ANALYSIS OF DNA OBTAINED FROM WIRELESS ELECTRONIC
TRIGGERING MECHANISMS USED WITH IMPROVISED EXPLOSIVE
DEVICES

By
Scott Russell Grammer

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTERS OF SCIENCE
School of Criminal Justice
2012

ABSTRACT
ANALYSIS OF DNA OBTAINED FROM WIRELESS ELECTRONIC TRIGGERING
MECHANISMS USED WITH IMPROVISED EXPLOSIVE DEVICES
By
Scott Russell Grammer
In recent years IEDs have been used both domestically and internationally for
unconventional warfare and terrorism. Some of the more advanced IEDs use a wireless
triggering mechanism typically composed of a cell phone, two-way radio, or other small
electronic device that can receive a signal from great distances. In past research the
feasibility of obtaining a genetic profile directly from the explosive of an IED following
handling and deflagration was examined. Due to the poor state of DNA in shed skin cells
along with extreme temperatures of the deflagration, only highly degraded DNA is
generally recovered from the resultant bomb fragments, decreasing the chance of
obtaining a genetic profile of the assembler. Focusing on the trigger mechanism instead
of the explosive may result in increased potential for obtaining a complete genetic profile,
mainly from longer handling during assembly and its distance from the deflagration. In
this study participants were asked to handle components of a mock electronic trigger.
Steel or PVC pipes were filled with smokeless powder and affixed to the trigger, then
detonated by fuse. Pieces of the mechanism were collected and DNA was isolated from
the individual components, quantified, and analyzed using miniSTRs. Allele assignments
were made blind before comparison to references. Results indicate that the success in
identifying an individual who handled the IED by analyzing DNA from the triggering
mechanism is higher than identification using the explosive device fragments.

ACKNOWLEDGEMENTS

There are many individuals that have spent much of their own time helping me
complete this thesis that I would like to thank. First, I would like to give my thanks to
Dr. Foran for all of his guidance and knowledge while I attended Michigan State
University. I would also like to thank all of my committee members who have spent
much of their own time reviewing my work and giving me direction.
I am very grateful for the help of Lt. Shawn Stallworth and Sgt. Timothy Ketvirtis
of the Michigan State Police Bomb Squad who coordinated the deflagrations and the
Lansing Fire Department for providing a safe facility. If it wasn‟t for them this research
would have never taken place. I also extend my gratitude to the volunteers who showed
an interest in science and were patient enough to spend some of their time after class to
handle all of the IED components, to Kamila Gomez who helped facilitate this process,
and to my other classmates who have helped me out along the way.
Last, but certainly not least, I would like to thank my family and my loving wife
whose words of encouragement and support were invaluable for the completion of this
project.

iii

TABLE OF CONTENTS
LIST OF TABLES...............................................................................................................v
LIST OF FIGURES............................................................................................................vi
INTRODUCTION...............................................................................................................1
DNA as evidence in IED investigations........................................................................4
Low copy number and degraded DNA..........................................................................6
miniSTR analysis...........................................................................................................7
Quantification and detection of PCR inhibitors.............................................................9
Research objectives......................................................................................................11
MATERIALS AND METHODS.......................................................................................13
Collection and cleaning of mock trigger components.................................................13
Preparation of pipe bombs and containers...................................................................14
Assembly and handling of IEDs..................................................................................15
Deflagration and recovery of IEDs..............................................................................17
DNA isolation..............................................................................................................17
Quantification of DNA and determination of PCR inhibition.....................................20
Amplification of STRs.................................................................................................20
Determination of genetic profiles and assignations.....................................................21
Amplification using 1 µl DNA....................................................................................23
Statistical calculations..................................................................................................24
RESULTS..........................................................................................................................25
Post deflagration and DNA isolation observations......................................................25
DNA quantification......................................................................................................27
Detection of PCR inhibition.........................................................................................30
Analysis of STR electropherograms............................................................................32
Re-amplification of DNAs after clean-up using Microcon YM-100 spin columns.....33
Level of trigger fragmentation and pipe bomb type compared to STR results............34
Comparison of STR results among trigger components..............................................35
STR amplification using 1 µl DNA and the second set of triggers.............................36
DISCUSSION....................................................................................................................39
CONCLUSION..................................................................................................................50
APPENDIX A....................................................................................................................52
APPENDIX B....................................................................................................................57
REFERENCES..................................................................................................................67

iv

LIST OF TABLES

Table 1.

Type of components included in each IED......................................................16

Table 2.

Letter designators assigned to each swab set...................................................18

Table 3.

Degree of fragmentation for each mock IED trigger.......................................26

Table 4.

Quantity of DNA isolated from each trigger...................................................29

Table 5.

DNA obtained compared to trigger damage classification..............................30

Table 6.

STR analysis of inhibited samples...................................................................31

Table 7.

Evaluation of consensus profiles from initial amplifications..........................33

Table 8.

Comparison of STR results when DNAs were amplified following clean-up
with Microcon YM-30 spin columns versus Microcon YM-100....................34

Table 9.

STR results compared to damage classification and pipe bomb type..............35

Table 10. STR analysis compared to individual mock trigger components....................36
Table 11. Evaluation of consensus profiles from initial amplifications and reamplifications of 1 µl DNA combined............................................................38

v

LIST OF FIGURES

Figure 1. Example of an STR............................................................................................8
Figure 2. Loci targeted by Minifiler..................................................................................9
Figure 3. Detection of the TaqMan probe.......................................................................10
Figure 4. Areas of the mock trigger targeted for DNA collection..................................19

vi

INTRODUCTION

Improvised explosive devices (IEDs) have been used extensively during
Operation Iraqi Freedom and Operation Enduring Freedom, killing thousands of people
and wounding even more (Department of Defense personnel and military casualty
statistics 2011). Coalition forces and civilian infrastructure including police stations,
markets, and mosques have been successfully attacked using IEDs, mainly because they
are ideal weapons for an ambush (Wilson 2007). IEDs are easily concealed, use multiple
methods of detonation, and are relatively hard to counter or defend against. Further, the
components that make up an IED are often cheap and easy to obtain, and their
employment tactics and procedures are very flexible. For many of these same reasons,
IEDs pose a serious threat in the United States. In addition, the risk of IED attacks in
America is continuing to increase due to several factors including involvement in the
global war on terrorism, individuals capable of carrying out IED attacks are already
living in the United States, the vulnerability of many targets, and the ease in which bomb
designs and tactics can be spread over the internet, making it simple for almost anyone to
learn how to make and effectively use an IED in an attack (Broun et al. 2009).
Supervisory Special Agent Barbara Martinez, a senior official at the FBI‟s Critical
Incident Response Group, said that "Terrorists' use of IEDs cannot be extrapolated into
anything other than a major threat to this country," (Hsu and Sheridan 2007).
According to the Department of Defense Dictionary of Military and Associated
Terms (2001), an IED is “a device placed or fabricated in an improvised manner
incorporating destructive, lethal, noxious, pyrotechnic, or incendiary chemicals and

1

designed to destroy, incapacitate, harass, or distract. It may incorporate military stores,
but is normally devised from nonmilitary components.” The basic components of an IED
are an initiation system, explosive fill, detonator, possibly a power supply for the
detonator, and a container or other means of concealment (Globalsecurity.org 2005).
There are almost limitless means by which to deploy an IED, with three main methods of
detonation: 1) time delay, which is provided by mechanical or electronic timers, fuses, or
chemicals, 2) action, which is a mechanical system that can be initiated by pushing,
pulling, or applying pressure, and 3) command, by either an electrical or wireless signal
(Bolz et al. 2005). The advantage of command detonation is that the attack can take
place at an opportune moment when the amount of damage can be maximized; however,
there must be line-of-site with the target in order to coordinate the attack, which may put
the triggerman in a vulnerable position. Modern electronics such as long range cordless
phones, key fobs, cell phones, and two way radios have helped to alleviate this drawback
by allowing a greater stand-off distance from the IED, which is why these types of
triggers are being used more often (Bolz et al. 2005).
With the great diversity in IED types and tactics comes an increasingly difficult
task of defeating attacks. Countermeasures currently in place to help mitigate the threat
of an IED attack in Iraq and Afghanistan are electronic jammers, X-ray equipment, radar
systems, robotic explosive ordnance disposal equipment, and armor for vehicles and
personnel (Wilson 2006). These methods have been engineered to protect personnel
overseas and can be modified for domestic use as well; however, even with cutting edge
research and development in IED defense, there is no way security forces can eliminate
attacks completely. Further action needs to be taken to ensure the public‟s safety from

2

the IED threat. These actions are outlined in the Homeland Security Presidential
Directive/HSPD-19 issued on February 12, 2007 which states:
It is the policy of the United States to counter the threat of explosive attacks
aggressively by coordinating Federal, State, local, territorial, and tribal
government efforts and collaborating with the owners and operators of critical
infrastructure and key resources to deter, prevent, detect, protect against, and
respond to explosive attacks, including the following:
(a) applying techniques of psychological and behavioral sciences in the
analysis of potential threats of explosive attack;
(b) using the most effective technologies, capabilities, and explosives search
procedures, and applications thereof, to detect, locate, and render safe
explosives before they detonate or function as part of an explosive attack,
including detection of explosive materials and precursor chemicals used to
make improvised explosive or incendiary mixtures;
(c) applying all appropriate resources to pre-blast or pre-functioning search
and explosives render-safe procedures, and to post-blast or post-functioning
investigatory and search activities, in order to detect secondary and tertiary
explosives and for the purposes of attribution;
(d) employing effective capabilities, technologies, and methodologies,
including blast mitigation techniques, to mitigate or neutralize the physical
effects of an explosive attack on human life, critical infrastructure, and key
resources; and
(e) clarifying specific roles and responsibilities of agencies and heads of
agencies through all phases of incident management from prevention and
protection through response and recovery.

As stated in the directive, it is important to continue developing methods of determining
who is responsible for an attack. Current procedures of analyzing IED evidence include
class characterization of the IED components such as size, weight, materials, methods of
assembly, etc., trace analysis of hairs, fibers, or any other microscopic evidence, and
chemical tests of the explosive charge. Fingerprints are generally the only

3

individualizing evidence collected; however, they may be unrecoverable after
deflagration (Shachtman 2007).

DNA as evidence in IED investigations
Van Oorshot and Jones (1997) showed that accurate genetic profiles can be
obtained from shed epithelial cells left behind on objects that have been handled, which is
referred to as “touch DNA”. The likelihood of getting a complete profile depends on the
quantity and quality of DNA collected. Some factors that seem to affect DNA deposition
are the amount of handling, time since last hand wash, and the substrate handled
(Kisilevsky and Wickenheiser 1999; Phipps and Petricevic 2007). The techniques used
to obtain touch DNA are also important. The double swab technique (Sweet et al. 1997)
may offer advantages over traditional swabbing methods where only one swab is used
(Pang and Cheung 2007). Cotton swabs are also recommended over synthetic because
they are hydrophilic and have an irregular surface which can increase the tendency of
cells to adhere to their surface (Jobin and DeGouffe 1999 as cited by Wickenheiser
2002).
Currently the use of DNA in IED investigations is limited. For regular use, and to
establish much needed standards of operation, research must be conducted on the
collection and analysis of DNA from deflagrated IEDs. Research involving the analysis
of DNA obtained from exploded IEDs began with a study by Esslinger et al. (2004)
wherein conventional short tandem repeat (STR) analysis was used for DNA isolated
from steel or PVC pipe bombs following deflagration. The success of determining the
handler is this study was minimal, where a profile with both alleles present at all 10 loci

4

using AmpFlSTR Profiler Plus (Applied Biosystems, Foster City, CA) was obtained for
only 1 of 20 bombs, likely due to the limited amount and condition of DNA. Work was
continued by Foran et al. (2009) who analyzed the hypervariable regions of
mitochondrial DNA (mtDNA) collected from deflagrated pipe bombs. Eighteen of 38
bombs were correctly assigned to their respective handler. However, mtDNA analysis is
not individualizing. Further work by Kremer (2008) involved the analysis of DNA
obtained from deflagrated pipe bombs using two sets of miniSTRs, which offer
advantages over conventional STRs when dealing with low amounts of degraded DNA
(see below), together with the analysis of mtDNA. Combining the two techniques, the
handler of 8 bombs was determined out of a set of 34, while 9 were assigned to a subset
of handlers.
Since there are limitations in analyzing DNA from the explosive charge, focusing
on other components of IEDs may be the key to increasing success in determining the
handler‟s profile. In a study by Hoffmann et al. (2011), backpacks, a common container
for IEDs, were given to individuals who used them for 11 days. A steel or PVC pipe
bomb was then placed inside each backpack. After deflagration of the IED, the remnants
of the backpack were analyzed using an AmpFlSTR Minifiler PCR Amplification Kit.
Genetic profiles from different areas of the backpacks were combined to create a single
consensus profile, which was then compared to reference samples to assess profile
accuracy. As a result, a full profile of the handler was obtained from every backpack
except at a single locus, providing a feasible method to analyze deflagrated IEDs.
However, due to variability in IED design, where a container may not be used, and since
the handler of the backpack may not be the same individual who was responsible for the

5

IED‟s construction, other methods for collecting/analyzing DNA from deflagrated IEDs
must be explored.

Low copy number and degraded DNA
In traditional STR analysis 1 ng of nuclear DNA is recommended (Applied
Biosystems 2001); however, it is unlikely that that amount will be obtained from touch
DNA where only a limited number of shed epithelial cells remain. Using current
polymerase chain reaction (PCR) techniques a genetic profile can sometimes be obtained
from 100 pg or less of DNA, which is referred to as low copy number (LCN) (Gill et al.
2000). In a single human cell there is around 5 pg of DNA, therefore as few as 20 cells
worth is used in LCN analysis. There are, however, several difficulties in analyzing LCN
DNA such as stutter, unbalanced peak heights, drop-out (missing alleles) due to
stochastic sampling effects, drop-in, and higher susceptibility to contamination (Balding
and Buckleton 2009). Approaches to help alleviate these problems include reducing the
total PCR volume, using filtration of the PCR product to remove ions that compete with
the DNA during injection into the capillary, using low conductivity formamide with
increased PCR product added, and increasing injection time (Budowle et al. 2001).
Increasing the PCR cycle number has also been mentioned (van Hoofstat et al. 1998; Gill
2001) but was cautioned against by Budowle et al. (2001) because of the risk of over
amplifying exogenous DNA. Gill et al. (2000) recommended analyzing LCN DNA by
amplifying in replicates and only calling alleles that are present more than once.
The quality of the DNA from deflagrated IEDs can also be problematic. The
DNA obtained from epithelial cells found on the surface of the IED has likely been

6

subjected to environmental stresses such as direct sunlight, moisture, and bacterial and
fungal contamination, which can cause DNA degradation (Pang and Cheung 2007). In
addition, the DNA is exposed to intense heat and pressure from deflagration of the IED.
In a study by Threadgold and Brown (2003) DNA from wheat seeds degraded at
temperatures above 200ºC. The temperature DNA is subjected to during deflagration is
not known but is undoubtedly far greater than this.

miniSTR analysis
STRs are made up of short stretches of DNA (between 2 and 6 base pairs (bp) in
length) that are repeated in tandem and exhibit variation in length (based on the number
of repeats) among individuals (Figure 1). Many of these regions found throughout the
human genome can be utilized for analysis of DNA obtained during criminal
investigations. The STR analysis starts with a PCR that is used to make many copies of
the DNA. Fluorescently labeled primers are used in the PCR to target STR loci where
amplification of the DNA occurs. The amplified DNA is then separated by size using
capillary electrophoresis and the fragments are detected based on their fluorescent
primers. By using a size standard, which is composed of many DNA fragments of known
size, and an allelic ladder, which contains all of the common alleles, it is possible to
determine the number of repeat units at each STR locus of the DNA obtained from
evidence or reference samples, thereby constructing a genetic profile for the contributing
individual.

7

Figure 1. Example of an STR

GATA

GATA

GATA

GATA

GATA

GATA

An STR is made up of a short stretch of DNA that is repeated. Shown is an STR
(GATA) that has six repeats.

Conventional STR kits such as AmpFlSTR Identifiler PCR Amplification Kit by
Applied Biosystems or PowerPlex 16 System by Promega work well when analyzing
higher quality DNA; however, when the DNA is degraded, some of the larger loci may
drop-out resulting in a partial profile. By moving the primers to a location that closely
flanks the repeat region, resulting in an overall decrease in amplicon size (known as
miniSTRs), low quantities of highly degraded DNA can be more successfully typed
(Butler et al. 2003). In 2007 Applied Biosystems released the AmpFlSTR Minifiler PCR
Amplification Kit (Minifiler) which, in one PCR reaction, amplifies eight of the larger
loci included in the Identifiler kit (D13S317, D7S820, D2S1338, D21S11, D16S539,
D18S51, CSF1PO and FGA) along with the sex determining marker amelogenin
(Applied Biosystems 2007). The kit was designed for genotyping degraded DNA that
may or may not contain PCR inhibitors, with amplicons ranging from 70 to 283 bp in
length (Figure 2). In a validation study by Mulero et al. (2008), results using Minifiler
showed performance improvements over Identifiler in the analysis of degraded DNA and
samples containing PCR inhibitors. Given these advantages, Minifiler may be better
suited for amplifying DNA recovered from deflagrated IEDs, which is likely to be highly
degraded.

8

Figure 2. Loci targeted by Minifiler

The size ranges of all 10 loci amplified by the Minifiler multiplex are shown with their
corresponding dye color. (Product description, AmpFlSTR Minifiler PCR Amplification
Kit, Applied Biosystems)

Quantification and detection of PCR inhibitors
As a quality control requirement for casework samples, any PCR-based assay
must be preceded by quantification of nuclear DNA (nDNA) (DNA Advisory Board
2000). In 2006 Applied Biosystems released the Quantifiler Human DNA Quantification
Kit (Quantifiler) which is a real time PCR quantification assay that is capable of detecting
well below 100 pg of DNA (Green et al. 2005). The primers target the human telomerase
reverse transcriptase locus located on chromosome 5, resulting in a 62 bp amplicon
(Applied Biosystems 2006). In order to detect amplification in real time, a TaqMan
probe is used (Figure 3). It is sequence specific and incorporates a fluorescent dye and a
quencher that suppresses detection of the dye when the probe is intact. During the

9

annealing step of PCR the probe binds to a specific sequence within the amplicon. Taq
polymerase then degrades the probe during extension which causes a release of the
quencher from the dye. Once the dye has been separated from the quencher it can be
detected. With each additional cycle of PCR there is an exponential buildup of detectable
dye. Eight DNA standards of known concentrations are analyzed in the same manner to
generate a standard curve to which the dye intensity of the unknown sample is compared
to determine its DNA concentration.

Figure 3. Detection of the TaqMan probe
Denaturation

TaqMan Probe

Primer
Template DNA

Annealing

Extension
Polymerase

Shown are the three steps of real time PCR. The encircled “R” and “Q” represent the
reporter and quencher of the TaqMan probe. During the extension phase, polymerase
degrades the probe which releases the reporter from its quencher, resulting in
fluorescence/detection.

10

A beneficial aspect of Quantifiler is the ability to detect PCR inhibition using its
internal PCR control (IPC). The IPC consists of synthetic DNA with primers and a
TaqMan probe that are specific to it. Since the concentration of synthetic DNA is
constant, it should amplify to the same degree in all samples. Inconsistencies in the
amplification rate of an unknown sample‟s IPC as compared to the standard‟s IPC
indicate the presence of PCR inhibitors within the unknown. Though undocumented, it is
likely that there are many potential inhibitors of PCR associated with residue from the
deflagrated bombs or other components or chemicals that make up the IED, which could
co-extract with the DNA. Methods used to overcome inhibition include chemical
additives such as bovine serum albumin (BSA) (Comey et al. 1994) or betaine (Al-Soud
and Rådström 2000), decreasing sample DNA volume to dilute inhibitors, increasing the
amount of Taq polymerase (Bessetti 2007), using different types of polymerase (Eilert
and Foran 2009), or separating the DNA from the inhibitors using filtration (Comey et al.
1994).

Research objectives
The focus of this study was to determine if the success in identifying the handler
of an IED after deflagration can be increased by analyzing other components of an IED,
such as electronic triggering mechanisms. Focusing on the triggering device may result
in a greater potential of obtaining a complete genetic profile, for a variety of reasons.
First, an electronic triggering device incorporates multiple components that require
assembly and thus longer handling, potentially resulting in a greater accumulation of
touch DNA. Second, DNA on the triggering mechanism may not experience the same

11

heat as the explosive charge, and therefore is less degraded. Third, the triggering
mechanism may only separate into its individual components (wireless device, battery,
circuit board, etc.), instead of fragmenting into many small pieces as does an explosive
charge casing, making their recovery much easier. Fourth, depending on how the
triggering mechanism is attached to the detonator of the IED, there can be substantial
separation or obstacles between the two, resulting in decreased damage during the blast.
Participants in this study were asked to mock assemble an electronic IED
triggering mechanism. Pipes were filled with smokeless powder and affixed to the mock
triggers, then deflagrated by fuse in a controlled environment, after which all pieces of
the mechanism were collected and swabbed. Following extraction, the DNA was
quantified and analyzed using miniSTRs. Allele assignments were made blind before
comparison to reference samples from the volunteers to determine the frequency of which
the handler‟s profile could be accurately obtained.

12

MATERIALS AND METHODS

Collection and cleaning of mock trigger components
Equipment for 18 mock triggering mechanisms included ten used cell phones
(various models), eight two way radios (approximately 3 x 5 inches), circuit boards from
laptop computers (approximately 3 x 4 inches), AA batteries, wire (single, coated,
approximately eight inches in length), clamps (PO-35 or HI-988, Bulldog Hardware,
Memphis, TN), Velcro, screws (#6, 1/2 inch, Alma Bolt Company, Alma, MI), and 3/8
inch plywood (cut to approximately 11 1/2 x 6 1/4 inches). All components were soaked
for one hour in a 10% bleach solution, scrubbed thoroughly on all surfaces, and rinsed
with de-ionized water (except the battery, which was only scrubbed, and the Velcro,
which was unhandled out of the packaging). They were then placed in a Spectrolinker
XL-1500 UV Crosslinker (Spectronics Corporation, Westbury, NY) for 15 minutes each
side (approximately 7.5 J/cm2).
To ensure the donated phones were free of DNA after being cleaned, three were
separately swabbed front and back. A double swab technique (Sweet et al. 1997) was
used, where one swab (25-806 2PC, Puritan Medical Products Co. LLC, Guilford, ME),
after being UV irradiated for five minutes, was moistened with 150 μl of digestion buffer
(10 mM Tris, 20 mM EDTA, 0.1% SDS, pH 7.5) then passed over the targeted surface,
immediately followed by a dry swab. The swab sets from the three phones (along with a
clean swab set that served as a substrate blank) were placed separately into 1.5 ml
microcentrifuge tubes with 350 µl of digestion buffer and 6 µl of proteinase K (20
mg/ml) and incubated overnight at 55oC. The swabs were then removed and placed into

13

spin baskets with 2 ml collection tubes and centrifuged for 1 minute at 13,000 rpm and
discarded. The extracted liquid was pipetted back into the original tubes. Five hundred
microliters of phenol were added followed by vortexing and centrifugation at 13,000 rpm
for 6 minutes. The aqueous layers were pipetted into clean (autoclaved and UV
irradiated) 1.5 ml microcentrifuge tubes followed by the addition of 500 µl of
chloroform. The tubes were vortexed and centrifuged at 13,000 rpm for 6 minutes. The
aqueous layers were pipetted into Microcon YM-30 spin columns (Millipore Corporation,
Billerica, MA), and 100 μl of TE (10 mM Tris, 1 mM EDTA, pH 7.5) was added. The
columns were centrifuged at 14,000 x g for 12 minutes. The flowthrough was discarded
and 200 µl TE was added to the samples, followed by centrifugation at 14,000 x g for 8
minutes. Twenty microliters of TE was added and left for 5 minutes. The columns were
then inverted into clean tubes and centrifuged at 1,000 x g for 3 minutes. The extracts
were amplified using primers specific to human amelogenin (Kiley 2009), 0.1 µl
AmpliTaq Gold DNA polymerase with 1 µl buffer (Applied Biosystems), 200 µM
dNTPs, 2.5 mM MgCl2, and 4 µl water. Parameters for thermal cycling included a 94ºC
hold for 12 minutes, 38 cycles of 94ºC for 30 seconds, 58ºC for 30 seconds, and 72ºC for
one minute, and a 72ºC hold for two minutes. Post amplification products were
electrophoresed on a 1.5 % agarose gel.

Preparation of pipe bombs and containers
Nine sections of PVC and galvanized steel pipe (one foot in length, one inch in
diameter), with two end caps for each pipe, were obtained for use as explosive casings.
Nine small cardboard boxes and backpacks (LEED‟S, Pittsburgh, PA) were obtained for

14

IED containers. A 1/4 inch hole was drilled into the center of one of the end caps for
each pipe. The pipes and end caps were cleaned by soaking in a 10% bleach solution for
one hour, rinsed with de-ionized water and UV irradiated for five minutes on each side.
The outside of the pipes and the entire surface of the end caps were wiped with
ELIMINase (Decon Laboratories Inc., Bryn Mawr, PA) and rinsed with de-ionized water.
The PVC end caps lacking a drilled hole were glued to each of the PVC pipes using PVC
cement. The cardboard boxes and backpacks were autoclaved and UV irradiated on all
surfaces for 15 minutes.

Assembly and handling of IEDs
Velcro was used to attach the cell phone or radio, battery, and circuit board to the
plywood base, and screws were used to anchor the clamp that held the pipe bomb in
place. The 18 containers, pipes, and mock triggers were placed separately into brown
paper bags and then assigned a number from 13 – 30 (1 – 12 were used in a previous
study). The cardboard boxes were assigned an even number and the backpacks were
assigned an odd number. The steel pipe assignations ranged from 13 – 21 and the PVC,
22 – 30. Mock triggers that incorporated a cell phone versus a radio were assigned
numbers so that there was an even distribution of each among the different combinations
of cardboard box to backpack and steel to PVC (Table 1).
Eighteen volunteers randomly drew a number ranging from 13 – 30 that was used
by them to identify which container, pipe, and mock trigger they were assigned. They
also drew a letter that was recorded next to their number on a sheet of paper maintained
by the principal investigator. Buccal swabs were obtained from each participant as DNA

15

reference samples and labeled with their respective letter designator. The cardboard
boxes and pipes were handled for roughly 30 seconds to 1 minute. The backpacks were
utilized in place of the participant‟s own for ten days. The mock triggers, which were the
focus of this study, were removed from the brown paper bags, disassembled, handled
briefly, and then re-assembled (resulting in the handling of each component for roughly
20 seconds).

Table 1. Type of components included in each IED
Device
number
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

Explosive case
material
Steel
Steel
Steel
Steel
Steel
Steel
Steel
Steel
Steel
PVC
PVC
PVC
PVC
PVC
PVC
PVC
PVC
PVC

Cell Phone/Radio
Cell Phone
Cell Phone
Radio
Radio
Cell Phone
Cell Phone
Radio
Radio
Cell Phone
Cell Phone
Cell Phone
Radio
Radio
Cell Phone
Cell Phone
Radio
Radio
Cell Phone

Container
type
Box
Backpack
Box
Backpack
Box
Backpack
Box
Backpack
Box
Backpack
Box
Backpack
Box
Backpack
Box
Backpack
Box
Backpack

Each component of the IED was handled by the same individual. The mock trigger,
explosive, and container were all analyzed separately, with the analysis of the mock
trigger being the focus of this study.

16

Deflagration and recovery of IEDs
Deflagrations took place at the Lansing Fire Fighting Training Facility‟s
(Lansing, MI) smoke room. At that location, 1.5 ounces of Green Dot Smokeless
Shotshell Powder (Alliant Powder Co., Radford, VA) were added to the pipes, the second
end cap was set into place, and a 45 second fuse was inserted through the hole drilled in
the end cap. The pipe bomb was affixed to the mock trigger and then placed inside the
container with the fuse showing. The IED was placed inside a metal crate designed to
allow pressure from the blast to escape while containing the larger pieces of debris. After
deflagration the remains of the container, pipe bomb, and mock trigger were collected
separately and placed in brown paper bags for transport to the laboratory. The level of
damage done to each was assessed based on the following 0 – 5 scale:
0. No components were badly damaged or fragmented
1. At least one or two components were moderately damaged, but mostly intact
2. At least two or three components were moderately damaged with some
fragmentation
3. At least three or four components were moderately damaged with some
fragmentation
4. At least three or four components were moderately damaged with high
degrees of fragmentation
5. At least four or five components were highly damaged/fragmented

DNA isolation
The components of the mock-triggering mechanisms were swabbed separately
and labeled with their respective device number and a lettered designator, a – j, specific
to the location the swab was taken (Table 2). Three sets of swabs were used to cover all
surfaces of the front, back, and sides of the cell phones or radios, two sets of swabs were
used for the front and back of the circuit boards and plywood, and one set of swabs was

17

used for the entire surface of the batteries and wire (Figure 4). Since some of the areas
that were being swabbed were large, extra digestion buffer was applied to the first swab
to keep it moist throughout the process. The swabs were placed into a 2 ml tube with 600
µl of digestion buffer (400 µl for reference samples) and 6 µl of proteinase K (5 µl for
reference samples) and incubated overnight at 55ºC. The swabs were then placed into
spin baskets with collection tubes and centrifuged for 1 minute at 13,000 rpm and
discarded. The extracted liquid was pipetted back into the original tubes. The organic
extraction was carried out as above using an equal volume of phenol and chloroform as
extracted liquid (650 µl for unknowns and 400 µl for reference samples).

Table 2. Letter designators assigned to each swab set
Letter designator
a
b
c
d
e
f
g
h
i
j

Trigger component
Phone/Radio front
Phone/Radio back
Phone/Radio sides
Circuit board front
Circuit board back
Battery
Clamp
Wire
Plywood front
Plywood back

Each swab set was labeled with the IED number followed by the respective letter
designator.

Preliminary tests showed the use of Microcon YM-100 spin columns, instead of
YM-30 spin columns, resulted in more complete PCR amplification of the unknown
DNAs so they were used for the remainder of this study (except for reference samples).
18

Five hundred microliters of the aqueous layer from the organic extractions were added to
the spin column and centrifuged for 25 minutes at 500 x g. The flowthrough was
discarded and the remainder of the aqueous layer was added to the spin column followed
by centrifugation for 20 minutes at 500 x g. Two hundred microliters of TE were added
and centrifuged through the column at 500 x g until almost all of the liquid had passed
through (around 12 minutes). Roughly 5 µl of TE remained against the wall of the spin
column so 15 µl of TE was added to bring the total volume up to 20 µl. The column was
left for 3 minutes then inverted into a clean tube and centrifuged for 1 minute at 1,000 x
g. The reference DNA was cleaned as above and eluted at a final volume of 50 µl.

Figure 4. Areas of the mock trigger targeted for DNA collection

Ten swabs were taken; however, the plywood samples were not used in the analysis.
19

Quantification of DNA and determination of PCR inhibition
A Quantifiler Human DNA Quantification Kit was used to quantify DNA and
determine if PCR inhibitors were present. Amplification was performed and detected on
an iCycler thermal cycler with an iQ5 multi-color real-time PCR detection system (BioRad, Hercules, CA). The reaction was carried out in 0.2 ml dome cap tubes (Dot
Scientific, Burton, MI) and was set up according to the manufacturer‟s protocol at a final
volume of 15 µl (7.5 µl reaction mix, 6.3 µl primer mix, and 1.2 µl DNA). The kit‟s 200
ng/µl standard DNA was serially diluted per the manufacturer‟s protocol providing eight
standards ranging from 50 ng/μl to 0.023 ng/μl. Parameters for thermal cycling included
a 95ºC hold for 10 minutes followed by 40 cycles of 95ºC for 15 seconds and 60ºC for
one minute. Total DNA was calculated by multiplying the concentration of DNA (ng/µl,
determined by quantification) by the sample volume (µl). This value was reported as the
DNA quantity. Sample inhibition was determined by comparing the IPC cycle threshold
(Ct) of an unknown sample to the standards. If the Ct of an unknown IPC was more than
one cycle higher than the standards, then that sample was noted as possibly containing
PCR inhibitors.

Amplification of STRs
An AmpFlSTR Minifiler PCR Amplification Kit was used to amplify DNA
isolated from the mock triggers. The total volume in the reaction was reduced to 10 µl,
including 4 µl of the AmpFlSTR Minifiler Master Mix, 2 µl of the AmpFlSTR Minifiler
Primer Set, and 4 µl of sample DNA or 0.5 µl of the control DNA (0.1 ng/µl) plus 3.5 µl
of sterile water. Parameters for thermal cycling included an initial 95ºC hold for 11

20

minutes followed by 33 cycles of 94ºC for 20 seconds, 59ºC for 2 minutes, and 72ºC for
1 minute, ending with a final 60ºC hold for 45 minutes. Select DNAs that yielded only a
partial or no profile were re-amplified using 1 µl if the quantification indicated sufficient
DNA or presence of inhibitors in those samples. Amplifications using 30 and 35 cycles
were also tested during optimization. Results were improved using a higher cycle
number; however, 35 resulted in an increase in artifacts. Re-amplifications of some
inhibited samples were set-up using 250 or 500 ng/µl bovine serum albumin (BSA).
There were no improvements over the first amplifications so the use of BSA was not
continued.
PowerPlex 16 System (Promega) was used to amplify reference DNAs. The total
volume of the reaction was 20 µl and included 0.3 µl AmpliTaq Gold DNA polymerase,
1 µl Gold ST★R 10X buffer, 1 µl PowerPlex 16 10X Primer Pair Mix, 7 µl sterile water,
and 1 µl of a 1:10 dilution of reference DNA. Parameters for thermal cycling included
one cycle of 95ºC for 11 minutes, 10 cycles of 94ºC for 30 seconds, 60ºC for 30 seconds,
and 70ºC for 45 seconds, 22 cycles of 90ºC for 30 seconds, 60ºC for 30 seconds, and
70ºC for 45 seconds, and one cycle of 60ºC for 45 minutes.

Determination of genetic profiles and assignations
Two microliters of unknown amplified DNA (1.5 µl for the amplified control
DNA and Minifiler allelic ladder) were combined in a 0.5 ml tube with 0.5 μl of
GeneScan 500 LIZ Size Standard (Applied Biosystems) and 24.5 µl of deionized
formamide. Following centrifugation, the samples were incubated at 95ºC for 3 minutes

21

and immediately put on ice for 3 minutes. The tube lids were cut off and one drop of
mineral oil was added.
Amplified DNA was electrophoresed on an ABI PRISM 310 Genetic Analyzer
(Applied Biosystems) with GS STR POP4 (1 ml) G5 v2.md5 as the run module (GS STR
POP4 (1 ml) A.md4 for the reference samples), performance optimized polymer 4
(Applied Biosystems), and 1X buffer with EDTA (Applied Biosystems). Electrophoresis
parameters for DNAs amplified with Minifiler included a 15 kV injection for 5 seconds
and a 15 kV run for 28 minutes at 60ºC. To increase peak heights of some samples that
were well below threshold, amplified DNAs were cleaned using Millipore Montage Spin
Columns and injected for 20 seconds and 60 seconds. The STR results were not
improved so the standard injection conditions detailed above were used for all samples.
GeneMapper ID software v3.2.1 (Applied Biosystems) was used to analyze the
data using Minifiler_GS500_HID_v1 as the analysis method, Minifiler_GS500_v1 as the
panel, CE_G5_HID_GS500 as the size standard, and DS-33 Matrix 7-12-07 as the
matrix. A threshold of 50 relative fluorescence units (RFU) was used for data
interpretation. Parameters for reference DNAs amplified with PowerPlex included a 15
kV injection for 3 seconds and a 15 kV run for 30 minutes at 60ºC. The GeneMapper ID
settings used for data analysis were: Analysis PP 16 SH as the analysis method,
PowerPlex_16_ID3.1.0 as the panel, ILS600 Advanced as the size standard, and Matrix –
UMSTest as the matrix.
DNA profiles from the unknown samples were compared to reference profiles and
each locus was categorized on a scale from A – F (Appendix A):
A. Presence of both handler alleles without others
B. Presence of both handler alleles with others
22

C.
D.
E.
F.

One single handler allele present
One handler allele with others present
Amplification but of unexpected alleles
No callable alleles

A consensus genetic profile was produced by comparing allele calls at each locus of the
eight DNA samples obtained from the mock triggers. All interpretations of the unknowns
were done before seeing reference profiles. If two major alleles (or one allele for
homozygotes) were present at least twice, then those were recorded as the consensus for
that locus, otherwise all possible alleles were recorded. The consensus profile was then
compared to the reference profile and results for each locus were placed into one of four
categories:
1.
2.
3.
4.

Both alleles were consistent with the handler with no other calls included
Both handler alleles were present among other possible calls
Only one handler allele was present among other possible calls
Neither handler allele was called

Amplification using 1 µl DNA
Based on the Quantifiler internal PCR controls and the STR results, PCR
inhibition and/or degradation was most likely the cause of incomplete or absence of STR
amplification for many of the DNA samples. Select DNAs whose quantification results
indicated the presence of human DNA but had little or no amplification were re-amplified
using 1 µl of DNA in an attempt to dilute any inhibitors. This also helped determine
whether these samples were affected more by inhibition or degradation. A second
consensus profile was generated (as described above) using data from the reamplifications and the initial amplifications combined.

23

An additional six IEDs (numbered 31 – 36) were prepared and deflagrated as
above (with the exception of a slightly increased handling time). DNA quantities in the
sample extracts were not determined. Amplifications were set up in duplicate with 1 µl
and 4 µl of DNA. Data from these amplifications were interpreted as above.

Statistical calculations
Minitab 16 software (Minitab Inc., State College, PA) was used for statistical
calculations. The normality of a set of data was determined first using a protocol in
Minitab 16 called “Normality”. Since data sets were not normally distributed their P
values were determined using the Mann-Whitney test. An alpha of 0.05 was used.

24

RESULTS

Post deflagration and DNA isolation observations
There was a wide range of damage among triggers, wherein some were minimally
affected by the blast and others were completely destroyed. The damage and
fragmentation was much higher with the triggers that incorporated a steel pipe bomb than
those with a PVC pipe bomb (Table 3). In some instances the steel pipe bombs caused
the lid of the crate to fly open allowing components of the triggers to be strewn about the
containment room. The crate remained secured during deflagration of PVC pipe bombs,
where typically the only debris that escaped were pieces small enough to fit through the
holes in the crate, including many fragments of the pipe itself. PVC bombs caused more
charring, and increased unburned and partially burned smokeless powder. In most cases
there was black residue on the plywood base, and of those, about half had partially burnt
powder imbedded into some of the trigger components that left small yellow/brown
marks. The amount of residue/unburned powder varied among the mock triggers and did
not seem to relate to the amount of overall damage observed.

25

Table 3. Degree of fragmentation for each mock IED trigger
Steel

PVC

Device # Phone/Radio Classification

Device #

Phone/Radio Classification

13

Phone

1

22

Phone

0

14

Phone

3

23

Phone

1

15

Radio

3

24

Radio

3

16

Radio

4

25

Radio

1

17

Phone

3

26

Phone

1

18

Phone

5

27

Phone

0

19

Radio

4

28

Radio

0

20

Radio

4

29

Radio

1

21

Phone

5

30

Phone

1

Each mock trigger was rated as to how much damage was incurred during deflagration.
A mock trigger was rated a 0 when there was virtually no damage, and 5 if almost all
components of the device were damaged. Devices 13 – 21 were deflagrated using a steel
pipe bomb while PVC was used for devices 22 – 30.

There were also trends in the amount of damage done to the individual
components. Generally the phone or radio suffered the greatest amount of visible
damage, followed by the plywood and then the metal clamp. Most of the batteries and
wires appeared relatively unaffected, except in some cases where the battery shell or wire
coating was stripped off. The least damaged component was the circuit board; in only
two cases was it obviously affected by deflagration.
Recovery of the individual trigger components for subsequent DNA isolation
depended on their level of fragmentation. Generally all of the components were
recovered in their entirety. When components were highly fragmented, some of the
26

pieces were usually not recovered, especially in cases where the lid to the containment
crate was forced open; however, at least some fragments from every component were
recovered for each trigger.
Swabbing the components collected visible residue on their outer surfaces. As a
result, most swabs were black in color and some had solid pieces of adhering smokeless
powder. The residue was mostly separated during phenol/chloroform extraction;
however, a very fine black residue was visible on the Microcon filters of some samples.
The swabs of the plywood base were black or yellow/brown in color. The black residue
was removed as above, but a yellow discoloration remained in the aqueous layer after
organic extraction. During Microcon filtration the yellow substance did not filter
through, but remained with the DNA. Initial amplification of these DNAs (from the first
few triggers processed) failed due to high levels of PCR inhibition, and analysis of
plywood samples was halted at that point.

DNA quantification
The total amount of DNA isolated from swab pairs of the trigger components
ranged from 0.0 – 5.22 ng (not including the negative control), with an average of 0.497
± 0.80 ng (Table 4). There were 17 swab pairs that contained greater than 1 ng of DNA,
67 with greater than 0.1 ng but less than 1 ng, and 52 with less than 0.1 ng. The negative
control swab pairs contained no DNA except for the front of the phone and the battery,
which had 0.452 ng and 0.075 ng, respectively. All reagent negative controls contained
no DNA. Total DNA isolated from each mock trigger ranged from 0.302 – 9.081 ng. Of
the 67.62 ng of DNA recovered, 24.485 ng (36.2%) came from the cell phones and

27

radios, 17.146 ng (25.4%) from the circuit boards, 10.456 ng (15.5%) from the clamps,
8.159 ng (12.1%) from the wires, and 7.312 ng (10.8%) from the batteries.
The total amount of DNA isolated from devices that incorporated a steel pipe
bomb was not significantly different than the amount from triggers with a PVC pipe
bomb (p = 0.736) (Table 5). Thirty and three tenths nanograms of DNA were obtained
from 7 triggers (average of 4.3 ± 0.84 ng, median of 3.78) that utilized a steel pipe bomb,
and 37.3 ng DNA (average of 4.7 ± 0.77 ng, median of 3.04) were obtained from 8
triggers where a PVC bomb was used. There did not seem to be any correlation between
the amount of trigger damage and DNA recovered, where the average quantity of DNA
isolated from category 0 was 5.8 ng, 1 was 3.7 ng, 3 was 5.0 ng, 4 was 1.6 ng, and 5 was
3.5 ng (no mock triggers were in category 2).

28

Table 4. Quantity of DNA isolated from each trigger

Device
number

Amount of DNA (ng) recovered from each trigger component
Phone/radio Phone/radio Phone/radio Circuit
Circuit
front
back
sides
board front board back Battery

Clamp

Wire

Total

13**

0.452

0.000

0.000

0.000

0.000

0.075

0.000

0.000

0.527

14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Total

5.22
0.714
0.103
0.000
0.393
0.278*
0.000*
0.122
0.052
0.000
0.188
0.338
0.000*
2.19
0.000*
0.041
0.173
9.81

0.122
0.515
0.575
0.458
0.000*
0.145*
0.000
0.000*
0.798
0.384
0.240
0.446
0.025*
1.74*
0.042
0.030
0.521
6.04

0.117
1.48
0.414
0.417
0.309
0.878*
0.077*
0.000
0.084
0.558
0.192
0.825
0.015
2.45
0.000*
0.079
0.752
8.64

0.522
1.18
0.000*
0.263*
3.63*
0.024*
0.000*
0.000*
0.222
0.570
0.000
0.941
0.000*
1.56
0.162
0.079
1.68
10.8

0.050*
0.000
0.000*
0.534
0.000*
0.000
0.000
0.000*
0.144
0.050
0.216
1.08
0.549
0.000*
0.618
0.000
3.08
6.31

0.000*
0.297
0.197
1.58
0.000*
0.311
0.095
0.516
0.744
0.737
0.027
0.804
0.336
0.518
0.107
0.000
1.11
7.38

1.34
0.836
0.353
0.821
0.630
0.061
0.000
0.006
0.149
0.066
0.126
0.000
0.365
0.504
4.46
0.000
0.750
10.5

0.488
0.794
1.05
0.798
0.000
0.000
0.147
1.44
0.626
0.674
0.395
0.573
0.887
0.125
0.000
0.074
0.093
8.16

7.85
5.81
2.69
4.87
4.96
1.70
0.319
2.08
2.82
3.04
1.38
5.00
2.18
9.08
5.38
0.302
8.16
67.6

The amount of DNA isolated from each swab set ranged from 0.00 ng – 5.22 ng with an average of 0.497 ± 0.80 ng. Total DNA for
each component (not including quantities from the negative control) and trigger are also shown. Numbers in bold indicate samples
where both handler alleles were obtained from at least 2 loci when amplified with Minifiler. * Internal PCR Control indicated the
presence of inhibition ** Device served as a negative control
29

Table 5. DNA obtained compared to trigger damage classification
Steel

PVC

Device #
13*
14
15
17
16
19
20
18
21

Damage
Total DNA
Obtained (ng) Classification
0.527
1
7.85
3
5.81
3
4.87
3
2.69
4
1.70
4
0.319
4
4.96
5
2.08
5

Device #
22
27
28
23
25
26
29
30
24

Damage
Total DNA
Obtained (ng) Classification
2.82
0
9.08
0
5.38
0
3.04
1
5.00
1
2.18
1
0.302
1
8.16
1
1.38
3

The amount of damage done to the mock triggers during deflagration was rated on a scale
of 0 to 5, with 0 being the lowest and 5 being the highest. The average amount of DNA
obtained from triggers in each damage category was 5.76 ± 3.15 ng for category 0, 3.73 ±
3.06 ng for 1, 4.98 ± 2.7 ng for 3, 1.57 ± 1.19 ng for 4, and 3.52 ± 2.04 ng for 5.
* Device served as a negative control

Detection of PCR inhibition
Twenty-six DNAs showed PCR inhibition (Table 6). Twenty of those were from
triggers deflagrated with a steel pipe bomb and seven were from PVC IEDs. Twelve of
the inhibited DNAs were from the phone/radios, 12 from circuit boards, 2 from batteries,
and 0 from the metal clamps or wires. Ten of the inhibited samples quantified as having
some DNA, ranging from 0.024 – 3.63 ng, of which only three produced STR profiles of
five or more loci where both handler alleles were obtained. The remaining 16 inhibited
DNAs quantified at 0, four of which had STR profiles where both handler alleles were
obtained from more than five loci.

30

Table 6. STR analysis of inhibited samples

D13
D7
Amel.
D21
D16
D18
CSF
FGA

14e*
F
F
F
F
C
F
F
F

14f*
F
F
F
F
C
F
F
F

16d*
F
F
F
F
F
F
F
F

16e*
F
F
F
F
C
F
C
F

17d
F
F
F
F
F
F
A
F

18b**
B
A
A
A
C
A
B
F

18d
F
F
F
F
F
F
F
F

18e**
B
F
A
B
C
A
A
A

18f**
F
F
F
F
F
F
F
F

19a
A
F
A
A
F
A
A
C

19b
E
F
C
C
F
B
A
F

19c
F
F
A
F
F
A
A
F

19d
F
F
F
F
F
F
F
F

D13
D7
Amel.
D21
D16
D18
CSF
FGA

20a**
F
F
C
C
E
A
E
F

20c
D
E
C
F
D
D
B
F

20d**
F
F
F
F
F
F
F
F

21b**
F
F
F
F
F
F
F
F

21d**
F
F
F
F
F
F
C
F

21e**
F
F
F
F
F
F
F
F

26a**
F
F
F
F
F
F
F
F

26b**
F
F
F
F
F
F
F
F

26d**
F
F
F
F
F
F
F
F

27b
A
A
B
A
D
B
A
A

27e
A
A
A
F
A
A
A
C

28a**
F
F
F
F
F
F
F
F

28c**
F
F
F
F
F
F
F
F

Twenty-six DNAs had indication of PCR inhibition based on Quantifiler. A scale from A – F was used to represent the number of
handler‟s alleles obtained from STR analysis, where A represents the presence of both handler alleles without others, B, both handler
alleles present with others, C, one handler allele with no others, D, one handler allele with others, E, non-handler alleles only, or F, no
callable alleles. *250 ng/µl BSA used **500 ng/µl BSA used

31

Analysis of STR electropherograms
Of the 1088 loci analyzed from initial amplifications (Appendix A) the number
that fell into each category was: A – 229 (21%), B – 120 (11%), C – 77 (7%), D – 43
(4%), E – 29 (3%), and F – 590 (54%). Thirty-two had no indication of DNA, 9 of which
produced a partial profile containing at least two loci with both handler‟s alleles. A full,
clean profile of the handler was not obtained from any DNA sample; however, there were
14 where both handler alleles were present at all loci (with some loci having other
alleles). Consensus profiles generally provided much more information than did any
profile by itself. There were 68/136 (50%) consensus loci that fell into category 1,
20/136 (15%) in category 2, 3/136 (6%) in category 3, and 40/136 (29%) in category 4
(Table 7). Based on the consensus, 10/17 triggers had a profile where at least 3 or 4 loci
had only the handler‟s alleles present, 4 of which had only the handler‟s alleles called at
all loci.

32

Table 7. Evaluation of consensus profiles from initial amplifications

14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

D13
4
2
1
1
1
1
2
1
2
1
3
1
4
1
4
4
4

D7
4
2
1
1
1
4
2
1
1
1
4
1
4
1
4
3
4

Amel.
1
1
1
1
1
1
1
1
1
1
4
1
4
1
4
2
1

D21
4
2
1
1
2
1
2
1
1
1
4
1
4
1
4
3
4

D16
4
2
1
2
2
4
2
2
2
1
4
1
4
1
4
3
4

D18
4
2
1
1
1
1
2
1
1
1
4
1
4
1
4
1
4

CSF
4
1
1
1
1
1
2
1
2
1
1
1
4
1
4
3
1

FGA
4
1
1
1
2
3
3
3
4
1
4
1
4
1
4
4
4

A scale from 1 – 4 was used to represent the number of handler‟s alleles obtained using
the consensus profile, where 1 represents the presence of both handler alleles only
(including homozygotes), 2, both handler alleles with others present, 3, one handler allele
by itself, and 4, no handler alleles were obtained. These are based on results from the
original amplifications using 4 µl of sample DNA.

Re-amplification of DNAs after clean-up using Microcon YM-100 spin columns
The three sets of DNAs that were re-cleaned with Microcon YM-100 columns and
re-amplified yielded more complete profiles than previous amplifications (Table 8).
Twenty three of twenty four (96%) loci had both handler alleles present when DNAs
were cleaned with YM-100 columns as compared to only 8 of 24 (33%) with YM-30
columns. No handler alleles were obtained from 13 of 24 (54%) loci analyzed after
33

initial amplification, while at least one handler allele was obtained from every locus as a
result of re-cleaning and amplification.

Table 8. Comparison of STR results when DNAs were amplified following clean-up
with Microcon YM-30 spin columns versus Microcon YM-100

D13
D7
Amel.
D21
D16
D18
CSF
FGA

Microcon YM-30
23g
27b
27c
B
F
F
A
F
F
A
F
A
C
F
F
D
F
F
E
F
B
C
A
A
A
F
F

Microcon YM-100
23g
27b
27c
B
A
A
A
A
B
A
B
B
A
A
A
A
D
B
B
B
B
B
A
A
A
A
A

Original STR data from DNAs concentrated using Microcon YM-30 columns after
extraction yielded little information. Select samples were re-purified with Microcon YM100 columns and amplified again. A scale from A – F was used to represent the number
of handler‟s alleles obtained from STR analysis, where A represents the presence of both
handler alleles without others, B, both handler alleles present with others, C, one handler
allele with no others, D, one handler allele with others, E, non-handler alleles only, or F,
no callable alleles.

Level of trigger fragmentation and pipe bomb type compared to STR results
There was no indication that the amount of damage to the triggers correlated with
STR analysis outcomes. Triggers at the highest damage classification yielded handler
alleles while some with low damage had few or no handler alleles (Table 9). All triggers
from IEDs deflagrated with steel pipe bombs had a consensus profile where at least one
locus had only the handler‟s alleles, as opposed to seven of nine triggers from IEDs
deflagrated with PVC pipe bombs. Full consensus profiles were obtained from three
triggers associated with PVC and one with steel.
34

Table 9. STR results compared to damage classification and pipe bomb type
Steel

PVC

Total number Damage
Device # of loci
Classification
1
22
4
0
3
27
8
0
7
28
0
0
8
23
8
1
5
25
8
1
1
26
0
1
5
29
1
1
6
30
2
1
24
1
3
The amount of damage done to the mock triggers during deflagration was rated on a scale
from 0 to 5, 0 being the lowest and 5 the highest. These damage classifications were
compared to the total number of loci in the consensus profile for each trigger where only
the handler‟s alleles were called.
Total number
Device # of loci
14
15
17
16
19
20
18
21

Damage
Classification
3
3
3
4
4
4
5
5

Comparison of STR results among trigger components
Based on the scale from A – F that was used to rate each locus for the presence of
the handler‟s alleles, the cell phones/radios had an average of 56 loci (of 136) (41%) in
categories A and B, the clamp 47 (35%), the battery 36 (26%), the circuit board (average
of front and back) 35 (26%), and the wire 29 (21%) (Table 10). No handler alleles were
obtained from 66 (49%) of the cell phone or radio DNAs (average of the three swabs
taken), 74 (54%) of the clamps, 81 (60%) of the batteries, 86 (63%) of the circuit boards
(average of the two samples), and 94 (69%) of the wires.

35

Table 10. STR analysis compared to individual mock trigger components

Total A
Total B
Total C
Total D
Total E
Total F

Phone
or
Radio
Front
28
25
12
6
6
59

Phone
or
Radio
Back
32
21
5
7
4
67

Phone
or
Radio
Sides
38
23
7
6
4
58

Circuit Circuit
Board Board
Front
Back Battery
20
29
25
10
11
11
7
19
10
1
3
9
1
6
4
97
68
77

Clamp
35
12
9
6
1
73

Wire
22
7
8
5
3
91

A scale of A – F was used to represent the number of handler‟s alleles obtained, where A
represents the presence of both handler alleles without others, B, both handler alleles
present with others, C, one handler allele with no others, D, one handler allele with
others, E, non-handler alleles only, or F, no callable alleles.

STR amplification using 1µl DNA and the second set of triggers
Seven of ten triggers (devices 14, 15, 19, 24, 26, 28, and 30) showed an
improvement in STR results when re-amplified using 1 µl of DNA, with an average
increase of 33% (13% – 46%) in the number of loci that fell into categories A and B.
Two triggers, 26 and 28, had no STR amplification using 4 µl of DNA, while reamplification resulted in 15% and 46%, respectively, of loci falling into categories A and
B. DNAs from the remaining 3 mock trigger DNAs amplified using 1 ul (triggers 20, 22,
and 29) gave an average decrease of 17% of loci in categories A and B.
Combining data from the amplifications of 1 and 4 µl DNA to build a consensus
profile gave better overall results than using only the initial amplifications (Table 11).
Ninety-one of one hundred thirty six (67%) loci fell into category 1, 33/136 (24%) in
category 2, 11/136 (8%) in category 3, and 1/136 (1%) in category 4. Fourteen of
seventeen triggers had a profile in which at least 3 or 4 loci had only the handler‟s alleles
36

present, 11/17 had a profile with at least 5 or 6 loci, 8/17 had at least 7, and 5/17 had only
the handler‟s alleles called at all loci.
The amount of damage to triggers 31 – 33, which used a steel pipe bomb, was
similar to previous trials; however, much less damage was done to 34 – 36, which used
PVC, due to the end cap of the pipe coming off during deflagration. STR analysis (based
on consensus profiles) resulted in 38/48 (79%) of loci falling into category 1, 8/48 (17%)
in category 2, 1/48 (2%) in category 3, and 2/48 (2%) in category 4. Five of six triggers
had a profile where at least 6 loci had the alleles present, 4 of which had at least 7, with 2
of those having only the handler‟s alleles called at all loci.

37

Table 11. Evaluation of consensus profiles from initial amplifications and reamplifications of 1 µl DNA combined

14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

D13
2
1
1
1
1
1
2
1
1
1
1
1
3
1
1
3
1

D7
1
1
1
1
1
1
2
1
2
1
3
1
3
1
2
2
1

Amel.
1
1
1
1
1
1
1
1
1
1
3
1
1
1
1
3
1

D21
3
1
1
1
2
1
2
1
1
1
2
1
2
1
2
2
1

D16
2
1
1
2
2
1
2
2
2
1
1
1
3
1
2
2
2

D18
3
2
1
1
1
1
2
1
2
1
2
1
3
1
2
1
1

CSF
1
1
1
1
1
1
2
1
1
1
2
1
2
1
2
2
1

FGA
2
1
1
1
1
1
2
3
1
1
1
1
4
1
1
1
1

31
32
33
34
35
36

1
1
1
1
1
1

2
3
1
1
1
2

1
1
1
1
1
1

1
1
1
1
1
2

1
1
1
1
4
2

1
1
1
1
2
2

1
1
1
1
1
2

1
1
1
1
1
2

A scale from 1 – 4 was used to represent the number of handler‟s alleles obtained using
the consensus profile, where 1 represents the presence of both handler alleles only, 2,
both handler alleles with others present, 3, one handler allele only, and 4, no handler
alleles. The data include results from the original amplifications using 4 µl of DNA
combined with selected samples that were re-amplified using 1 µl. Also shown are the
second set of mock triggers (31 – 36) that were amplified using both DNA volumes.

38

DISCUSSION

Previous analyses of IEDs have focused mainly on class characterizations of the
components used, analyzing trace evidence, chemical testing of the explosive charge, and
examination for possible fingerprints (Shachtman 2007). With the increase in both
international and domestic IED threat, it is important that the investigative techniques
used for analyzing attacks continue to develop. In recent studies, DNA obtained from the
explosive charge has shown some success in providing a genetic profile of individuals
that may have come into contact with the device (Esslinger et al. 2004; Kremer 2008;
Foran et al. 2009); however, the amount and quality of DNA is generally insufficient to
reliably obtain the handler‟s profile on a case by case basis. The focus of this study was
to determine if the chance of identifying the handler of an IED after deflagration could be
increased by analyzing other components of the IED, such as electronic triggering
mechanisms. There are several reasons why the trigger may produce more reliable and
consistent results as compared to the explosive charge itself. One is the increased
distance of the trigger components to the explosive charge where the heat and pressure
from deflagration would have less of an impact on DNA. In this study a sort of “worst
case scenario” was tested with the trigger components being positioned within 6 inches of
the explosive device. As a result of their close proximity, many of the mock triggers
were extensively damaged. It is likely that DNA on the components was also affected,
which may be one of the reasons that only a partial or no profile was obtained from many
of the trigger component DNAs, as opposed to the analysis of DNA from IED containers
where a full profile of the handler was almost always obtained (Hoffmann et al. 2011);

39

however, there was still an increase in success over previous studies that focused on the
explosive charge alone (Esslinger et al. 2004; Kremer 2008; Foran et al. 2009). Petrovick
and Harper (2011) carried out a similar study where approximately 10 ng of extracted
buccal cell DNA was deposited on IED triggers that were then placed 1 – 5 meters from
an explosive device (using C4, Composition B, and ammonium nitrate) to determine if
the effects of deflagration on DNA varied at different distances. They found that the
amount of DNA lost during deflagration decreased as the distance of the trigger to the
explosive increased and that at 1 meter approximately 49% of the handler‟s alleles were
obtained while at 5 meters it increased to 95%. Given the drop-off in success from 5 to 1
meter, it is not surprising that even fewer alleles were detected in this study when the
trigger was placed very close to the explosive, particularly considering that shed
epithelial cells were the source of DNA in this study, rather than high molecular weight
DNA. If the mock triggers were placed further away from the pipe bombs it is likely that
there would have been an increase in success of determining the handler‟s profile.
Another potential advantage of analyzing electronic triggers results from their
construction. Since the mechanisms are made of multiple individual components, there
may be less fragmentation of each as they separate during deflagration, resulting in easier
recovery and swabbing. In most cases the components in the current study remained
intact but separated from the plywood base, however many times they were completely
destroyed. The tendency for the phones and radios to fragment more than the other
components may have been due to their size and complexity in that they were the largest
elements and consisted of many smaller parts that separated during deflagration. This
was especially true for the radios, which were made with a brittle plastic. The other

40

components were smaller and generally comprised of a single solid piece and constructed
of metal (clamps, batteries, and wire) or tougher plastic (circuit boards). Since
deflagration was done in a metal crate rather than out in the open some of the
fragmentation potentially occurred when the components collided with the container. It
is difficult to determine the ease of „real world‟ trigger component recovery compared to
the explosive device since both remained inside of the containment crate in this study.
Generally large pieces of the steel pipes were recovered while both large and small pieces
of the triggers remained. Collecting pieces of the PVC pipe was difficult considering that
it fragmented into numerous small pieces during deflagration.
A third advantage of analyzing the IED trigger over the explosive charge was that
trigger assembly might require longer handling time resulting in more accumulated DNA.
In a study by Gomez (2009), multiple pipe bombs (PVC and steel) were swabbed after
deflagration and an average of 0.023 ± 0.022 ng (median of 0.007 ng) DNA was obtained
from each pipe. A significantly higher amount of DNA was obtained per swab set from
the triggers used in this study, with an average of 0.497 ± 0.800 ng (median of 0.206 ng;
p = < 0.01); however, it was originally thought that even more DNA would be obtained
considering increased handling of the trigger components. Kisilevsky et al. (1999)
showed that DNA transferred through sloughed epithelial cells persists on rougher
surfaces longer than smooth surfaces. All of the components incorporated in the mock
triggers had smooth surfaces (except for the unanalyzed plywood bases), which may have
affected the retention of DNA after handling. The only rough areas on any of the triggers
were the mounted electrical components on some circuit boards. Swabbing these areas
was extremely difficult since the cotton from the swab often became snagged. Petrovick

41

and Harper (2011) recommend the use of foam swabs for rough surfaces, and while not
used in this study, they may provide advantages over cotton swabs for the circuit boards
or other rough/sharp areas. Lowe et al. (2002) proposed that some people leave behind
more DNA than others and different conditions such as hand washing, length of handling,
and time after contact affect DNA deposition as well. It is possible that a combination of
these factors had an impact on the number of skin cells that remained on the triggers. In
this study participants were asked to handle each of the components for roughly 20
seconds. Had they been handled for a longer period of time, which would closer
resemble actual assembly of an IED that incorporated a triggering mechanism, more
cells/DNA would presumably have been deposited. This would have likely increased
overall success in determining the handler‟s profile. For the second set of mock IEDs
that were used to address this, participants handled the components for an extended
period of time. As a result of longer handling and multiple amplifications (discussed
below) there was an increase in success of obtaining the handler‟s profile.
One of the goals of this project was to determine which IED trigger components
were the best for analysis. Petrovick and Harper (2011) recovered very little DNA from
wire and asserted that the probability of obtaining a profile from it is low. In this study,
similar quantities of DNA were isolated from the wire as the battery and clamp, and
partial profiles were obtained from each. Therefore, it seems that all components of the
trigger can retain enough DNA for analysis and should not be overlooked, although the
greatest amount of DNA and highest number of handler‟s alleles originated from the
phones and radios, perhaps making them the “best” component for analysis.

42

It was originally thought that by using multiple swabs to cover larger surfaces,
more DNA would be recovered, consequently three swab sets were used for the
phones/radios (front, back, and sides) and two for the circuit boards (front and back);
however, it is possible that more handler alleles could have been obtained if the DNA
was collected onto one swab rather than being separated onto multiple swabs. While a
comparison of using one swab vs. multiple swabs to collect the handler‟s DNA was not
included in this study, work by Richert (2011) showed that using one swab to cover all
surfaces of a firearm was more advantageous than using multiple swabs. Had DNA from
the phones/radios and circuit boards been combined, it is possible that more complete
profiles could have been obtained from those two components. On average each sample
from the front, back, and sides of the phones/radios yielded 0.40 ng human DNA while
the average from the front and backs of the circuit boards was 0.52 ng (excluding the
negative control and device #14 which had an unusually high amount of DNA on the
phone‟s front, presumably from contamination), so generally less than 0.5 ng was being
amplified since the entire extract was never consumed for one amplification. If the DNA
had been collected together, the average amount of total human DNA would have likely
been around 1.2 ng for the phones/radios and 1.04 ng for the circuit boards, which would
have resulted in more DNA available for amplification. A potential disadvantage of
using one swab is that multiple handlers would result in a mixture. Using multiple swabs
in these circumstances may be beneficial in differentiating individuals. The effects of
this would not have been seen in this study because the components were cleaned in
advance and only one volunteer handled them.

43

Since there was no statistical difference in the amount of DNA obtained from
triggers when a steel or PVC pipe bomb was used, it is possible that the pipe material
does not play a role in DNA retention or recovery from IED triggers; however, more
damage was done to the mock triggers of IEDs that incorporated a steel pipe bomb, even
though the same type of propellant was used. Due to the increased strength of steel over
PVC there was much more pressure built-up during deflagration and subsequently greater
concussion on objects around the explosive, with more physical damage done. Further,
there was more partially burned and unburned powder on the components when PVC was
used, which stemmed from the weaker casing separating before all of the powder was
completely consumed. Other evidence of this comes from video of one of the mock IED
deflagrations that used a PVC pipe: substantial flames came out of the metal container
during deflagration indicating that there was a great deal of burning powder being
expelled from the casing.
The quality of DNA isolated from the IED triggers was likely affected by
deflagration. Initial STR analysis of DNA from trigger 23 components resulted in almost
no usable data, even though quantification indicated there was enough DNA to obtain a
genetic profile and PCR inhibition was not detected. This discrepancy may have resulted
from DNA degradation. The Quantifiler amplicon size is 62 bases (Quantifiler User
Guide) whereas the Minifiler size range is roughly 75 – 285 bases (Minifiler User Guide).
The smaller amplicon will be less influenced by degradation, so there was possibly a
reduced effect on quantification compared to STR amplification. To test this, selected
samples were re-cleaned using Microcon YM-100 columns, which have a nucleotide
cutoff of 125 bases (as opposed to the YM-30 cutoff of 50 bases). Though these samples

44

were not re-quantified for a direct comparison to the STR amplification, by filtering out
the smaller DNA fragments more complete profiles were obtained.
The black residue on the swabs from the trigger components was removed during
extraction; however, it is possible that other contaminants remained. The IPC included in
Quantifiler indicated inhibition in 26 samples, 19 of which originated from mock triggers
incorporating a steel pipe bomb. Based on these findings it seems possible that inhibition
from the steel pipe bombs was greater, even though there were larger amounts of black
residue on the trigger components of IEDs using PVC.
Minifiler provides improved amplification of DNA containing inhibitors as
compared to standard STR kits (Mulero et al. 2008) still, many of the trigger DNAs had
little or no STR amplification, even when quantification results indicated the presence of
DNA. This could have resulted from the amount of DNA/inhibitor being added to each
reaction. One and two tenths microliters of template DNA was added to Quantifiler
reactions (15 µl total volume) while 4 µl was added to Minifiler reactions (10 µl total
volume). If inhibitors were present, they would have a higher impact on the Minifiler
reactions where the DNA sample makes up 40% of the total reaction volume compared to
8% of the Quantifiler reaction. Additional evidence of PCR inhibition comes from the
success of re-amplifications using less template DNA, where decreasing the input from 4
to 1 µl in the STR PCR resulted in more complete profiles from 7 of 10 triggers tested.
For the re-amplifications in which profile success declined, the already low amount of
DNA was likely decreased too much. Combining data from 1 and 4 µl amplifications
resulted in an increase of overall success in determining the handler‟s profile.
Subsequent findings supported this, where some amplifications from the second set of

45

IEDs gave better results using 1 µl DNA, while 4 µl was more suitable for others.
Conversely, 32 samples quantified as having 0 ng of DNA, 9 of which produced a partial
profile of two or more loci with the handler‟s alleles. This discrepancy may be attributed
to a number of things, including the higher amount of DNA added to the Minifiler
reaction. If stochastic sampling effects influenced both Minifiler and Quantifiler assays,
Minifiler, with nine loci targeted, would have a higher chance of some loci amplifying
than would Quantifiler, which targets only one locus. In addition, PCR inhibitors could
have affected the Quantifiler reaction more than the Minifiler reaction. The buffer used
with Minifiler was improved for amplification of low quality DNAs with inhibitors
(Minifiler User Guide), including a “carrier protein” that is not found in the Quantifiler
buffer. It is also possible that there was a mutation at the binding site of a Quantifiler
primer or the TaqMan probe in some samples, which would decrease or even prevent
amplification/detection of DNA. Either way, the data show that a zero quantification
value from Quantifiler does not always indicate the absence of human DNA, which
should be considered when determining whether to amplify a particular sample or not.
Rarely did analysis of trigger DNA result in a pristine profile. Often, degraded
DNA profiles appear on an electropherogram as a “ski slope”, where the peaks of the
smaller amplicons are higher, and decrease in height as the size of the amplicons increase
(Bessetti 2007). Sometimes this can lead to drop-out of the larger loci, resulting in a
partial profile. Though this was occasionally observed, especially at FGA and D21S11,
all loci seemed to be susceptible to drop-out. This indicates greater effects due to PCR
inhibition than DNA degradation.

46

More than two peaks were present at several loci, making it difficult to determine
which were the handler‟s alleles. Though contamination at any step could not be ruled
out, it seems unlikely since there was very little DNA obtained from the negative control
(device #13) indicating that cleaning prior to handling was effective and that methods
used for analysis did not introduce extraneous DNA. Furthermore, there was no evidence
of alleles matching lab personnel or any of the bomb squad members who may have
come into contact with the device. The swabs taken from the front of the negative
control phone recovered 0.45 ng DNA, which could have been carryover from its original
owner. This might explain extra peaks in some samples from donated phones, though
elimination standards were not obtained to confirm this. Benschop et al. (2011) found
that increased forward (+1 repeat from the parent allele) and back (-1 repeat from the
parent allele) stutter contributed to many of the drop-in peaks when multiple LCN DNAs
were analyzed. They also noted that when the number of PCR cycles was increased, so
did the ratio of stutter to the parent allele. The recommended PCR cycle number for
Minifiler is 30 (Minifiler User Guide), while in this study 33 were used. In many of the
samples analyzed in this study, increased stutter may have accounted for some of the
extra alleles. This made interpretation of these profiles difficult, where in some cases a
homozygote with high stutter was interpreted as a heterozygote. A similar problem was
encountered with high heterozygote peak imbalances, which may also be in part
attributed to the increased number of PCR cycles (Bright et al. 2010). However, in many
of the interpretable profiles the peak heights were low. If the cycle number was reduced
those peaks would likely have dropped out. This occurred in initial amplifications of

47

samples from device 23 using 30 cycles; however, an extensive study comparing cycle
numbers was not undertaken.
The use of consensus profiles increased the reliability of typing the handler‟s
DNA and was effective for excluding many drop-in alleles, including stutter. In addition,
complete (or nearly complete) handler profiles were determined for some of the
triggering mechanisms. In contrast, interpretation of individual profiles from each of the
component DNAs resulted in elevated drop-in alleles and few or no full handler profiles.
In many instances drop-out of handler alleles occurred at different loci for separate
component‟s DNA amplifications. If those profiles were interpreted by themselves the
result would be a partial profile. A more conservative approach was taken to build the
consensus profiles in this study by including alleles that amplified from 2 or more trigger
component DNAs, although this did result in some extra alleles. If an allele had to
amplify three times to be included in the consensus, more of the extraneous peaks could
be eliminated; however, fewer handler alleles would also have been called. This can be
demonstrated by re-interpreting the data from the initial amplifications of 4 µl of DNA.
Increasing the number of replicate alleles to three would have resulted in no change for
98/136 (72%) loci, drop-out of one handler allele for 16/136 (12%) of loci, drop-out of all
alleles for 18/136 (13%) of loci, and inclusion of only the handler‟s alleles (where other
extraneous alleles were previously included) for 4/136 (3%) of loci.
A potential drawback to the consensus approach used here is that by comparing
DNA profiles isolated from different components of the trigger, there is an assumption
that the same individual‟s DNA resides on each. That assumption is accurate for this
study because all trigger components were cleaned in advance and only handled by one

48

individual. In a “real world” scenario the same may not be the case, and certainly they
are unlikely to have been cleaned in advance. A better approach may be to amplify
DNAs of each component in replicates (possibly using different volumes of template
DNA) and determine a consensus profile using only those amplifications.

49

CONCLUSION

With an ever-increasing threat of IEDs, both domestically and internationally, it
has become essential that investigative techniques into attacks be improved. Using the
sensitivity of today‟s DNA analysis technologies, it is possible to use biological evidence
from exploded IEDs to help investigators identify the perpetrator. However, there are
many challenges associated with such analyses, considering the effects deflagration has
on DNA.
Analysis of IED trigger components yielded more DNA and more complete
genetic profiles than the explosive device; however, heat and pressure from deflagration
still have a large impact on the quality of DNA isolated. Considering that IEDs are
generally fabricated by hand, any of the components may retain enough DNA for analysis
and should not be overlooked. In this study the handler‟s DNA was obtained from all
trigger components in some instances, including a cell phone or radio, circuit board,
battery, pipe bomb clamp, and wire. Since the amount of DNA recovered from IED
trigger components is generally very small, LCN techniques such as the use of miniSTRs,
increased PCR cycling, and amplifying in replicates with different volumes of DNA may
be necessary. Finally, developing a consensus profile from multiple DNAs taken from
the same triggering device results in a more complete profile of the handler.
Though there are still challenges that must be overcome when dealing with DNA
samples taken from deflagrated IEDs, the analysis of triggering devices may be an
important tool for investigating IED attacks, and depending on the circumstances of the
attack could give investigators the best chance of determining the assembler. Due to the

50

increased complexity of IEDs today it is probable that bombs will include such a device,
making them a worthwhile focus for DNA collection in future IED investigations.

51

APPENDIX A. By locus categorization of allele calls for each mock trigger

In order to evaluate the success in determining the expected allele calls from the
interpretation of the unknown DNA samples, after comparison to the reference samples
taken from the IED handlers, each locus was categorized on a scale from A – F:
A - Presence of both handler alleles without others
B - Presence of both handler alleles with others
C - One single handler allele present
D - One handler allele with others present
E - Amplification but of unexpected alleles
F - No callable alleles
Below are the classifications for every locus analyzed from the initial amplification of
DNA taken from each mock IED trigger, except for 13 which served as a negative
control.

D13
D7
Amel.
D21
D16
D18
CSF
FGA

14a
F
F
F
F
F
F
F
F

14b
F
F
F
F
F
F
F
F

14c
F
F
F
F
F
F
F
F

14d
F
F
F
F
F
F
B
F

14e
F
F
F
F
C
F
F
F

14f
F
F
F
F
C
F
F
F

14g
F
F
F
F
F
F
F
F

14h
F
F
F
F
F
F
F
F

D13
D7
Amel.
D21
D16
D18
CSF
FGA

15a
B
F
A
A
F
B
B
A

15b
F
F
F
F
F
B
F
F

15c
B
A
A
A
B
F
A
A

15d
C
F
F
F
F
F
C
F

15e
F
F
C
F
F
F
C
F

15f
F
F
F
F
F
F
F
F

15g
F
F
F
F
F
F
A
F

15h
F
F
F
F
F
F
F
F

52

D13
D7
Amel.
D21
D16
D18
CSF
FGA

16a
B
B
A
F
B
A
B
D

16b
A
A
B
C
A
A
A
B

16c
B
B
A
A
B
B
B
B

16d
F
F
F
F
F
F
F
F

16e
F
F
F
F
C
F
C
F

16f
C
D
A
F
F
B
B
A

16g
B
B
A
A
B
A
D
A

16h
F
F
F
F
F
A
A
F

D13
D7
Amel.
D21
D16
D18
CSF
FGA

17a
A
F
A
F
F
F
A
C

17b
F
F
F
F
F
F
A
F

17c
B
A
A
D
A
B
A
B

17d
F
F
F
F
F
F
A
F

17e
C
F
A
A
C
A
A
C

17f
F
F
F
F
F
F
A
F

17g
B
A
A
A
A
A
A
A

17h
F
F
F
F
F
F
A
F

D13
D7
Amel.
D21
D16
D18
CSF
FGA

18a
D
F
A
A
C
A
B
A

18b
B
A
A
A
C
A
B
F

18c
A
A
A
A
C
C
B
A

18d
F
F
F
F
F
F
F
F

18e
B
F
A
B
C
A
A
A

18f
F
F
F
F
F
F
F
F

18g
A
B
A
B
A
A
A
B

18h
F
F
F
F
F
F
F
F

D13
D7
Amel.
D21
D16
D18
CSF
FGA

19a
A
F
A
A
F
A
A
C

19b
E
F
C
C
F
B
A
F

19c
F
F
A
F
F
A
A
F

19d
F
F
F
F
F
F
F
F

19e
F
F
F
F
F
F
F
F

19f
F
F
F
F
F
F
A
F

19g
F
F
F
F
F
F
F
F

19h
F
F
F
F
F
F
F
F

53

D13
D7
Amel.
D21
D16
D18
CSF
FGA

20a
F
F
C
C
E
A
E
F

20b
D
E
A
D
B
E
B
C

20c
D
E
C
F
D
D
B
F

20d
F
F
F
F
F
F
F
F

20e
A
F
C
A
E
D
A
F

20f
F
F
C
F
F
E
F
F

20g
B
B
A
C
E
C
A
D

20h
D
F
F
F
A
D
A
F

D13
D7
Amel.
D21
D16
D18
CSF
FGA

21a
A
A
A
D
A
B
A
E

21b
F
F
F
F
F
F
F
F

21c
A
F
A
C
B
A
C
E

21d
F
F
F
F
F
F
C
F

21e
F
F
F
F
F
F
F
F

21f
A
C
A
A
A
D
A
D

21g
F
F
F
F
A
A
C
F

21h
A
C
A
F
B
A
A
E

D13
D7
Amel.
D21
D16
D18
CSF
FGA

22a
A
D
C
C
E
D
B
C

22b
B
B
A
B
F
B
A
A

22c
F
F
F
F
F
F
A
F

22d
A
F
C
A
B
A
B
F

22e
B
A
A
C
B
C
A
C

22f
B
A
A
D
D
D
B
D

22g
B
F
A
C
F
A
A
A

22h
F
F
F
F
F
D
A
F

D13
D7
Amel.
D21
D16
D18
CSF
FGA

23a
F
E
F
F
F
C
F
F

23b
B
A
A
A
A
A
A
A

23c
A
A
A
C
A
B
A
A

23d
F
A
D
B
A
B
A
C

23e
F
F
A
F
D
C
A
F

23f
A
C
A
A
A
A
A
B

23g
B
A
A
A
A
A
B
F

23h
A
C
A
C
A
C
B
B

54

D13
D7
Amel.
D21
D16
D18
CSF
FGA

24a
F
F
F
F
F
F
B
F

24b
F
F
F
F
F
F
F
F

24c
F
F
F
F
F
F
F
F

24d
F
F
F
F
F
F
F
F

24e
D
F
F
F
F
F
B
F

24f
F
F
F
F
F
F
F
F

24g
F
F
F
F
F
F
F
F

24h
F
F
F
F
F
F
F
F

D13
D7
Amel.
D21
D16
D18
CSF
FGA

25a
B
B
A
B
B
B
B
B

25b
B
A
A
B
B
B
B
A

25c
B
B
A
B
B
A
A
B

25d
B
B
A
B
A
A
A
B

25e
B
B
A
A
B
A
A
B

25f
B
B
A
B
B
A
A
B

25g
A
D
A
F
D
D
C
D

25h
A
B
A
A
A
A
B
A

D13
D7
Amel.
D21
D16
D18
CSF
FGA

26a
F
F
F
F
F
F
F
F

26b
F
F
F
F
F
F
F
F

26c
F
F
F
F
F
F
F
F

26d
F
F
F
F
F
F
F
F

26e
F
F
F
F
F
F
F
F

26f
F
F
F
F
F
F
F
F

26g
F
F
F
F
F
F
F
F

26h
F
F
F
F
F
F
F
F

D13
D7
Amel.
D21
D16
D18
CSF
FGA

27a
B
B
A
A
B
B
B
B

27b
A
A
B
A
D
B
A
A

27c
A
B
B
A
B
A
A
A

27d
B
A
A
A
A
A
A
A

27e
A
A
A
F
A
A
A
C

27f
A
C
A
F
C
A
C
C

27g
C
C
A
F
A
F
C
F

27h
F
C
A
C
A
C
A
F

55

D13
D7
Amel.
D21
D16
D18
CSF
FGA

28a
F
F
F
F
F
F
F
F

28b
F
F
F
F
F
F
F
F

28c
F
F
F
F
F
F
F
F

28d
F
F
F
F
F
F
F
F

28e
F
F
F
F
F
F
F
F

28f
F
F
F
F
F
F
F
F

28g
F
F
F
F
F
F
F
F

28h
F
F
F
F
F
F
F
F

D13
D7
Amel.
D21
D16
D18
CSF
FGA

29a
D
F
B
E
C
A
C
C

29b
E
D
B
D
D
A
D
A

29c
E
D
A
C
E
F
F
D

29d
F
F
A
F
C
F
E
C

29e
F
C
A
E
E
C
C
F

29f
F
D
F
F
E
D
C
F

29g
F
C
F
F
F
F
F
F

29h
D
B
B
F
E
E
D
C

D13
D7
Amel.
D21
D16
D18
CSF
FGA

30a
F
F
F
F
F
F
F
F

30b
F
F
F
F
F
F
F
F

30c
F
F
F
F
F
F
F
F

30d
F
F
F
F
F
F
A
F

30e
E
E
A
C
B
B
A
E

30f
E
F
A
F
B
F
A
E

30g
F
F
F
F
F
F
A
F

30h
F
F
F
F
F
F
F
F

56

APPENDIX B. STR amplification using 1 µl of DNA compared to 4 µl
Below is a side-by-side comparison of the STR results obtained from separate amplifications of 4 µl and 1 µl DNA with the
total number and percentage of each classification, A – F, used to determine the success in the interpretation. A represents the
presence of both of the handler‟s alleles without others, B, both handler alleles present with others, C, one handler allele with no
others, D, one handler allele with others, E, amplification but of unexpected alleles, or F, no callable alleles.

Amplification of 4 µl DNA

D13
D7
Amel.
D21
D16
D18
CSF
FGA
Tot. A
0
0%

14a
F
F
F
F
F
F
F
F

14c
F
F
F
F
F
F
F
F

Tot. B
1
3%

Amplification of 1 µl DNA
14d
F
F
F
F
F
F
B
F

Tot. C
0
0%

14g
F
F
F
F
F
F
F
F
Tot. D
0
0%

14h
F
F
F
F
F
F
F
F
Tot. E
0
0%

D13
D7
Amel.
D21
D16
D18
CSF
FGA
Tot. F
39
98%

Tot. A
11
28%

57

14a
B
D
A
E
E
E
B
E
Tot. B
6
15%

14c
F
F
A
C
E
E
A
E

14d
B
B
A
D
D
D
B
C

Tot. C
4
10%

14g
A
C
A
F
A
C
A
D
Tot. D
5
13%

14h
A
A
B
F
E
F
A
F
Tot. E
8
20%

Tot. F
6
15%

Amplification of 4 µl DNA

D13
D7
Amel.
D21
D16
D18
CSF
FGA
Tot. A
9
19%

15a
B
F
A
A
F
B
B
A
Tot. B
6
13%

15b
F
F
F
F
F
B
F
F

15c
B
A
A
A
B
F
A
A

Tot. C
0
0%

Amplification of 1 µl DNA
15f
F
F
F
F
F
F
F
F
Tot. D
0
0%

15g
F
F
F
F
F
F
A
F
Tot. E
0
0%

15h
F
F
F
F
F
F
F
F

D13
D7
Amel.
D21
D16
D18
CSF
FGA
Tot. A
19
40%

Tot. F
33
69%

58

15a
C
A
A
A
A
D
B
A
Tot. B
13
27%

15b
D
F
A
A
B
D
A
D

15c
B
B
A
B
B
A
B
B

Tot. C
6
13%

15f
D
C
C
B
B
D
A
D
Tot. D
8
17%

15g
B
D
A
A
A
C
C
C
Tot. E
1
2%

15h
A
B
A
B
A
E
A
A
Tot. F
1
2%

Amplification of 4 µl DNA

D13
D7
Amel.
D21
D16
D18
CSF
FGA

19a
A
F
A
A
F
A
A
C

Tot. A
10
31%

Tot. B
1
3%

19b
E
F
C
C
F
B
A
F
Tot. C
3
9%

Amplification of 1 µl DNA
19c
F
F
A
F
F
A
A
F

19f
F
F
F
F
F
F
A
F

Tot. D
0
0%

D13
D7
Amel.
D21
D16
D18
CSF
FGA
Tot. E
1
3%

Tot. A
17
53%

Tot. F
17
53%

59

19a
C
B
A
D
A
A
C
A
Tot. B
5
16%

19b
D
C
A
F
C
C
A
F

19c
B
A
A
A
B
B
A
A

Tot. C
6
19%

19f
A
A
A
C
A
A
A
B
Tot. D
2
6%

Tot. E
0
0%

Tot. F
2
6%

Amplification of 4 µl DNA

D13
D7
Amel.
D21
D16
D18
CSF
FGA

20a
F
F
C
C
E
A
E
F

Tot. A
6
15%

Tot. B
5
13%

20b
D
E
A
D
B
E
B
C
Tot. C
6
15%

Amplification of 1 µl DNA

20c
D
E
C
F
D
D
B
F

20g
B
B
A
C
E
C
A
D

Tot. D
8
20%

20h
D
F
F
F
A
D
A
F
Tot. E
6
15%

D13
D7
Amel.
D21
D16
D18
CSF
FGA
Tot. A
3
8%

Tot. F
9
23%

60

20a
F
F
F
F
F
F
F
F
Tot. B
2
5%

20b
C
F
C
D
F
F
B
E

20c
F
F
A
E
F
F
F
C

Tot. C
3
8%

20g
F
F
F
F
B
F
F
F
Tot. D
1
3%

20h
F
F
F
F
A
E
A
F
Tot. E
3
8%

Tot. F
28
70%

Amplification of 4 µl DNA

D13
D7
Amel.
D21
D16
D18
CSF
FGA
Tot. A
13
27%

22a
A
D
C
C
E
D
B
C
Tot. B
11
23%

22b
B
B
A
B
F
B
A
A

22d
A
F
C
A
B
A
B
F

Tot. C
7
15%

Amplification of 1 µl DNA
22e
B
A
A
C
B
C
A
C
Tot. D
7
15%

22f
B
A
A
D
D
D
B
D
Tot. E
1
2%

22h
F
F
F
F
F
D
A
F

D13
D7
Amel.
D21
D16
D18
CSF
FGA
Tot. A
5
10%

Tot. F
9
19%

61

22a
F
F
F
C
E
D
F
F
Tot. B
10
21%

22b
B
D
A
A
B
B
B
B
Tot. C
5
10%

22d
A
F
C
F
B
F
A
F

22e
F
F
C
F
B
F
A
F
Tot. D
3
6%

22f
B
C
C
F
B
E
E
D
Tot. E
4
8%

22h
F
F
F
F
F
E
B
F
Tot. F
21
44%

Amplification of 4 µl DNA

D13
D7
Amel.
D21
D16
D18
CSF
FGA

24a
F
F
F
F
F
F
B
F

Tot. A
0
0%

Tot. B
2
4%

24b
F
F
F
F
F
F
F
F

24c
F
F
F
F
F
F
F
F

Tot. C
0
0%

Amplification of 1 µl DNA
24e
D
F
F
F
F
F
B
F
Tot. D
1
2%

24f
F
F
F
F
F
F
F
F
Tot. E
0
0%

24g
F
F
F
F
F
F
F
F

24h
F
F
F
F
F
F
F
F

Tot. F
53
95%

D13
D7
Amel.
D21
D16
D18
CSF
FGA
Tot. A
6
11%

62

24a
C
F
C
C
C
F
A
C
Tot. B
1
2%

24b
A
F
F
F
C
C
A
C

24c
D
F
C
C
C
C
B
A

Tot. C
19
34%

24e
E
C
C
E
C
C
A
D
Tot. D
2
4%

24f
F
F
F
F
C
F
E
F
Tot. E
5
9%

24g
F
F
C
F
F
F
F
F

24h
A
F
C
F
E
F
E
F

Tot. F
23
41%

Amplification of 4 µl DNA

D13
D7
Amel.
D21
D16
D18
CSF
FGA

26a
F
F
F
F
F
F
F
F

Tot. A
0
0%

Tot. B
0
0%

26e
F
F
F
F
F
F
F
F

Amplification of 1 µl DNA
26f
F
F
F
F
F
F
F
F

Tot. C
0
0%

26g
F
F
F
F
F
F
F
F

Tot. D
0
0%

26h
F
F
F
F
F
F
F
F
Tot. E
0
0%

D13
D7
Amel.
D21
D16
D18
CSF
FGA
Tot. A
4
10%

Tot. F
40
100%

63

26a
F
F
F
F
F
F
F
F
Tot. B
2
5%

26e
E
F
C
E
C
D
E
F

26f
F
E
C
B
E
E
B
F

Tot. C
8
20%

26g
C
C
C
E
A
C
A
F
Tot. D
1
3%

26h
F
F
C
A
F
F
A
F
Tot. E
7
18%

Tot. F
18
45%

Amplification of 4 µl DNA

D13
D7
Amel.
D21
D16
D18
CSF
FGA
Tot. A
0
0%

Amplification of 1 µl DNA

28a
F
F
F
F
F
F
F
F

28c
F
F
F
F
F
F
F
F

28d
F
F
F
F
F
F
F
F

28e
F
F
F
F
F
F
F
F

28g
F
F
F
F
F
F
F
F

Tot. B
0
0%

Tot. C
0
0%

Tot. D
0
0%

Tot. E
0
0%

D13
D7
Amel.
D21
D16
D18
CSF
FGA

Tot. F
40
100%

28c
A
F
A
A
F
F
B
C

28d
A
C
A
F
F
E
E
C

28e
E
F
F
A
A
E
A
E

28g
A
B
A
B
B
B
B
A

Tot. A
11
28%

64

28a
E
F
F
F
E
B
F
E
Tot. B
7
18%

Tot. C
3
8%

Tot. D
0
0%

Tot. E
8
20%

Tot. F
11
28%

Amplification of 4 µl DNA

D13
D7
Amel.
D21
D16
D18
CSF
FGA
Tot. A
2
8%

Amplification of 1 µl DNA

29a
D
F
B
E
C
A
C
C

29c
E
D
A
C
E
F
F
D

29h
D
B
B
F
E
E
D
C

Tot. B
3
13%

Tot. C
5
21%

Tot. D
5
21%

D13
D7
Amel.
D21
D16
D18
CSF
FGA
Tot. E
5
21%

65

29c
E
E
F
F
C
F
F
F

29h
F
F
F
F
D
E
F
F

Tot. A
1
4%

Tot. F
4
17%

29a
F
F
A
F
F
F
F
F
Tot. B
0
0%

Tot. C
1
4%

Tot. D
1
4%

Tot. E
3
13%

Tot. F
18
75%

Amplification of 4 µl DNA

D13
D7
Amel.
D21
D16
D18
CSF
FGA

30a
F
F
F
F
F
F
F
F

Tot. A
6
11%

Tot. B
3
5%

30b
F
F
F
F
F
F
F
F

30c
F
F
F
F
F
F
F
F

Tot. C
1
2%

Amplification of 1 µl DNA
30d
F
F
F
F
F
F
A
F
Tot. D
0
0%

30e
E
E
A
C
B
B
A
E
Tot. E
5
9%

30f
E
F
A
F
B
F
A
E

30g
F
F
F
F
F
F
A
F

D13
D7
Amel.
D21
D16
D18
CSF
FGA
Tot. A
34
61%

Tot. F
41
73%

66

30a
A
F
A
F
A
A
A
F
Tot. B
5
9%

30b
A
C
A
A
A
A
A
F
Tot. C
7
13%

30c
A
A
F
A
B
D
B
F

30d
A
D
A
A
B
A
A
A

Tot. D
2
4%

30e
A
A
B
A
E
A
A
A
Tot. E
1
2%

30f
A
C
A
C
A
A
A
A
Tot. F
7
13%

30g
B
A
A
C
F
C
C
C

REFERENCES

REFERENCES

Al-Soud W, Rådström P. 2000. Effects of amplification facilitators on diagnostic PCR
in the presence of blood, feces, and meat. J Clinical Microbiology. 38:4463 – 4470.
Applied Biosystems. 2001. AmpFlSTR Identifiler PCR Amplification Kit User‟s
Manual. Foster City, CA.
Applied Biosystems. 2006. Quantifiler Human DNA Quantification Kit and
Quantifiler Y Human Male DNA Quantification Kit User‟s Guide. Foster City, CA.
Applied Biosystems. 2007. AmpFlSTR Minifiler PCR Amplification Kit User‟s Guide.
Foster City, CA.
Balding DJ and Buckleton J. 2009. Interpreting low template DNA profiles. Forensic
Sci Int Genetics. 4:1 – 10.
Benschop C, van der Beek C, Meiland H, van Gorp A, Westen A, Sijen T. 2011. Low
template STR typing: Effect of replicate number and consensus method on
genotyping reliability and DNA database search results. Forensic Sci Int. 5:316 –
328.
Bessetti J. 2007. An introduction to PCR inhibitors. Profiles in DNA. 10:9 – 10.
Bolz F, Dudonis K, Schulz DP. 2005. The counterterrorism handbook. CRC Press.
Boca Raton, FL.
Bright J, Huizing E, Melia L, Buckleton J. 2010. Determination of the variables
affecting mixed Minifiler DNA profiles. Forensic Sci Int Genetics. 5(5):381 – 385
Broun J, Halpain E, Kent J, Lorenz C. 2009. The IED threat in America. Urban Warfare
Analysis Center. Available at
http://understandterror.com/Newsletter/Articles/The%20IED%20Threat%20in%20A
merica.pdf
Budowle B, Hobson DL, Smerick JB, Smith JAL. 2001. Low copy number consideration and caution. Presented at Twelfth International Symposium on Human
Identification. Biloxi, MS, USA.
Butler JM, Shen Y, McCord BR. 2003. The development of reduced size STR
amplicons as tools for analysis of degraded DNA. J Forensic Sci. 48(5):1054 – 1064.
Comey C, Koons B, Presley K, Smerick J, Sobieralski C, Stanley D, Baechtel F. 1994.
DNA extraction strategies for amplified fragment length polymorphism analysis. J
Forensic Sci. 39:1254 – 1269.
68

Department of Defense Dictionary of Military and Associated Terms. April 12, 2001 as
amended through October 17, 2008. Joint publication 1 – 02.
DNA Advisory Board. 2000. Quality assurance standards for forensic DNA testing
laboratories (approved July 1998). Forensic Sci Communications. 2:(3)9.4.2.1
DOD personnel and military casualty statistics. October 7, 2001 through September, 6
2011. Casualty summary by reason. Available at
http://siadapp.dmdc.osd.mil/personnel/CASUALTY/gwot_reason.pdf
Eilert K, Foran D. 2009. Polymerase resistance to polymerase chain reaction inhibitors
in bone. J Forensic Sci. 54(5):1001 – 1007.
Esslinger KJ, Siegel JA, Spillane H, Stallworth S. 2004. Using STR analysis to detect
human DNA from exploded pipe bomb devices. J Forensic Sci. 49:481 – 484.
Foran D, Gehring M, Stallworth S. 2009. The recovery and analysis of mitochondrial
DNA from exploded pipe bombs. J Forensic Sci. 54(1): 90 – 94.
Gill P, Whitaker J, Flaxman C, Brown N, Buckleton J. 2000. An investigation of the
rigor of interpretation rules for STRs derived from less than 100 pg of DNA.
Forensic Sci Int. 112:17 – 40.
Gill P. 2001. Application of low copy number DNA profiling. Croatian Medical J.
42:229 – 232
Globalsecurity.org. 2005. Improvised explosive devices (IEDs) / Booby traps.
Available at http://www.globalsecurity.org/military/intro/ied.htm
Gomez K. 2009. Recovery and quantification of nuclear and mitochondrial DNA from
improvised explosive devices using comparative soaking and swabbing techniques.
Master‟s thesis. Michigan State University.
Green RL, Roinestad IC, Boland C, Hennessy LK. 2005. Developmental validation of
the Quantifiler real-time PCR kits for the quantification of human nuclear DNA
samples. J Forensic Sci. 50:809 – 831.
Hoffmann S, Stallworth S, Foran D. 2011. Investigative studies into the recovery of
DNA from improvised explosive device containers. J Forensic Sci. 10.1111/j.15564029.2011.01982.x
Homeland Security Presidential Directive/HSPD – 19. Issued on February12, 2007.
Subject: Combating terrorist use of explosives in the United States. Available at
http://www.albany.edu/ncsp/docs/HSPDs/HSPD-19.pdf

69

Hsu S, Sheridan M. 2007. IEDs seen as rising threat in the U.S. Washington Post. A01.
Available at http://www.washingtonpost.com/wpdyn/content/article/2007/10/19/AR2007101902703.html?hpid=topnews
Jobin R, DeGouffe M. 1999. The persistence of seminal constituents on undershorts
after laundering: significance of investigations of sexual assaults. Proceedings of the
Annual Meeting of the Canadian Society of Forensic Science, Edmonton, Alberta.
Kiley B. 2009. A highly sensitive sex determination assay for low quality DNA.
Master‟s thesis. Michigan State University.
Kisilevsky A, Wickenheiser R. 1999. DNA PCR profiling of skin cells transferred
through handling. Proceedings of the Annual Meeting of the Canadian Society of
Forensic Science, Edmonton, Alberta.
Kremer S. 2008. The use of miniSTRs and mitochondrial DNA to identify handlers of
pipe bombs. Master‟s thesis. Michigan State University.
Lowe A, Murray C, Whitaker J, Tully G, Gill P. 2002. The propensity of individuals to
deposit DNA and secondary transfer of low level DNA from individuals to inert
surfaces. Forensic Sci Int. 129:25 – 34.
Petrovick M, Harper J. 2011. MIT Lincoln Laboratory Report: ANDE Forensic
Signature Science Results. Submitted to Ken Kroupa ASD(R&E).
Mulero J, Chang C, Lagac R, Wang D, Bas J, McMahon J, Hennessy L. 2008.
Development and validation of the AmpFlSTR Minifiler PCR Amplification Kit: A
miniSTR multiplex for the analysis of degraded and⁄or PCR inhibited DNA. J
Forensic Sci. 53(4):838 – 852.
Pang B, Cheung B. 2007. One-step generation of degraded DNA by UV irradiation.
Analytical Biochemistry. 360:163 – 165
Phipps M, Petricevic S. 2007. The tendency of individuals to transfer DNA to handled
items. Forensic Sci Int. 168:162 – 168.
Richert, N. 2011. Swabbing firearms for handler‟s DNA. J Forensic Sci. 56(4):972 –
975.
Shachtman N. 2007. CSI vs. IEDs: Inside Baghdad's forensic bomb squad. Wired News.
Available at http://blog.wired.com/defense/2007/09/post.html
Sweet D, Lorente M, Lorente JA, Valenzuela A, Villanueva E. 1997. An improved
method to recover saliva from human skin: the double swab technique. J Forensic
Sci. 42:320 – 322.

70

Threadgold T, Brown T. 2003. Degradation of DNA in artificially charred wheat seeds.
J Archaeological Sci. 30:1067 – 1076
Van Hoofstat D, Deforce D, Brochez V, De Pauw I, Janssens K, Mestdagh M,
Millecamps R, Van Geldre E, Van Den Eeckhout E. 1998. DNA typing of
fingerprints and skin debris: sensitivity of capillary electrophoresis in forensic
applications using multiplex PCR. Promega Corporation Proceedings of the 2nd
European Symposium of Human Identification, Innsbruck, Austria. 131 – 137.
Van Oorschot RAH, Jones MK. 1997. DNA fingerprints from fingerprints. Nature.
387:767.
Wickenheiser R. 2002. Trace DNA: a review, discussion of theory, and application of
the transfer of trace quantities of DNA through skin contact. J Forensic Sci.
47(3):442 – 450.
Wilson C. 2006. Improvised explosive devices in Iraq and Afghanistan:
Effects and countermeasures. CRS Report for Congress. Available at
http://www.usis.it/pdf/other/RS22330.pdf
Wilson C. 2007. Improvised explosive devices in Iraq and Afghanistan:
Effects and countermeasures. CRS Report for Congress. Available at
http://assets.opencrs.com/rpts/RS22330_20071121.pdf

71