actually removes cells that contain DNA from the skin (Brouwer et al., 2000).
One would expect that immediately after hand washing, an individual would ‘shed’ very
little DNA onto a surface or object if contact was made. However, the production of
epidermal cells is rapid and continuous. Skin cells are constantly proliferating and dying
and glands are producing oil and sweat, possibly presenting a high amount of cells
containing DNA. van Oorschot et al. (1997) produced genetic profiles using one genetic
marker from swabs taken directly from the hand; the average yield was 2 to 150
nanograms of DNA. However, they observed that dry hands, or hands that had
previously been washed, had a lesser yield.
A further consideration is the variation in amounts of DNA shed by different
people. Lowe, et al. labeled donors of DNA to handled objects as either ‘good’ shedders
or ‘poor’ shedders. This was determined by assessing how many alleles of their DNA
were typed from touched items fifteen minutes post hand washing. Individuals that left a
full profile fifteen minutes post hand washing were labeled ‘good’ shedders, while those
leaving only partial profiles were labeled as ‘poor’ shedders. Of those sampled, 60%
were labeled ‘good’ shedders (Lowe, et al. 2002). In contrast, Phipps and Petricevic
(2006) determined that it is difficult to label an individual as a ‘good’ shedder or ‘poor’
shedder because an individual cannot be relied upon to shed a consistent amount of DNA
over time. In Phipps and Petricevic’s study, it was found that after 15 minutes post-hand
washing and 4 hours post-hand washing an individual did not consistently shed the same
amount. The amount of DNA shed by an individual can vary on a day to day basis or
25
even an hour to hour basis. In fact, it was found that an individual could either shed less
or more over time depending on the individual. These results suggest labeling
individuals as ‘good’ or ‘poor’ shedders might be overly simplistic and that there are
more factors attributing to how much DNA is deposited, such as handling time, the
substrate or surface being contacted, the time of contact and environmental factors.
Goals of Project
The purpose of this study was to evaluate the suitability of DNA derived from
contact DNA on cartridge cases and gun grips for use in criminal investigations and also
to determine if the Minifiler™ amplification kit was suitable to amplify the amount of
DNA recovered for these types of samples. I aimed to address whether it is possible to
obtain DNA profiles from unfired and fired cartridge cases and gun grips using the
BioRobot®EZ1 for DNA extraction and the Minifiler™ amplification system, and finally
whether hand washing and handling time influences the amount of DNA recovered from
contact DNA samples.
26
MATERIALS AND METHODS
This project had two distinct aims. The first was to validate the BIORobot®EZ1
(Qiagen, Valencia CA) automated DNA extraction system at the Sacramento County
District Attorney’s Laboratory of Forensic Services (SCDALFS) and is described in Part
1 of the Materials and Methods and Results sections that follow. The validation was
performed as a service to the laboratory, but also to determine if I could use it to perform
the DNA extractions on the samples in Part 2 of my study. The laboratory has two
BioRobot®EZ1s and the validation was conducted on both robots. My plan was to
collect and analyze 690 swabs from cartridge cases and gun grips, and I hoped that I
would be able to utilize the largely automated BioRobot®EZ1 system rather than the
time-intensive organic extraction method that was being used by the laboratory at that
time.
The second aim was to perform a comprehensive study of the variables that affect
the recovery of contact DNA from common types of cartridge cases and gun grips and is
described in Part 2 of the Materials and Methods and Results sections.
Part 1: Validation of BioRobot®EZ1
Sample Collection
All samples were collected from employees of SCDALFS, as is standard for
validating new instruments and protocols there. Five volunteers were recruited, four men
and one woman. The samples collected included saliva, semen, blood, urine and hair.
27
Saliva samples were collected using sterile cotton swabs, with donors swabbing
the inside of their cheeks for 15 seconds. Each donor provided two cheek swabs. Blood
samples were collected using a sterile lancet device (Autolet®II, US PA 423118), and the
blood was then deposited on a blood card. Two mL of semen and 30 mL of urine were
collected from donors in 30 mL sterile tubes. Each individual also plucked two to three
scalp hairs and the hairs were examined microscopically to eliminate those that did not
contain an intact root. One cm of the root end of each hair was then excised and
combined with the root ends of the rest of the hair samples from that donor.
DNA Extraction
Blood, Saliva, and Urine
For organic extractions of DNA from the blood samples, a 3 mm x 3 mm cutting
was taken from each card and transferred into a microcentrifuge tube. For organic
extractions of DNA from the saliva samples, one half of each swab was transferred into a
microcentrifuge tube. Finally, for organic extractions of DNA from urine, two volumes
were taken for analysis, 50 µL and 200 µL, and placed in microcentrifuge tubes. The
DNA was then extracted according to the SCDALFS’s organic extraction protocols for
bodily fluids [See Appendix A]. For the organic extraction of blood, saliva and urine
samples a total of 20 extractions were conducted (5 blood, 5 saliva, and 10 urine).
Blood and saliva samples for DNA extractions using the BioRobot®EZ1 were
collected in the same manner as described above and processed using Qiagen’s EZ1
DNA Investigator kits according to the manufacturer’s trace protocol (EZ1 DNA
28
Investigator Handbook, 2006). At the end of the procedure, the DNA was eluted in TE
using 50 µL, 100 µL or 200 µL volumes so that the effect of elution volume on DNA
recovery could be studied. For blood and saliva extractions a total of 60 samples were
used (30 blood and 30 saliva). Urine samples were collected in the same manner as for
the organic extractions and eluted in 50 µL of TE at the end of the EZ1 extraction
procedure. A total of 20 urine samples were extracted (1 set at 50 µL, 1 set of 200 µL).
Hair
Organic extractions of DNA from the hair samples were performed according to
the standard procedures used by SCDALFS [See Appendix A]. A total of 5 samples were
extracted organically. The BioRobot®EZ1 DNA extractions were performed using EZ1
DNA Investigator kits according to the manufacturer’s trace protocol and the DNA was
eluted in a volume of 50 µL of TE. Ten samples were extracted (5 on each robot).
Semen
For organic extractions of DNA from semen samples, 50 µL of semen was
pipetted onto sterile cotton swabs. The swabs were then air-dried and half of each swab
was excised and transferred to a microcentrifuge tube containing 50 µL of sterile water.
To remove the sperm cells from the swabs, the tubes were first pulse-vortexed and
incubated at room temperature for 30 minutes. Next, the swabs were agitated with a
sterile stick to release the cells, the swabs were removed and discarded, and the tubes
were centrifuged for 1 minute on high speed to pellet the cells. Finally, all but 50 µL of
29
each supernatant was removed and discarded and the sperm cells were resuspended by
agitating the pellets with a sterile pipette tip.
To confirm that the spermatozoa had been successfully released from the swabs, 3
μL of each resuspended sperm sample was spotted onto a glass microscope slide and the
cells were fixed to the slide by incubating them in a 60° C oven for 15-30 minutes. The
cells were then visualized using Christmas Tree stain (SERI, Richmond CA).
Once the presence of sperm cells was confirmed, the organic DNA extractions
were performed according to the standard procedures used by SCDALFS [See Appendix
A] and the BioRobot®EZ1 DNA extractions were performed using EZ1 DNA
Investigator kits using the trace protocol and an elution volume of 50 µL of TE. For the
organic extractions, four semen samples were used. For the BioRobots, a total of eight
samples were extracted (replicate samples from each donor).
Mock Sexual Assault Samples
For the mock sexual assault samples, rectal, oral and vaginal swabs were
collected, in triplicate, from one of the female volunteers. Semen was also collected from
one of the male volunteers, and 50 µL was deposited onto each of the swabs collected
from the female to mimic swabs taken from rape kits. The DNA from two of each of the
rectal, oral, vaginal, and semen (6 swabs total) was then extracted using the
BioRobot®EZ1 trace protocol with an elution volume of 50 µL and the DNA from each
of the remaining swabs (3) was extracted using the SCDALFS’s organic method
[Appendix A].
30
Cross Contamination in the BioRobot®EZ1
To determine whether DNA extractions using the BioRobot®EZ1 are subject to
cross-contamination between samples analyzed side-by-side, several blood samples were
collected from one volunteer and the DNA was extracted using the BioRobot®EZ1 trace
protocol. During the procedure, blanks containing 200 uL of sterile water were inserted
in between each of the blood-containing samples to see whether any of the blanks became
contaminated with DNA [Figure 9]. A second examination was performed reversing the
samples and the blanks, as suggested by Montpetit, Fitch and O'Donnell (2005).
PCR Inhibition study
Seventeen different fabrics known to contain substances that inhibit downstream
PCR reactions following organic extractions were analyzed to see whether the
BioRobot®EZ1 DNA extraction method is able to remove them (Table 1). 50 μL of
blood and saliva was collected from three donors and aliquoted onto the fabrics. Three
sets of cuttings were made from the blood stain and saliva stains on each fabric (4x4 mm
to 6x6 mm). Two of the three sets were extracted via the BioRobot®EZ1 using the trace
protocol with an elution volume of 50 μL, while the third set was extracted via the
SCDALFS’s organic extraction method [Appendix A]. A total of 102 fabric samples
were extracted (68 for the robots and 34 organically).
Substances in cigar paper are also known to cause PCR inhibition, so the five
study volunteers were asked to deposit their saliva on cigars. Three 1 cm x 1 cm cuttings
31
of the paper surrounding the cigars were then excised and two from each cigar were
subjected to DNA extraction on the BioRobot®EZ1 using the trace protocol with an
elution volume of 50 µL The remaining cigar papers were extracted using the SCDALFS
organic method [Appendix A]. A total of 15 samples were extracted (10 for the robots
and 5 organically).
In all a total of 244 samples were analyzed in the Validations (176 on the robots
and 68 organic).
DNA Quantitation
The concentration of human DNA in the validation study extracts was determined
for two reasons. First, it allowed a comparison between the total DNA yields from the
organic and BioRobot®EZ1 DNA extraction methods. Second, the ABI AmpFℓSTR®
Identifiler™ PCR kit requires 0.5-1.25ng of DNA for optimal results during the
amplification process, and I wanted to ensure that when I amplified the samples for
genotyping I was adding the optimal amount of DNA to the reactions. Quantitation was
achieved using Quantifiler™ Human DNA Quantification kits (ABI, Foster City CA) on
an ABI PRISM® 7000 Sequence Detection platform followed by dilutions of the samples
to 0.1 ng/uL.
32
Figure 9. Arrangment for samples in the BioRobot®EZ1 contamination study.
33
Table 1. Fabrics used in the Inhibition study.
Number
1
2
Substrate
Suede-Black
Leather-Black
3
Leather-Brown
4
Leather-Tan
5
Denim-Black
6
Denim-Dark
7
Denim-Blue
8
Bandana-Black
9
Bandana-Red
10
Bandana-Maroon
11
Bandana-Pink
12
Bandana-Blue
13
Bandana-Royal Blue
14
Bandana-Orange
15
Bandana-Flag Print
16
Bandana-Camouflage
17
White gauze
34
Genotyping
10 µL of each extract (1 ng total) was amplified using ABI AmpFℓSTR®
Identifiler™ PCR kits in an ABI 9700 thermocycler. The samples were then genotyped
on an ABI 3130 Genetic Analyzer according the manufacturer’s standard protocols
[Appendix A] and using ABI Data Collection™ (v. 3.0) and Genemapper ID™ software.
Statistical Analyses
Quantitation values obtained using the organic and BioRobot®EZ1 methods were
compared using t-tests. P-values of less than or equal to 0.05 were considered
significant.
Part 2: Recovery of Contact DNA from Cartridge Cases and Gun Grips
Overview
Part 2 of my project examined whether it is possible to retrieve contact DNA from
cartridge cases and gun grips under a variety of conditions relevant to forensic casework.
Since contact DNA is usually present at very low levels, I used the highly sensitive
AmpFℓSTR® Minifiler™ system for most of my analyses. However, samples exhibiting
6 or more alleles in their AmpFℓSTR® Minifiler™ profiles were also subjected to
AmpFℓSTR Identifiler™ testing to see whether additional information could be obtained
from them.
35
Part 2 was performed in two phases. In Phase 1, ten volunteers were recruited
and screened for their ability to shed contact DNA onto brass .22 caliber cartridge cases.
Of these volunteers, five were selected to participate in Phase 2, including two who shed
multiple alleles onto the cartridges during handling. In Phase 2, the volunteers were
asked to handle cartridge cases and gun grips of different types under several different
conditions and the cases and grips were swabbed for DNA-bearing cells. The swabs
were then analyzed to determine whether contact DNA was present and, if so, how much
genetic information could be obtained from the DNA. I examined five variables to see
whether they affected DNA recovery from the cartridge cases: (1) the time elapsed
between hand-washing and handling, (2) the length of time the cartridges were handled,
(3) the caliber of the cartridges, (4) the metallic composition of the cartridges, and (5)
whether or not the cartridge had been fired from a gun. For the gun grips, I examined
three variables: (1) the time elapsed between hand-washing and handling, (2) the length
of time the gun grips were handled, and (3) the composition and texture of the grip.
One hundred and twenty swabs were analyzed during Phase 1 and 570 swabs
were analyzed during Phase 2. Therefore, I analyzed a total of 690 swabs in Part 2 of my
project.
Recruitment of Volunteers
It is standard practice for employees from the SCDALFS to submit samples for
research projects conducted in the laboratory. Therefore, laboratory employees were
36
recruited as volunteers for the study. For Phase 1, 7 men and 3 women were recruited.
Of these, three men and two women were chosen to continue as volunteers for Phase 2.
Sample collection
Phase 1
As discussed in the Introduction, some people shed contact DNA more easily onto
objects than others. In particular, Lowe e al. found that 60% of the population sheds
DNA well (i.e. are “good shedders”) as defined by the ability to shed a full DNA profile
on a plastic tube after handling it for 15 minutes. Since most people are good shedders
and I wanted to ensure that my study results accurately reflected what forensic analysts
are likely to encounter during routine casework, I initially screened 10 persons for their
ability to shed DNA onto .22 caliber brass cases. My reasoning was that if I chose only
one volunteer, there was a 40% chance of not choosing a good shedder. However, if I
screened 10 volunteers, there was only a (0.4)10 (approximately 1 in 10,000) chance that I
would not choose at least one good shedder.
Lowe et al. performed their contact DNA studies with the ABI AmpFℓSTR®
SGM Plus™ system. However, since I planned to use the AmpFℓSTR®Minifiler™
system, which is much more sensitive, I decided to screen my volunteers using shorter
handling times. Volunteers were asked to wash their hands and then immediately handle
three pristine (direct from the manufacturer) unfired .22 caliber brass cartridge cases: one
for 15 seconds, one for 30 seconds, and one for 60 seconds in succession (volunteers did
not wash their hands in between handling times). The process was then repeated, but
37
volunteers were asked to wash their hands and engage in normal activities for one hour
prior to handling the cases. After handling, the cases were placed in individual envelopes
and stored at room temperature prior to swabbing and DNA analysis.
To collect the DNA-bearing cells, each cartridge case was swabbed using the
efficient “double swab” method (Sweet, et al. 1997). This involves first dampening a
sterile cotton swab with 50 µL of sterile water and gently rolling it across the entire
surface of an object. Then a second, dry sterile swab is rolled across the surface to
collect any remaining cells. The swabs are then air-dried and packaged together prior to
DNA extraction and analysis.
As a final step in the collection of samples for the Phase 1 experiments, I repeated
the process described above but asked the volunteers to load the cartridge cases into a
gun after handling them and to fire the weapon. The expelled cases were then collected,
swabbed, and analyzed using the same method as for the unfired cases. Since I planned
to study the effect of firing on other types of cartridge cases in Phase 2, adding this to the
Phase 1 experiments allowed me to avoid repeating the .22 brass caliber experiments in
Phase 2. A flow diagram for the sample collection for the Phase 1 experiments is shown
in Figure 10.
38
10 volunteers
Wash hands
Immediately
handle cases
15”
Colle
30”
Collect
FIRE
and
collec
t
15”
60”
Collect
ct
FIRE
and
collec
t
Wait one hour prior to
handling cases
FIRE
and
collec
t
Collec
t
30”
Collect
FIRE
and
collec
t
60”
Collect
FIRE
and
collec
t
FIRE
and
collec
t
Figure 10. Flow chart for the collection of samples for Phase 1 of Part 2. Ten volunteers
handled three 0.22 brass caliber cases either immediately after washing their hands or one
hour after washing their hands. Each case was handled for either15 seconds, 30 seconds, or
60 seconds, and then either collected immediately or collected after the gun was fired. 10
volunteers x 2 handling conditions x 3 handling times x 2 collection protocols = 120
samples
39
Phase 2
In Phase 2, the Phase 1 experiments were repeated for 7 additional types of
cartridge cases: brass, nickel, and aluminum .38 caliber and brass, nickel, aluminum and
steel 9 mm. (Five volunteers x 7 cartridge types x 2 handling conditions x 3 handling
times x 2 collection protocols = 420 samples.)
The volunteers were also asked to handle 5 different types of gun grips under the
same conditions as for the cartridge cases except that the effect of firing the gun on the
collection of contact DNA from the grip was not examined.
Gun grips were cleaned with a 10% bleach solution prior to handling. Three gun
grips commonly used in crimes were included, each made of different material – wood
(textured and smooth), plastic (textured and smooth) and textured rubber. (Five
volunteers x 5 gun grip types x 2 handling conditions x 3 handling times = 150 samples.)
DNA Extraction
The DNA was extracted from the swabs using the BioRobot®EZ1 instrument.
First, the wet and dry swabs from each sample were excised and combined into a 2 mL
sample tube. Then the samples were pretreated following the manufacturer’s protocol for
forensic surface and contact swabs. Finally, the DNA in the samples was extracted using
the BioRobot®EZ1 trace protocol with an elution volume of 50 uL.
PCR Amplification
After DNA extraction, the samples were subjected to PCR using the
AmpFℓSTR® Minifiler™ system (ABI, Foster City, CA). Ten µL of each extract was
40
amplified in a total reaction volume of 25 µl on an ABI GeneAmp 9700 thermocycler,
following the manufacturer’s standard protocol for sample set-up and thermocycling.
Samples with at least 6 alleles in the AmpFℓSTR® Minifiler™ profiles were then
subjected to qPCR to determine whether there was sufficient DNA to proceed with ABI
AmpFℓSTR® Identifiler testing. qPCR was performed using ABI’s Quantifiler™ Human
DNA Quantification kits (Applied Biosystems, Foster City, CA) on an ABI Prism®
7000 Sequence detection platform , and the information from the reactions was used to
calculate how much of each extract should be added to the Identifiler ™ reactions. The
Identifiler ™ reactions were then performed according to the manufacturer’s standard
protocols (Applied Biosystems, Foster City CA).
Sample Genotyping
Minifiler™ and Identifiler ™ reaction amplicons were separated by automated
capillary gel electrophoresis on an ABI 3130xl Genetic Analyzer. Raw data was collected
using ABI Data Collection™ software v3.0 and analyzed using ABI GeneMapper ID™
(v.3.2) software.
Identification of Contaminated Samples and Withdrawal from the Study
After Minifiler™ analysis, the profiles of samples containing at least one allele at
or above 75 RFUs were compared against the known profiles of the donors. Any samples
41
that had alleles that could not have been contributed from the donor were then labeled as
“Contaminated” and withdrawn from the study.
Identification of Samples with Allelic Drop Out
Comparisons of the samples containing at least one allele at or above 75 RFU
with the donors’ profiles also enabled me to identify samples in which complete allelic
drop-out had occurred. Such samples present as homozygous at a locus when, in fact, the
donor is a heterozygote. This is because the second peak cannot be detected above
background “noise.” While such samples provide information for forensic casework, they
can also be misleading if they are not interpreted with caution. It is important for the
forensics community to know how many contact DNA samples are likely to contain
allelic drop out using the Minifiler™ system and under what conditions. Therefore, the
study tracked them and reported their frequency.
Classification of Genetic Profiles
All non-contaminated samples (including those with allelic drop out) were
classified into one of the following three categories:
(1) Full profile. The electropherogram contained at least one allele at or above
75 RFUs (relative fluorescent units) at all 8 AmpFℓSTR® Minifiler™™ STR loci or at
all 15 AmpFℓSTR® Identifiler™ STR loci
42
(2) Partial profile. The electropherogram contained at least one AmpFℓSTR®
Minifiler™ or AmpFℓSTR® Identifiler™ allele at or above 75 RFUs but did not contain
alleles at or above 75 RFUs at all STR loci in the system.
(3) No profile. The electropherogram did not contain any alleles at or above 75
RFUs.
Statistical Analyses and Reporting Methods
DNA recovery was assessed by counting the number of alleles deposited on each
cartridge case and gun grip. Differences across samples in the same group (e.g. caliber
type, amount of time handled, etc.) were compared using ANOVA tests. P values of less
than or equal to 0.05 were considered significant.
The number of samples falling into the each of the three broad categories (“Full
Profiles”, “Partial Profiles”, and “No Profile”) was divided by the total number of noncontaminated swabs analyzed, and the results were reported as percentages. Samples
falling into the “Contaminated” and “Allelic Drop Out” categories were reported in
absolute numbers
43
RESULTS
Part 1: Validation of BioRobot®EZ1
DNA Yields
Saliva
The BioRobot®EZ1 extraction method produced DNA yields comparable to
those obtained using the organic extraction method. The mean yields for the samples
extracted via the BioRobot®EZ1 were 365.0 ng (50 µL elution volume), 506.0 ng (100
µL elution volume), and 82.0 ng (200 µL elution volume). The mean yield for samples
extracted organically was 530.0 ng. There was no significant difference between the
mean DNA yields obtained at any of the elution volumes for the BioRobot®EZ1 and the
mean yield for the samples extracted organically (p=0.50 at 50 µL, p=0.98 at 100 µL and
p=0.07 at 200 µL df=4) [Figure 11].
Blood
The DNA yields from blood samples indicate that the BioRobot®EZ1 extracts
DNA from samples as effectively as the organic method. The mean DNA yield for
organic extracts was 48.9 ng. The mean DNA yields for the BioRobot®EZ1 extracts
were 41.6 ng (50 µL elution volume), 30.2 ng (100 µL elution volume), and 171.0 ng
(200 µL elution volume). The p values were as follows: p=0.78 for 50 µL, p=0.40 for
100 µL, and p=0.11 for 200 µL [Figure 12].
44
Figure 11—Histogram comparing the mean DNA yields from saliva samples using the organic
extraction method (n=5) and the BioRobot®EZ1 at three different elution volumes (50, 100, and
200 µL, n=30).
45
Figure 12. Histogram depicting the mean DNA yields from blood samples using the organic
extraction method(n=5) and the BioRobot®EZ1 extraction method at three different elution
volumes (50, 100 and 200 µL, n=30)
46
Semen
DNA yields were also compared between the BioRobot®EZ1 and organic
extraction methods for semen. However, in this case, only one volume (50 uL) was used
to elute the DNA in the BioRobot®EZ1 samples. The mean DNA yield for samples
extracted organically was 557.0 ng and the mean DNA yield for samples extracted with
the BioRobot®EZ1 was 1534.0 ng. The BioRobot®EZ1 DNA yields for semen samples
were significantly higher than those produced by the organic method (p=0.002).
However, the yields for both extraction methods were still well above the lower limit set
for downstream amplification with Identifiler™ (0.5-1.25 ng of DNA) [Figure 13].
Hair
DNA yields were compared for hair samples extracted by the BioRobot®EZ1 and
the organic extraction method. The mean DNA yield for the samples extracted
organically was 2.22 ng and samples extracted by the BioRobot®EZ1 eluted in 50 µL
was 17.4 ng. DNA yields of samples extracted via the BioRobot®EZ1 resulted in
slightly higher DNA yields that those extracted organically (p=0.04) [Figure 14].
47
Figure 13. Histogram depicting the mean DNA yields from neat semen samples using the organic
extraction method (n=4)and the BioRobot®EZ1 extraction method at an elution volume of 50 µl
(n=8).
48
Figure 14. Histogram depicting the comparison of DNA yields from hair samples (root) extracted
using the organic method (n=5) and the BioRobot®EZ1 extraction method at an elution volume
of 50 uL(n=10)..
49
Urine
The DNA yields from urine samples extracted by the BioRobot®EZ1 and the
organic method for two different input volumes of urine, 50 µL and 200 µL, were
compared. For 50 µl urine samples, the mean DNA yield for samples extracted
organically was 0.006 ng and the mean DNA yield for the samples extracted via the
BioRobot®EZ1 was 0.19 ng [Figure 15]. The 200 µL urine samples extracted
organically had a mean DNA yield of 0.04 ng and the mean DNA yield for the
BioRobot®EZ1 samples eluted in 50 µL of TE Buffer was 0.74 ng [Figure 16]. For 200
uL input samples, the BioRobot®EZ1 robot yielded slightly more DNA than the organic
method (p=0.04). However, for 50 µL input samples the difference between yields was
not significant (p=0.09). It should be noted that the DNA yields from both extraction
systems was very low and was insufficient for downstream analysis using the standard
DNA typing methodologies used in forensic analyses.
Inhibition Studies
Blood and Saliva on Fabrics
An inhibition study was conducted using seventeen different fabrics in order to
determine if the BioRobot®EZ1 was better at removing PCR inhibitors than the organic
50
Figure 15. Histogram depicting the mean DNA yields for the organic extraction method (n=5) and
the BioRobot®EZ1 for 50 µL of urine (n=10).
51
Figure 16—Histogram depicting the mean DNA yields for the organic extraction method (n=5)
and BioRobot®EZ1 for 200 µL of urine (n=10).
52
extraction method. After extraction, a visual examination of the extracts suggested that
the BioRobot®EZ1 is better at removing dyes from some substrates (black suede, black
leather, red bandana and maroon bandana) since the organically extracted samples were
slightly colored whereas the BioRobot®EZ1 samples were clear.
Inhibition was detected by examining the internal control CT values obtained
during qPCR with the ABI Quantifiler™ Human DNA Quantification system. Inhibition
was observed after organic extraction from some substrates [Table 2]. However, PCR
inhibition was not observed in extracts generated using the BioRobot®EZ1, suggesting
that the BioRobot®EZ1 is a better method, overall, for removing PCR inhibitors from
blood and saliva stains on fabrics.
Inhibited samples were subjected to additional analysis using the ABI AmpFℓSTR
Identifiler™ system to see whether they could be genotyped. Interestingly, full DNA
profiles could still be obtained from some extracts inhibited during qPCR. This indicates
that high CT values of the internal control during qPCR may not be a good indicator of
the performance of the extract during downstream genotyping.
53
Table 2. Substrates containing PCR inhibitors that could not be removed during organic extraction. Some
samples that showed inhibition during qPCR could still be used to generate full profiles in downstream
Identifiler™ reactions.
Substrate
DNA Concentration
(ng/µL)
Inhibited
IPC CT
STR Data
Tan Leather
Undetermined
Yes
Undetermined
No profile
Black Leather
0
Yes
Undetermined
No profile
Suede Black
2.34
Yes
35.01
Full Profile
Royal Blue
Bandana
5.51
Yes
31.43
Full Profile
Dark Denim
3.66
Yes
31.94
Full Profile
Camouflage
6.76
Yes
33.19
Full Profile
54
Cigars
None of the cigar DNA extracts contained PCR inhibitors after extraction. Four of
the five organically extracted samples produced full profiles in downstream genotyping,
while one produced a partial profile. The BioRobot®EZ1 yielded full DNA profiles for
all samples [Table 3].
Cross-Contamination Study
Cross contamination can occur due to the close proximity of samples during
analysis and could be especially problematical for the BioRobot®EZ1 because sample
tubes remain open during the extraction process. However, no DNA cross-contamination
was detected.
Mock Sexual Assault Study
The mock sexual assault samples contained a mixture of female DNA from oral,
rectal and vaginal swabs and semen from a single male. Mixtures like these are
commonly encountered during forensic casework and it is important that the female and
male DNA fractions be separated as effectively as possible to prevent mixed profiles in
downstream genotyping.
The organic extraction method proved to separate the female and male fractions
efficiently. However, all female extracts generated from the BioRobot®EZ1 contained
mixed DNA profiles [Figure 17]. Therefore, the organic DNA extraction method is
preferable for stains containing a mixture of semen and female epithelial cells.
55
Table 3. Mean DNA yields and genotyping success of saliva stains on cigar paper following DNA
extraction with the organic and BioRobot®EZ1 methods. Samples 1-5: Organic extraction. Samples 1Q5Q: BioRobot®EZ1 extraction.
Samples
DNA Yields (ng)
Item-1
Item-1Q
Item-2
Item-2Q
Item-3
Item-3Q
Item-4
Item-4Q
Item-5
Item-5Q
2.77
9
3.14
10
1.34
3.25
0.65
3
0.13
2.83
STR Data
Partial STR Profile
Full STR Profile
Full STR Profile
Full STR Profile
Full STR Profile
Full STR Profile
Full STR Profile
Full STR Profile
Full STR Profile
Full STR Profile
56
A)
B)\
Figure 17. Comparison of organic and BioRobot EZ1™ DNA extraction methods in separating male and
female DNA fractions in a mock sexual assault sample. A) Organic extraction. One minor allele from the
male appears in the female’s profile. B) EZ1 extraction. Several alleles from the male appear in the
female’s profile, and the peak heights of the male alleles are higher than those observed for the same
sample extracted organically.
57
Phase 1
Part 2: Recovery of Contact DNA from Cartridge Cases and Gun Grips
The results from Phase 1 are shown in Table 4. Of the ten volunteers screened,
two (Volunteers 7 and 8) shed more DNA than the others. Both shed one allele onto a
case immediately after they washed their hands and shed many alleles onto the cases
when they handled them one hour post-hand washing. Interestingly, only three of the
other volunteers (Volunteers 3, 4 and 5) shed any alleles onto the cases, regardless of the
handling conditions, and each shed so little DNA that only one or two alleles could be
detected above the75 RFU threshold.
The two donors who shed the most DNA were selected to continue as volunteers
for Phase 2. In addition, three other donors were chosen, according to convenience, as
additional volunteers for Phase 2.
Phase II
Cartridge Cases: Identification of Contaminated Samples and Withdrawal from the Study
Thirty-nine out of the 540 swabs analyzed were contaminated and withdrawn
from the study. Therefore, all downstream analyses were performed on only 501 samples.
Identification of Samples with Allelic Drop Out
Thirty-one of the samples contained at least one locus that exhibited total drop out
of one of the alleles at one or more heterozygous loci. These samples were not removed
from the study and underwent all steps of the downstream analyses. However, they were
tracked in the study.
58
Table 4. DNA shed onto .22 caliber brass cases by 10 different donors as detected by AmpFlSTR®
Minifiler™. Donors handled cases for 3 different time periods (15 sec, 30 sec, or 60 sec) either
immediately after washing their hands or 1 hour after washing their hands. Volunteers 7 and 8 shed much
more DNA than the other donors. Donors 2, 5, 6, 7, and 8 were chosen to continue as volunteers for Phase
2. Alleles detected above a threshold of 75 RFU are emboldened.
Immediately after Washing Hands
Handling time
Volunteer 1
Volunteer 2
Volunteer 3
Volunteer 4
Volunteer 5
Volunteer 6
Volunteer 7
Volunteer 8
Volunteer 9
Volunteer 10
15 sec
0
0
0
0
0
0
0
1
0
0
30 sec
0
0
0
0
0
0
0
0
0
0
60 sec
0
0
0
0
0
0
1
0
0
0
1 Hour After Washing Hands
15 sec
0
0
0
1
0
0
12
6
0
0
30 sec
0
0
0
0
2
0
2
11
0
0
60 sec
0
0
1
0
0
0
2
16
0
0
59
Effect of Caliber Type on DNA Recovery from Cartridge Cases
A comparison between the total number of alleles recovered from three calibers,
.22, .38 and 9mm cartridge cases, did not show a significant difference in the recovery of
DNA (p=0.24). No profiles were obtained for 90% of samples for .22 cartridge cases
(n=114), 83% of samples for .38 cartridge cases (n=161) and 85% of samples for 9mm
cases (n=226). Partial profiles were observed for 9.0% of .22 samples, 13% of .38
samples and 13% of 9mm samples. Full profiles were observed in 1.0% of .22 samples,
4.0% of .38 samples and 2.0% of 9mm samples [Figure 18].
Contamination was observed in 6 of the samples from .22 caliber cases, 19 of the
samples from .38 caliber cases, and 14 samples from 9mm cases. These samples were
excluded from the study.
Effect of Firing on DNA Recovery from Cartridge Cases
Of the 270 unfired case samples, 20 were contaminated and were withdrawn from
the study. Of the 270 fired case samples, 19 were contaminated and were withdrawn from
the study. This left a total of 250 samples from unfired cases and 251 from fired cases.
A comparison of unfired versus fired cartridge cases showed that there was a highly
significant difference in the amount of DNA recovered (p<0.001). More DNA was
recovered from unfired cases than from fired cases, which was expected. No profiles
were obtained for 80% of unfired samples and 92% of fired samples. Partial profiles
were obtained for 15% of unfired and 8% of fired. Full profiles were observed for 5% of
unfired and 0% of fired [Figure 19].
60
Figure 18. Histogram depicting the percentage of samples with “Full Profiles”, “Partial Profiles” or “No
Profiles” of different caliber types (n=501)
61
Figure 19.Percentof samples with full profiles partial profiles, and no profiles from unfired and fired
cartridge cases (n=501).
62
Effect of Handling Time on DNA Recovery from Cartridge Cases
Of the 180 samples handled by donors for 15 seconds, 14 were contaminated and
were withdrawn from the study. Of the 180 samples handled by donors for 30 seconds,
11 were contaminated and were withdrawn from the study. Of the 180 samples handled
by donors for 60 seconds, 14 were contaminated and were withdrawn from the study.
This left a total of 166 handled for 15 seconds, 169 handled for 30 seconds, and 166
handled for 60 seconds.
Full profiles were obtained from 2% of the samples handled for 15 seconds, 3%
of the samples handled for 30 seconds, and 3% of the samples handled for 60 seconds.
Partial profiles were obtained from 11% of the samples handled for 15 seconds, 13% of
the samples handled for 30 seconds, and 13% of the samples handled for 60 seconds. No
profiles were obtained from 87% of the samples handled for 15 seconds, 84% of the
samples handled for 30 seconds, and 84% of the samples handled for 60 seconds [Figure
20].
When the total number of alleles recovered from the cases was compared, no
difference was observed (p=0.85). Therefore, the amount of time a person handles a
cartridge case, at least within the 15/30/60 second window examined in this study, makes
no difference in the amount of contact DNA they shed on the cases. Since it is unlikely
that a person loading a gun would handle a case for more than about 1 minute, these
results are probably a good predictor of the results analysts can expect from casework
samples.
63
Figure 20. Percent of samples with “Full Profiles”, “Partial Profiles”, and “No Profiles”
generated from cartridge cases handled for three different lengths of time (n=501).
64
Effect of Case Material on DNA Recovery from Cartridge Cases
Of the 240 brass cartridge case samples, 13 were contaminated and were
withdrawn from the study. Of the 120 aluminum cartridge case samples, 6 were
contaminated and were withdrawn from the study. Of the 120 nickel cartridge cases, 20
were contaminated and were withdrawn from the study. Of the 60 steel cartridge cases,
none were contaminated, so all were included in the study. Full profiles were obtained
from 1.0% of the brass cartridge cases, 3% of the aluminum cartridge cases, 5.0% of the
nickel cartridge cases, and 0% of the steel cartridge cases. Partial profiles were obtained
from 7.0% of the brass cartridge cases,
21% of the aluminum cartridge cases, 11% of the nickel cartridge cases, and 18%
of the steel cartridge cases. No profiles were obtained from 92% of the brass cartridge
cases, 75% of the aluminum cartridge cases, 84% of the nickel cartridge cases, and 82%
of the steel cartridge cases [Figure 21].
When the total number of alleles recovered from the cases was compared, no
difference was observed (p=0.11). Therefore, metal composition does not appear to make
a significant difference in the amount of contact DNA shed onto cartridge cases.
Effect of Time Since Hand Washing on DNA Recovery from Cartridge Cases
Of the 270 cases handled immediately after hand washing, 13 were contaminated
and were withdrawn from the study. Of the 270 cases handled one hour after hand
washing, 26 were contaminated and were withdrawn from the study.
65
Figure 21. Percent of samples with “Full Profiles”, “Partial Profiles”, and “No Profiles”
generated from cartridge cases comprised of 4 different types of metals (n=501)..
66
“Full Profiles” were obtained from 2.0% of the cases handled immediately after hand
washing and 3% of the samples handled one hour after hand washing. “Partial Profiles”
were obtained from 6% of the samples handled immediately after hand washing and 18%
of samples handled one hour after hand washing. “No Profiles” were obtained from 92%
of samples handled immediately after hand washing and 79% of samples handled one
hour after hand washing [Figure 22].
When the total number of alleles recovered from the cases was compared, a
significant difference was observed. The DNA recovery was much higher when a person
waited one hour after hand washing to handle a cartridge case than if a person handled a
case immediately after hand washing (p=0.001).
AmpFℓSTR Identifiler™ Typing
Samples containing three or more alleles from the donor were quantitated to
determine if they were candidates for additional genotyping using the less sensitive but
more informative AmpFℓSTR Identifiler™ system. A minimum of about 0.5 ng of DNA
is needed to generate reproducible Identifiler™ genotypes free of peak height
imbalances, allele drop out and other stochastic effects. Therefore, samples containing
less than 0.1 ng of DNA were not tested with this method. Forty-seven samples
67
Figure 22—Comparison of percentage of samples at two wash times with no profile, a partial
profile and a full profile for cartridge cases amplified with Minifiler™ (n=501).
68
contained three or more alleles in the Minifiler™ analyses and were quantitated. Of
these, 11 contained at least 0.1 ng of DNA and were amplified with Identifiler™.
One of the 11 samples run through Identifiler™, one yielded a “Full Profile” in
and yielded “Partial Profiles”. Only one of the “Full Profiles” was complete (contained
all the alleles carried by the donor). Therefore, the vast majority of non-contaminated
cartridge cases in this study (496/501) either contained too little DNA for Identifiler™
analysis or failed to produce profiles when the analysis was performed.
Summary of Cartridge Case Study Results
In total, 540 cartridge cases were swabbed. Of these, 39 were contaminated and
removed from the study. Therefore, the results from 501 samples were analyzed.
Of the 501 uncontaminated samples, “Full Profiles” were obtained from 2.4%
(12/501) of the samples, “Partial Profiles” were obtained from 10.2% (51/501) of the
samples, and “No Profiles” were obtained from 87.4% (438/501) of the samples.
Very few of the “Full Profile” samples contained the complete DNA profile of the
donor in either Minifiler™ or Identifiler™ testing. Only 10 samples tested with
Minifiler™ and only one sample tested with Identifiler™ contained every allele from the
donor at a level of at least 75 RFU.
69
Gun Grips
Identification of Contaminated Samples and Withdrawal from the Study
Twelve of the 150 samples taken from gun grips were contaminated and were
withdrawn from the study. Therefore, all downstream analyses were performed on only
138 samples.
Identification of Samples with Allelic Drop Out
Nineteen of the samples contained at least one locus that exhibited complete drop
out of one of the alleles at one or more heterozygous loci. These samples were not
removed from the study and underwent all steps of the downstream analyses. However,
they were tracked in the study.
Effect of Grip Type on the Recovery of DNA from Gun Grips
Of the samples taken from smooth wood gun grips, 5 were contaminated.
Therefore, 25 were analyzed. Of the samples taken from textured wood grips, 3 were
contaminated. Therefore, 27 were analyzed. Of the samples taken from smooth plastic
grips, 1 was contaminated. Therefore, 29 were analyzed. Of the samples taken from
textured plastic grips 0 were contaminated. Therefore, 30 were analyzed. Finally, of the
samples taken from textured rubber grips, 3 were contaminated. Therefore, 27 were
analyzed.
The percent of full profiles, partial profiles, and no profiles obtained from each
grip type are shown in Figure 23. When the total number of alleles deposited on the
70
different grip types was compared, no significant difference in DNA recovery was
observed (p=0.06)
Effect of Handling Time on the Recovery of DNA from Gun Grips
Of the samples taken from the donors after they handled the gun grips for 15
seconds, 5 were contaminated. Therefore, 45 were analyzed. Of the samples taken from
the donors after they handled the gun grips for 30 seconds, 4 were contaminated.
Therefore, 46 were analyzed. Of the samples taken from the donors after they handled the
gun grips for 60 seconds, 3 were contaminated. Therefore, 47 were analyzed.
Data on the percent of “full profiles”, “partial profiles” and “no profiles” obtained
at different handling times is shown in Figure 24. When the total number of alleles
recovered from the grips at different handling times was compared, no significant
difference was observed (p=0.45).
Effect of Hand washing on the Recovery of DNA from Gun Grips
Of the samples taken from the donors who handled the gun grips immediately after hand
washing, 5 were contaminated. Therefore, 70 were analyzed. Of the samples taken from
the donors who handled the gun grips one hour after hand washing, 7 were contaminated.
Therefore, 68 were analyzed. Data of the percent of “full profiles”, “partial profiles” and
“no profiles” obtained at immediately after and one hour post-hand washing in shown in
Figure 25.
71
Figure 23. Effect of grip material on the percent of “Full Profiles,” “Partial Profiles,” and “No
Profile” recovered from gun grips. Amplifications were performed with AmpFlSTR Minifiler™
(n=138).
72
Figure 24. Effect of handling time on the percent of “Full Profiles,” “Partial Profiles,” and “No Profile”
recovered from gun grips. Amplifications were performed with AmpFlSTR Minifiler™ (n=138).
73
When the number of alleles recovered from the grips was compared, a significant
difference was observed. More alleles could be recovered from grips handled one hour
after hand washing than those handled immediately after hand washing (p=0.002). These
results are similar to those in the cartridge case study, providing further evidence that
time since hand washing is an important factor influencing the recovery of contact DNA
from objects.
AmpFlSTR Identifiler Typing
Fifty-nine of the gun grip samples yielded Minifiler™ profiles with three or more
alleles at or above 75 RFU and were subjected to downstream quantitation. Thirty-four
did not have sufficient levels of human DNA for Identifiler™ typing. However, 25
produced profiles when they underwent Identifiler™ analysis. Six produced a full profile
and 14 produced partial profiles. All six of the full profile samples contained all of the
alleles carried by the donor and none exhibited the complete drop out of alleles.
Summary of Gun Grip Study Results
In total, 150 gun grips were swabbed. Of these, 8.0% (12/150) were contaminated
and removed from the study. Therefore, the results from 138 samples were analyzed.
74
Figure 25. Percent of gun grips for which “No Profiles” “Partial Profiles” or “Full profiles” were observed
when amplified with Minifiler™ (n=138).
75
Full profiles were obtained from 18.8% (26/138) % of the samples, partial profiles were
obtained from 33.3 % (46/138) of the samples, and no profiles were obtained from 47.8%
(66/138) of the samples when analyzed using Minifiler™. Of the samples containing full
profiles or partial profiles, 19 exhibited at least one locus with complete allelic drop out.
Of the full profile samples, 26 contained the complete genotype of the donor
when analyzed by Minifiler™ and 6 contained the complete Identifiler™ profile.
Comparison of Results from Cartridge Case and Gun Grip Studies
A flow chart comparing the results of the Cartridge Case and Gun Grip studies is
shown in the Figure 22. When the data is viewed from this perspective, five trends
emerge. First, under the same handling conditions, more alleles 641were shed onto the
gun grip samples than onto the cartridge cases 368 (p = < 0.001). Second, the quality of
the profiles obtained from the gun grips was higher than those obtained from the cartridge
cases. Fifty-two% (72/501) of the gun grip samples yielded genetic information, while
only 12.5% (63/138) of the cartridge case samples yielded information. Third,
downstream Identifiler™ testing was more successful on gun grips samples. Of the
samples analyzed in the Identifiler™ system, 29.9% (20/67) of the gun grip samples
yielded genetic information compared to only 10.6% (5/47) of the cartridge case samples.
Fourth, the Minifiler™ system, probably due to it greater level of sensitivity, produced
contaminated profiles while the Identifiler™ system did not. 7.4% (51/690) of the
Minifiler profiles were contaminated and had to be withdrawn from further analyses.
76
Finally, complete allelic drop out was absent from the Identifiler™ profiles, while 7.5%
(48/639) of the Minifiler™ samples exhibited complete drop out of one or more alleles.
A bias exists in the data concerning allelic drop out because only samples with
optimal levels of DNA were subjected to Identifiler™ analysis. However, complete
allelic drop out is rare in Identifiler profiles; usually, the second allele in a heterozygous
pair can still be detected above background in electropherograms. Therefore,
heterozygosity can be detected and used for inclusionary or exclusionary purposes.
77
Minifiler™
690 swabs
150 gun grips
540 cartridge cases
39
contaminate
d
438
None
501 analyzed
51 Partial
12 Full
138 analyzed
12
contaminated
46 Partial
26 Full
66 None
29 total
allelic drop
out
observed
2
incomple
te
10 complete
26 complete
2 total allelic drop
out observed
0 incomplete
19 total
allelic drop
out observed
0 total allelic drop
out observed
Quantifiler™
59 samples quantitated
47 samples quantitated
36 swabs
<0.1 ng
11 swabs >0.1 ng
25 swabs > 0.1 ng
34 swabs
<0.1 ng
Figure 26- Flow chart comparing the results obtained from the cartridge case and gun grip studies
continued on page 79).
78
Identifiler™
11 swabs >0.1
ng
0
Contaminate
d
6 None
4 Partial
25 swabs > 0.1 ng
1 Full
0 Contaminated
14 Partial
5 None
6 Full
0 total allelic drop
out observed
1
complete
0 _incomplete
6 complete
0 _incomplete
0 total allelic
drop out
observed
79
DISCUSSION
Validation
Validation studies are extremely important in forensic laboratories to determine
whether a new procedure or instrument performs as efficiently as the
procedure/instrument that it is replacing. Each laboratory is responsible for conducting
its own validation study before the instrument/procedure can be implemented. This
validation of the BioRobot®EZ1 provided data that demonstrated the capability of this
platform in performing DNA extractions as efficiently, in most cases, as the phenolchloroform DNA extraction method that was in use at the laboratory before this study
was performed.
The data generated in this study demonstrated that both extraction methods give
comparable and robust DNA yields from saliva and blood but that the BioRobot®EZ1
outperforms the organic method on semen and hair samples. For urine, the
BioRobot®EZ1yielded slightly more DNA when the input volume was 200 uL but
comparable yields when the input volume was 50 uL.
The BioRobot®EZ1 proved more effective at removing PCR inhibitors than the
organic extraction method. The DNA from samples containing known inhibitors and
extracted using the BioRobot®EZ1 showed no inhibition in qPCR. The BioRobot®EZ1
also proved to be much more successful in completely removing PCR inhibitors from
leather fabrics.
80
This study saw no PCR inhibition in DNA extracts from cigar samples. However
Montpetit et al., (2005) observed PCR inhibition in cigar samples extracted using the
organic method, while the majority of their cigar samples extracted using the
BioRobot®EZ1 yielded full DNA profiles with no inhibition. It is unclear why this study
obtained different results, but it could possibly be due to the types of cigars used or the
fact that Monpetit et al .used larger cuttings. They used one-third of the paper from the
filter region, where this study used only 1 cm x 1 cm cuttings. This may suggest that
taking smaller cuttings of cigars is a good idea because it limits the inhibition of PCR in
downstream applications. However, a more careful study would need to be performed to
confirm this finding.
There are two reasons that might explain why the BioRobot®EZ1 is more
effective at removing PCR inhibitors during DNA extraction. First, the magnetic bead
technology that is used allows for tight binding of the DNA to the silica beads and the
inhibitors likely do not bind, yielding a more pure sample. Secondly, samples are eluted
in a much higher volume, therefore the samples are far less concentrated, hence diluting
out the inhibitors. Prior to systems like the BioRobot®EZ1, it was common practice to
dilute samples or increase the number of PCR cycles in order to over come inhibition.
This study did not fully examine every sample that could contain potential PCR inhibitors
and it would be beneficial to conduct further experiments to determine what type of
inhibitors cannot be removed using the silica magnetic bead technology, if any.
The BioRobot®EZ1 platform contains sample and elution tubes that remain open
during the extraction process, which could lead to sample-sample contamination. To
81
control for this, we interspersed blank samples in between samples with blood. No DNA
was detected in any of the blank samples as shown by the quantitation of the samples as
well as the subsequent PCR amplification. The reagents are contained in single use
cartridges, and the extraction process occurs in the filter tip which does not cross over
any other samples. This greatly reduces the risk of contamination between samples. The
filter tips are also disposable, further preventing contamination of samples. Each sample
has its own filter tip and after each run, a new filter tip is used.
Comparisons of the STR profiles generated from the mock sexual assault samples
demonstrated that at this time the BioRobot®EZ1-based differential extraction does not
perform as well as the organic method. Non-sperm fraction samples extracted using the
BioRobot®EZ1 contained some carry over from the sperm cells as shown by the STR
profiles generated in all of the samples. There was also some carry over in the non-sperm
fraction seen in some organically extracted samples; however it was far less than what
was observed from the BioRobot®EZ1 samples. This is possibly due to the proteinase K
that is provided in the Qiagen kit. Qiagen does not release the details of the
concentration of the Proteinase K, but it is believed that it is at such a high concentration
that it lyses the sperm cells too early, thus making it harder to separate the two fractions.
Another theory is that the Qiagen Buffer G2 contains a high concentration of detergent,
which also increases the likelihood that the sperm will lyse early. It is recommended that
studies examining why the BioRobot®EZ1 does not perform as efficiently for these
samples continue to be investigated.
82
Contact DNA
Contact DNA has been important in some very high profile cases, such as the
JonBenet Ramsey case, in which Bode Technology was able to obtain a contact DNA
profile from the long johns she wore the night she was murdered (Bode Technology
2009). Contact DNA is not a new idea; however, recent improvements in the technology
have made it possible to generate human DNA profiles from much smaller quantities of
DNA. The goal of this project was to determine if it was possible to obtain human
genetic profiles from contact DNA shed onto cartridge cases gun grips, which are some
of the most common forms of evidence submitted to crime labs. The SCDALFS has
repeatedly been asked to examine these types of samples, and this study aimed to provide
evidence as to whether such an analysis is viable.
It was not the goal of this experiment to determine the frequency of ‘good DNA
shedders’ in the population. However, a preliminary experiment was used to determine
the five ‘best’ shedders out of ten volunteers. It was observed that of the five individuals
chosen, three ‘shed’ more DNA onto cartridge cases and gun grips than the other
volunteers throughout the remainder of the study. This is consistent with the Lowe, et al.
finding that approximately 60% of the population shed DNA well. Another interesting
observation was that two of the five volunteers in this study were women and all of them
shed less DNA than the men. Lowe et al., did not observe a male or female bias in their
sample size of 30 and gender was not considered for the Phipps et al. study (2003). It
would be interesting to further investigate if men, in general, are better shedders than
women and why this might be the case.
83
It was determined that for cartridge cases, the physical size of the case, the
material used to manufacture the case and the handling time did not influence the amount
of DNA recovered. It was expected that .38 cartridge cases would yield more DNA
compared to the other two calibers, because this caliber has a larger surface area.
However, this was not observed. It was found that the type of metal in the case (brass,
aluminum, nickel and steel), also did not affect the recovery of DNA.
The length of time an object was handled had no effect on the amount of DNA
recovered. However, the time intervals included in this study were very short and it
possible that if an individual were to hold a cartridge case or gun grip for longer periods
of time, more DNA would be recovered. A follow-up study, especially one focusing on
gun grips (which are much more likely to undergo long-term contact than cartridge cases)
would therefore be advisable.
Minimal amounts of DNA (alleles present at 1-4 loci) were recovered from fired
cartridge cases. One of the fired cartridge cases contained a partial profile consisting of
alleles present at six loci. These alleles were consistent with those of the volunteer but
may have been produced by inadvertent handling of the case by the volunteer after the
case was fired. It is likely that firing a gun degrades DNA on cases because of the heat to
which the case is exposed. However, Given et al (1976), in an examination of the effects
of firing on the recovery of latent prints from cases, concluded that is it is more likely the
effects of the internal pressure that removes material from the surface of the case. The
pressure causes the case to expand, which produces friction may transfer material from
84
the case to the chamber of the firearm. It might be interesting to swab the inside of the
chamber of a gun after it is fired to see whether any DNA can be recovered.
Unlike fired cases, it is possible to recover informative profiles from unfired cases,
even if they are handled only briefly. However, the quantity of DNA recovered from
these types of objects after a single contact seems to be limited to a few nanograms.
Whether an individual washed their hands prior to touching an object or waited an
hour after hand washing proved to be significant to the amount of DNA recovered, for
both gun grips and cartridge cases. Therefore, the longer an individual goes without
washing their hands the more DNA they are likely to transfer. This was also observed by
Phipps et al .and Lowe et al.
van Oorschot et al., (1997) were the first to show that an individual’s genetic
profile could be generated from swabs taken from objects they handled, such as a
briefcase handle, plastic knives, pens and mugs. They examined different handling times
as well as the effects of hand washing. Later studies recovered smaller amounts of DNA
from contact samples, including coffee mugs, phones and door handles (Ladd, et al.
1999, Lowe, et al. 2002, Esslinger, et al. 2004). The samples examined in this study
generated yields of 0.0045 ng up to 38 ng. However, when using Minifiler™, it was
possible to recover alleles from less than 0.0045 ng of DNA (the optimal amount is 0.5 0.75 ng). The wide range can be explained by the different types of samples used in this
study and the many variables that were tested.
This study focused solely on handguns and handgun ammunition, so additional
studies on other types of firearms, especially those that require large ammunition (e.g.
85
shotguns and rifles), would be beneficial. In addition, it would be helpful to know
whether areas of a gun other than the grip are a good source of contact DNA.
Complete allelic drop out was seen in many of the Minifiler™ profiles. At this
time, it is unclear why Minifiler™ is prone to this problem. It may result from the fact
that Minifiler™ is usually used only on very low copy number samples, and the PCR
reactions may therefore be subject to sampling error in the initial rounds of primer
binding and amplification. A full Minifiler™ profile is shown in Figure 27.
A profile of the same individual, but exhibiting complete allelic drop out, is shown in
Figure 28.
This study also indicates that Minifiler™ is more subject to contamination than
Identifiler™, and that the source of the contamination is not always easy to trace. Indeed,
several of the samples in this study contained alleles that were not carried by either the
donor or the person swabbing and analyzing the sample, indicating that tiny amounts of
DNA in the environment or in the reagents used to process the samples may amplify in
Minifiler™ reactions despite preventative measures.
The results of this study therefore support the idea that the Minifiler™ system is
more prone than Identifiler™ to producing profiles that are potentially misleading.
Contamination and complete allelic drop out can interfere with the accurate interpretation
of forensic DNA profiles and Minifiler™ profiles from contact DNA samples should
therefore be interpreted with caution. The greater sensitivity of the Minifiler ™ system
must be carefully weighed against its tendency to yield misleading results, and the choice
of system should reflect a careful consideration of the advantages and limitations of each.
86
Figure 27. Electropherogram of a full Minifiler™
87
Figure 28—Electropherogram demonstrating allelic drop out at two loci. This person is
heterozygous (29, 31.2) at D21S11, but only one peak is present, 29. At FGA, the individual is
heterozygous (18, 23), but only peak 23 is present.
88
Appendix A
Protocols from the Sacramento County District Attorney’s Office, Laboratory of Forensic
Services for DNA extraction, quantitation and amplification.
Methods:
1. Extraction (phenol-chloroform, organic)
Blood
Semen Swab
Oral Swab
Hair
Urine
2. Concentration (Centricon Purification)
3. Quantitation
a. qPCR (7000)
4. Amplification (PCR GeneAmp 9700)
ABI Identifiler™
ABI Minifiler™
1. Extraction (phenol-chloroform)
Sample Digestion-Blood
1. Cut a cm2 from a blood card with a sterile scalpel and place in a 1.5
mL microcentrifuge tube.
2. Add 0.5 mL of Digest Buffer to the tube with the sample.
3. Add 15 µL of Proteinase K Solution
4. Mix gently
5. Incubate in a heat block at 56ºC ± 1ºC for 1 hour.
6. Add 15 µL of Proteinase K Solution.
7. Incubate in a heat block at 56ºC ± 1ºC for 1 hour.
8. After digestion, remove substrate with a sterile stick and discard.
Sample Digestion-Semen Swab
1. Pipette 50 µL of liquid semen onto a sterile cotton swab, and let dry
over night in the fume hood in a swab rack.
2. Cut half of the swab tip with a sterile scalpel and place in a 1.5 mL
microcentrifuge tube.
3. Add 0.5 mL of Digest Buffer to sample tube.
4. Add 20 µL of 1M Dithiothreitol (DTT)
5. Add 15 µL of Proteinase K
6. Incubate in a heat block at 56ºC ± 1ºC for 1 hour.
89
7. Add 15 µL of Proteinase K.
8. Incubate in a heat block at 56ºC ± 1ºC for 1 hour.
9. Remove substrate from sample.
Sample Digestion-Oral Swab
1. Cut half of the swab tip with a sterile scalpel and place in a 1.5 mL
microcentrifuge tube.
2. Add 0.5 mL of Digest Buffer
3. Add 15 µL of Proteinase K Solution
4. Mix gently
5. Incubate the sample for 1 hour in a heat block at 56ºC.
6. Add 15 µL of Proteinase K Solution.
7. Incubate the sample at 56ºC for 1 hour.
8. After incubation, remove substrate with a fresh autoclaved stick and
discard.
Sample Digestion-Hair
1. Wash hair with 1 mL of sterile water in a 1.5 mL microcentrifuge tube.
2. Cut off 1 cm of proximal (root) end for digestion
3. Cut off 1 cm of the shaft adjacent to the root for separate analysis as a
control.
4. Add 0.5 mL of Digest Buffer
5. Add 20 µL of 1M Dithiothreitol (DTT)
6. Add 15 µL of Proteinase K Solution.
7. Incubate the samples at 56ºC for at least 6 hours, hair will usually
soften but not dissolve after this initial incubation.
8. Vortex samples for 30 seconds.
9. Add an additional 20 µL of DTT
10. Add an additional 15 µL of Proteinase K Solution
11. Incubate at 56ºC overnight until hair is completely dissolved.
12. Vortex for 30 seconds
13. Centrifuge the samples in a microcentrifuge for 1 minute on high
speed at room temperature to remove any pigment and particles
14. Transfer the supernatant to a new sterile tube.
Sample Digestion-Urine
1. Transferred 50 µL of urine to 1.5 mL microcentrifuge tube.
2. Add 0.5 mL of Digest Buffer to sample
3. Add 15 µL of Proteinase K Solution
4. Incubate samples at 56ºC for 1 hour.
5. Add 15 µL of Proteinase K Solution.
6. Incubate at 56ºC for an addition hour.
DNA Extraction
90
1. Add 0.5 ml of Buffered Phenol-Chloroform-Isoamyl Alcohol Solution
to sample.
2. Cap the tube and vortex for 15 seconds or until a white emulsion
forms.
3. Microcentrifuge on high speed for three minutes at room temperature.
4. Remove the lower phenol-chloroform layer
5. Repeat steps 1-4 three additional times and ensure nothing is visible at
the interface and the aqueous layer is clear.
2. DNA Concentration and Wash (Centricon Purification)
1. Assemble a Centricon concentration unit
2. Add 1.5 mL of TE Buffer to upper reservoir
3. Transfer the entire upper aqueous layer containing the DNA to the TE
Buffer in the unit
4. Centrifuge at room temperature for 20 minutes in a Hermle Z 323 at
2000 x g.
5. Discard the effluent in the lower reservoir.
6. Add 2 mL of TE Buffer to the concentrated DNA in upper reservoir
7. Centrifuge at room temperature for 20 minutes at 2000 x g.
8. Repeat steps 5-7 for a total of three washes.
9. Collect the concentrated DNA (~15-50 µL) by inverting the upper
reservoir into the retention cup and centrifuge for 5 minutes 500 x g.
10. Transfer entire sample to new tube and record volume.
11. Freeze sample until PCR is performed.
3. Quantitation
Prepare DNA Quantification Standards
1. Add 30 µL of TE Buffer to 1 tube, and 20 µL of TE Buffer to 7 tubes.
2. Prepare Standard 1: vortex and spin Quantifiler Human DNA Standard
and add 10 µL to tube 1, containing 30 µL TE Buffer.
3. Prepare Standards 2-8 by performing a serial dilution (add 10 µL of
Std 1 to 20 µL of TE Buffer for Std 2, mix, add 10 µL of Std 2 to 20
µL of Std 3, mix, etc. (Standard 1 generates 50.0 ng/μL of DNA.
Standards 2-8 contain the following range of concentrations: 16.7
ng/μL, 5.560 ng/μl, 1.850 ng/μl, 0.620 ng/μl, 0.210 ng/μl, 0.068 ng/μl,
and 0.023 ng/μl).
Prepare PCR Master Mix (Primer Mix + PCR Reaction Mix)
1. Thaw the Primer Mix, then vortex and spin
2. Place 10.5 of Quantifiler Human Primer Mix per reaction in a tube
(10.5µL x X= Y µL)
3. Swirl the Quantifiler PCR Reaction Mix gently
91
4. Add 12.5 µL of Quantifiler PCR Reaction Mix per reaction (12.5µL x
X = Y µL), vortex and spin briefly.
Prepare Reaction Tubes
1. Dispense 23 µL of the Master Mix into each reaction well on a 96-well
plate
2. Add 2 µL of samples, standards and controls in the appropriate wells
for plate setup, run all reactions in duplicate in adjacent wells
3. Tap to remove bubbles
4. Seal the reaction plate with an Optical Adhesive Cover
5. Centrifuge plate at 500 x g for approximately 20 seconds
6. Place the Compression Pad over the plate with the brown side up an
aligned with plate wells
7. Turn the power on the 7000 and open the program on the computer
8. Position the plate so that the well A1 is in the upper left position, and
the notched corner of the plate is in the upper right position.
9. Open the door on the instrument, place plate inside the holder and
close the door
10. Open the 7000 SDS Software
11. Open “New”
12. Change the template to the Quantifiler Human
13. Import sample setup
14. “Save As” a template document
15. Click the Instrument Tab
16. Click Start to begin analysis
17. Once qPCR is complete look at the computer generated data,
quantities are expressed in µg/µL. When samples are run in duplicate,
the two values should be averaged.
Thermal cycler parameters:
Pre-heat
40 cycles:
95º, 10 minutes
95º, 15 seconds
60º, 1 minute
4. Amplification (ABI GeneAmp® PCR System 9700 Thermal Cycler)
AmpFℓSTR®Identifiler™
Hold:



95ºC for 11 minutes (for one cycle), then:
denature 94ºC for 1 minute
anneal at 59ºC for 1 minute
92



extend at 72ºC for 1 minute (28 cycles)
final extension at 60ºC for 60 minutes (1 cycle)
soak at 25ºC for ∞
PCR control contains 7 µL of control DNA and 3 µL of TE Buffer.
Negative control contains 10 µL of TE Buffer.
VOLUME PER
REACTION
AmpFℓSTR® Reaction Mix
10.5 µL
AmpliTaq Gold DNA Polymerase
0.5 µL
AmpFℓSTR®Identifiler™ Primer Set
5.5 µL
Total Reaction Volume (minus sample)*
15 µL
Reaction volume (plus template DNA)
25 µL
* Master Mix contains enough sample to compensate for loss during pipetting.
PCR MASTER MIX COMPONENTS
AmpFℓSTR®Minifiler™






initial denature at 95ºC for 11 minutes (for one cycle)
denature at 94ºC for 20 seconds
anneal at 59ºC for 2 minutes
extend at 72ºC for 1 minute (30 cycles)
final extension, 60ºC for 45 minutes
soak at 4ºC for ∞
Positive control contains 5 µL of positive PCR control and 5 µL of TE
Buffer.
Negative control contains 10 µL of TE Buffer.
PCR MASTER MIX COMPONENTS
AmpFℓSTR® Reaction Mix
AmpFℓSTR®Minifiler™ Primer Set
Total Reaction Volume (minus sample)*
Reaction volume (plus template DNA)
* Master Mix contains enough sample to compensate
VOLUME PER
REACTION
10.5 µL
5.5 µL
15 µL
25 µL
93
Appendix B
Permission to use figures from Applied Biosystems and Elvesier.
From: "Benfield, Jacquelyn" <Jacquelyn.Benfield@lifetech.com>
To: Lisa Branch <ldbranch6@yahoo.com>
Sent: Tuesday, June 23, 2009 4:10:40 PM
Subject: RE: permission to use AB figures
Hi Lisa,
Our legal group took a look at the figures and said you just need to reference applied Biosystems
and if possible the source…user manual, microsite, etc.
Let me know if you have any other questions. And good luck.
Jacki
This is a License Agreement between Lisa Branch ("You") and Elsevier ("Elsevier"). The
license consists of your order details, the terms and conditions provided by Elsevier, and
the payment terms and conditions.
License number
Reference confirmation email for license number License date: Jun 23, 2009
Licensed content publisher: Elsevier
Licensed content publication: Genomics, Proteomics & Bioinformatics
Licensed content title: A Brief Review of Short Tandem Repeat Mutation
Licensed content author: Hao Fan and Jia-You Chu
Licensed content date: 2007
Volume number 5, Issue number1, Pages 8
Type of Use : Thesis / Dissertation
Portion: Figures/table/illustration/abstracts. Portion Quantity 1
Format : Both print and electronic
94
Elsevier VAT number: GB 494 6272 12
Billing type: Invoice Billing address
281 Candela Circle
Sacramento, CA 95835
United States
95
LITERATURE CITED
American National Standards Institute. International Standards
Organization/International Electrotechnical Commision. ISO/IEC Guide 25:1990, New
York: American National Standards Institute, 1990.
American Society of Crime Laboratory Directors/Laboratory Accrediation Board.
ASCLD/LAB Accreditationi Manuel. Garner, North Carolina: ASCLD/Lab, 2003.
Anslinger, K., B. Bayer, B. Rold, W. Keil, and W. Eisenmenger. "Application of the
BioRobot EZ1 in a forensic laboratory." Legal Medicine 7 (2005): 164-168.
Bayer, Birgit, Burkhard Rolf, Wolfgang Keil, Katja Anslinger, and Wolfgang
Eisenmenger. "Application of the BioRobot EZ1 in a forensic laboratory." Legal
Medicine 7 (2005): 164-168.
Biosystems, Applied. Minifiler Product Bulletin. 2008.
http://www3.appliedbiosystems.com/cms/groups/applied_markets_marketing/documents/
generaldocuments/cms_043658.pdf (accessed October 2008, 2008).
Board, DNA Advisory. "Federal Bureau of Investigations." 2007.
http://www.fbi.gov/hq/lab/fsc/backissu/july2000/codis2b.htm#Validation (accessed
2007).
Bode Technology. Touch DNA-Overview. 2009.
http://www.bodetech.com/technologies/touch-dna/touch-dna-overview (accessed April
27, 2009).
Brouwer, Derk H, Mark F Boeniger, and Joop Van Hemmen. "Hand Wash and Manual
Skin Wipes." American Occupational Hygene, 2000: 501-510.
Bureau of Justice Statistics. December 20, 2007.
http://www.ojp.gov/bjs/homicide/weapons.htm (accessed November 15, 2007).
Butler, J. M., Y. Shen, and B. R. McCord. "The development of reduced size STR
amplicons as tools for analsis of degraded DNA." Journal of Forensic Science 48 (2003):
1054-1064.
Butler, J. Short tandem repeat DNA internet database. October 02, 1997.
http://www.cstl.nist.gov/biotech/strbase/ (accessed July 1, 2007).
96
Butler, John M. Forensic DNA Typing: Biology, Technology, and Genetics of STR
markers. 2002.
—. Short Tandem Repeat DNA Internet Database. 10 02, 1997.
http://cstl.nist.gov/div831/strbase/index.htm (accessed April 4, 2008).
Chung, D. T., J. Drabek, K. L. Opel, J. M. Butler, and B. R. McCord. "A study on the
effects of degratation and template concentration on the amplification efficiency of the
STR miniplex primer sets." Journal of Forensic Sciences 51, no. 2 (2004): 733-740.
DNA Advisory Board, Federal Bureau of Investigations. "Forensic Science
Communications." Federal Bureau of Inviestigation Publication. July 2004.
http://www.fbi.gov/hq/lab/fsc/backissu/july2004/standards/2004_03_standards02.htm
(accessed September 1, 2007).
Esslinger, Kelly, Jay A. Siegel, Heather Spillane, and Shawn Stallworth. "Using STR
analysis to detect human DNA from exploded pipe bomb devices." Journal of Forensic
Sciences 49, no. 3 (2004): 481-484.
Fan, H., and J. Chu. "A brief review of short tandem repeat mutation." Genomic,
Proteomics and Bioinformatics 5, no. 1 (2007): 7-14.
Federal Bureau of Investigation DNA Advisory Board. "Quality assurance standards for
convicted offender DNA databasing laboratories." Forensic Science Communications.
1999. www.fbi.gov/hg/lab/fsc/backissu/july2000/codispre.htm.
Federal Bureau of Investigation DNA Advisory Board. "Quality assurance standards for
foresnic DNA testing laboratories." Forensic Science Communications. 1998.
www.fbi.gov/hg/lab/fsc/backissu/july2000/codispre.htm.
Federal Bureau of Investigation, Department of Justice. 2006 Crime in the United States.
September 2006.
http://www2.fbi.gov/ucr/cius2006/offenses/expanded_information/data/shrtable_10.html
(accessed November 11, 2007).
Findlay, I., A. Taylor, and P. Quirke. "DNA fingerprinting from single cells." Nature 389
(1997): 555-556.
Fisher, Barry A.J. Techniques of Crime Scene Investigation. 6th Edition. Florida: CRC
Press LLC, 2000.
97
Given, B. W. "Latent fingerprints on cartridges and expended cartridge casings." Journal
of Forensic Sciences 21, no. 3 (1976): 587-594.
Grubwieser, P., R. Muhlmann, B. Berger, H. Niederstatter, M. Pavlic, and W. Parson. "A
new "miniSTR-multiplex" displaying reduced amplicon lengths for the analsis of
degraded DNA." International Journal of Legal Medicine 120 (2006): 115-120.
Hill, C. R., et al. "Concordance study between the AmpFlSTR Minifiler PCR
amplification kit and conventional STR typing kits." Journal of Forensic Sciences 52
(2007): 870-873.
Jeffreys, Alec J., Victoria Wilson, and Swee L. Thein. "Hypervariable "minisatellite"
regions in human DNA." Nature 314, no. 6006 (1985): 67-73.
Justice, Department of. DNA Initiative: Advancing Criminal Justice Through DNA
technology. March 11, 2003. www.dna.gov (accessed November 20, 2007).
Kishore, R, R. Hardy, V. J. Anderson, N. A. Sanchez, and M. Buibcristiani.
"Optimization of DNA extraction from low-yield and degraded samples using the
BioRobot EZ1 and M48." Journal of Forensic Sciences 51, no. 5 (2006): 1055-61.
Levinson, Gene, and George A. Gutman. "Slipped Strand Mechanism: A major
mechanism for DNA sequences." Molecular Biology and Evolution 4, no. 3 (1997): 203221.
Lowe, Alex, Caroline Murray, Jonathan Whitaker, Gillian Tully, and Peter Gill. "The
propensity of individuals to deposit DNA and secondary transfer of low level DNA from
indiviiduals to inert surface." Forensic Science International, 2002: 25-34.
Migron, Yoelit, Gil Hocherman, Eliot Springer, Joseph Almog, and Daniel Mandler.
"Visualization of sebaceous fingerprints on fired cartridge casings: A laboratory study."
Journal of Forenisc Sciences 43, no. 3 (1998): 543-548.
Miller, C. R., P. Joyce, and L. P. Waits. "Assessing allelic dropout and genotype
reliabilty using maximum likelihoot." Genetics 160 (2002): 357-366.
Montpetit, S., I. T. Fitch, and P. T. O'Donnell. "Simple automated instrument for DNA
extraction in forensic casework." Journal of Forensic Sciences, 2005: 555-563.
Opel, K L, D. T. Chung, J. Drabek, N. E. Tatarek, L. M. Jantz, and B. R. McCord. "The
application of miniple primer sets in the analysis of degraded DNA from human skeletal
remains." Journal of Forensic Sciences 51 (2006): 351-356.
98
Petruska, J., M. J. Hartenstine, and M. F. Goodman. "Analysis of strand slippage in DNA
Polymerase expansions of CAG/CTG triplet repeat associate with neurodegenerative
disease." The Journal of Biochemistry 273, no. 9 (1998): 5204-5210.
Phipps, Matthew, and Susan Petrivevic. "The tendency of individuals to transfer DNA to
handled items." Forensic Science International, 2006.
Polley, D., et al. "An investigation of DNA recovery from firearms and cartridge cases."
Canadian Soceity of Forensic Sciences 39, no. 4 (2006): 217-228.
Qiagen. "EZ1 DNA Investigator Handbook." 2006.
Sacramento County District Attorney, Laboratory of Forensic Services.
"Ch3Procedures." DNA Procedures Manual. Sacramento, Ca, May 04, 2007.
Schehl, S.A. "U.S. Department of Justice, Federal Bureau of Investigation." Forensic
Science Communicaions. April 2000.
http://www.fbi.gov/hq/lab/fsc/backissu/april2000/schehl1.htm#Identifying (accessed
December 1, 2007).
Schiffer, L. A., E. J. Bajda, M. Prinz, J. Sebestyen, R. Shaler, and T. A. Caragine.
"Optimization of a simpe automatable extraction method to recover suffiecient DNA
from low copy number DNA samples for generation of short tandem repeat profiles."
Croatian Medical Journal 46 (2005): 578-586.
Schneider, P. M. "Basic issues in forensic DNA typing." Forensic Science International,
1997: 17-22.
Schulz, M. M., and W. Reichert. "Archieved or directly swabbed laten fingerprints as a
DNA source for STR typing." Forensic Science International 127 (2002): 128-13-.
Siegal, J. A., P. J. Saukko, and G. C. Knupfer, . Encyclopedia of Forensic Sciences. Vol.
2. London: Academic Press, 2000.
Sweet, D., M. Lorente, J. A. Lorente, A. Valenzuela, and E. Villanueva. "An improved
method to recover saliva from human skin: the double swab tecnique." Journal of
Forensic Science 42, no. 2 (1997): 320-322.
Technical Working Group on DNA Analysis Methods. "Guidelines for quality assuarance
program for DNA analysis." Crime Labortory Digest, 1995: 21-43.
99
User Bulletin: AmpFlSTR Identifiler PCR amplification kit.
Van Hoofstat, D., et al. "DNA typing of fingerprints and skin debris: sensitivity of
capillary electrophoresis in forensics applications using multiplex PCR." Proceedings of
the 2nd European Symposium of Human Idenfication. Innsbruck, Austria: Promega
Corporation, 1998. 131-137.
van Oorschsot, R. A., and M. K. Jones. "DNA fingerprints from fingerprints." Nature 387
(1997): 767.
Wickenheiser, R. A. "A review, discussion of theory, and application of the transfer of
trace quantites of DNA through skin contact." Journal of Forensic Science 47, no. 3
(2002): 442-450.
Wickenheiser, R. A., and C. M. Challoner. "Suspect DNA profiles obtained from the
handles of weapons recovered at crime scenes." 10th Annual Symposium. Promega. 1-10.
Wiegand, P., and M. Kleiber. "DNA typing o epithelial cells after strangulation."
International Journal of Legal Medicine 110 (1997): 181-183.
Wiegand, P., and M. Kleiber. "Less is more - length reduction of STR amplicons using
redesigned primers." International Journal of Legal Medicine 114 (2004): 285-287.
42 Code of Federal Regulations. "Chapter IV (10-1-05 Edition)." Health Care Financing
Administration, Health and Human Services