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RECOVERY AND QUANTIFICATION OF NUCLEAR AND
MITOCHONDRIAL DNA FROM IMPROVISED EXPLOSIVE
DEVICES USING COMPARATIVE SOAKING AND
SWABBING TECHNIQUES

presented by

KAMILA MARYAM GOMEZ

has been accepted towards fulfillment
of the requirements for the

MS. degree in CRIMINAL JUSTICE

 

 

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RECOVERY AND QUANTIFICATION OF NUCLEAR AND MITOCHONDRIAL
DNA FROM IMPROVISED EXPLOSIVE DEVICES USING COMPARATIVE
SOAKING AND SWABBING TECHNIQUES

By

Kamila Maryam Gomez

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTERS OF SCIENCE
Criminal Justice

2009

ABSTRACT

RECOVERY AND QUANTIFICATION OF NUCLEAR AND MITOCHONDRIAL
DNA FROM IMPROVISED EXPLOSIVE DEVICES USING COMPARATIVE
SOAKING AND SWABBING TECHNIQUES

By
Kamila Maryam Gomez

The use of improvised explosive devices (IEDs) in terror-related attacks has
become increasingly common worldwide, in large part because they are easily assembled
and disguised. Fingerprint and impression mark analysis are generally unsuccessful for
identifying manufacturers of IEDs, thus DNA has been examined as a new approach for
apprehending perpetrators. Previous researchers have used a double swab technique to
recover DNA from deflagrated pipe-bombs, which proved to be time consuming due to
the small bomb fragments. The purpose of this study was to compare the traditional
double swab technique and a novel soaking technique to ascertain which produced higher
quantity and quality DNA. Thirty-six volunteers handled either steel or PVC pipe nipples
and end caps for about 30 seconds. The pieces were assembled into pipe bombs and
deflagrated. Bomb fragments were either swabbed, or soaked at room temperature for 2
hours or overnight at 56 °C to recover the DNA. Subsequent quantitative real-time PCR
and STR analyses showed that DNA was recoverable from both types of pipe using both
techniques, although higher yields and better sample quality were obtained using the
swabbing technique. Full profiles were recovered from the DNA of swabbed fragments,
while soaked fragments generated partial or no profiles. The swabbing technique,

therefore, could be optimized for laboratory use in cases pertaining to IEDs.

ACKNOWLEDGEMENTS

A heartfelt thank you is extended to my committee members for taking the time to
invest in my work, for their invaluable input, and for graciously allowing me the extra
time that I needed to get the job done. To my advisor Dr. David F oran I extend a special
thank you, not for only being my advisor and guide, but for ensuring that I was always
well taken care of, whether it be financial aid, a summer job, or enquiring about whether
or not I had a place to stay at the times when family and friends mean the most. As an
international student it meant the world. To First Lt. Shawn Stallworth, my greatest
appreciation is extended to you and the team from the Michigan State Police Bomb
Squad for your dedication and commitment to the research conducted by the Michigan
State University Forensic Biology program, and for ensuring that we were able to
conduct our research safely. Finally, to Dr. Christina DeJong a gracious thank you for
showing enthusiasm for my work, for volunteering to participate and for your academic

assistance.

I would also like to thank my lab-mates Shane Hoffmann and Scott Grammer for
all their assistance toward my project. I extend a thank you, as well, to my other peers in
the lab who have willingly helped whenever I needed them and have kept me in good
spirits. To my C.S.A family, I will always be appreciative to you for making me a home
away from home. Last by not least, my sincerest gratitude is extended to my family and

loved ones, whose unwavering support kept me strong and focused in my times of need.

iii

TABLE OF CONTENTS

LIST OF TABLES ................................................................................... vi
LIST OF FIGURES ................................................................................. vii
INTRODUCTION .................................................................................... 1
Trends and Statistics on Improvised Explosive Devices ........................................ 2
Characteristics of Smokeless Powder ............................................................. 3
Nuclear and Mitochondrial DNA: the Two Human Genomes ................................. 5
DNA Profiles: Powers of Discrimination and Frequencies ..................................... 6
Touch Samples and Trace DNA .................................................................... 7
DNA Isolation from Touched Objects ............................................................. 9
DNA Amplification and Quantification .......................................................... 10
Previous Studies of DNA Isolation from Pipe Bombs .......................................... 12
Aim of the Current Study ........................................................................... 13
MATERIALS AND METHODS .................................................................. 14
Pipe Bomb Preparation ............................................................................. 14
Pipe Bomb Deflagration ........................................................................... 17
ProcessingPipeFragments... ...19
Optimization of Vivascience Ultrafiltration. Devices .......................................... 20
Pipe Bomb DNA Extraction ....................................................................... 23
Quantification of Pipe Bomb DNA ................................................................ 23
STR Analysis ........................................................................................ 25
Statistical Analysis .................................................................................. 26
RESULTS ............................................................................................ 28
Optimization of Vivascience Ultrafiltration Devices .......................................... 28
Post Deflagration and DNA Isolation Observations ............................................ 28
DNA Electropherograms from Swabbed and Soaked Bomb Fragments ..................... 29
Comparison of Timken et al. (2005) and Quantifiler Assays: Nuclear DNA ............... 40
DNA Quantity Relative to Pipe Material, Pipe Mass Recovered, and Isolation

Technique ............................................................................................. 40
Recovered Nuclear DNA Quantities and Levels of Pipe Fragmentation .................... 42
MtDNA Yields and Pipe Material, Pipe Mass and Isolation Technique ..................... 42
MtDNA Quantities and Levels of Pipe Fragmentation ......................................... 43
Recovered Nuclear DNA and MtDNA Quantities .............................................. 44
STR results for Swabbed and Soaked DNA samples .......................................... 47
DISCUSSION ....................................................................................... 51
CONCLUSION ...................................................................................... 63

iv

APPENDIX A: Nuclear and Mitochondrial Data ............................................... 64

APPENDIX B: STR Data .......................................................................... 74

REFERENCES .................................................... ‘ .................................. 77

LIST OF TABLES

Table 1. Percent nitrogen (N) contained in nitrocellulose versus the amount of heat released. . ....4

Table 2. Pipe Bombs with corresponding carrying container and/ or detonation device. . . . . 16
Table 3. Results from qPCR nuclear, mitochondrial, and STR analysis..................................38
Table 4. Comparison of nuclear DNA quantities in descending order to mtDNA quantities ....... 44
Table 5. Comparison of mtDNA quantities in descending order to nuclear DNA quantities ....... 46
Table A 1. Nuclear DNA and mtDNA quantities for each sample ...................................... 64
Table A2. Average amount of DNA in descending order ................................................ 65
Table A3. Quantity of DNA recovered in descending order by mass ................................. 67

Table A4. Average amount of DNA recovered from each fragmentation category .................. 68
Table A5. Average mtDNA quantity according to pipe material and isolation technique .......... 69
Table A6. Mitochondrial DNA copy number in descending order of mass ........................... 70
Table A7. Average number of copies recovered from each fragmentation category ................ 71

Table A8. MtDNA ranking v.s. corresponding nuclear DNA and Quantifiler ranking .............. 71
Table A9. Statistical differences of isolation techniques using t-tests ................................. 73

vi

LIST OF FIGURES

IMAGES IN THIS THESIS APPEAR IN COLOR

Figure 1. Steel and PVC pipe bombs ............................................................. 14
Figure 2. An assembled device with a two-way radio .......................................... 15
Figure 3. Metal box used to contain deflagrated pipe pieces ................................. 18
Figure 4. Pipe bomb with detonation device and carrying container ........................ 19
Figure 5. Plastic pouch containing PVC fragments for soaking .............................. 20
Figure 6. Amplification of DNA from swabbed steel fragments .............................. 28
Figure 7. Amplification of DNA from soaked steel fragments ............................... 31
Figure 8. Amplification of DNA from swabbed PVC fragments ............................. 32
Figure 9. Amplification of DNA from soaked PVC fragments before dilution ............ 33
Figure 10. Amplification of Quantifiler IPC showing partial inhibition ..................... 34
Figure 11. Amplification of Quantifiler IPC showing full inhibition ........................ 35

Figure 12. Amplification of DNA from soaked PVC fragments after DNA dilution ...... 36

Figure 13. Electropherogram depicting erroneous amplification curves ..................... 37
Figure 14. Electropherograms of STR categories B & C ...................................... 49
Figure 15. Electropherograms of STR categories D & F ...................................... 50

vii

Introduction

Prior to the 1980’s, terror-related incidents in the United States were mainly
perpetrated by domestic groups that were either anti-government, anti-legislation, or
extremists such as the Ku Klux Klan (FBI Terrorism Report 2002 - 2005). These trends
continued through the 19805, however at the time an increase in major terrorist events
against the US. was observed overseas. Between the early 1990’s and the beginning of
the 21St century, there was a change in the nature of terrorist attacks both domestically
and internationally (FBI Terrorist Report 2002 — 2005). Before the early 1990’s, terrorist
acts were more focused on destruction of property, but from 1993 — 2001 the main targets
were people. On February 26, 1993, the first attack on the World Trade Center was
executed by an international terrorist group using a vehicle, ammonium nitrate, and fuel
oil (Burke 2000). Most of the harm was property damage but the attack did send the
message that the United States was not immune to terrorist threats. The second attack, on
September 11, 2001, marked the pinnacle of fright and terror not only for the US, but
the world as a whole. It was the deadliest terrorist attack on US. soil, causing 2,972
deaths and over 12,000 injuries (FBI Terrorist Report 2000 — 2005). Worldwide, terrorist
assaults in the 21st century have become more frequent and elaborate; but while it is
impossible to actually stop the planning and execution of every attack, law enforcement
agencies can devise methods to prevent or reduce the scale of the problem by trying to
identify perpetrators postfacto.

Whether attacks are large- or small-scale, one common similarity is the use of
non-conventional bombs, or improvised explosive devices (IEDs), to cause injury and

destruction. As the name suggests, an [ED is a bomb that is often “homemade” and is

either assembled or detonated by means that differ from standard military munitions. The
bombs can be made from different types of material such as glass, plastic or metal, and
one of the most dangerous aspects of these devices is that they can be disguised or hidden
in ordinary containers like backpacks and mail packages. Using timers and electronic
signals, perpetrators have also exploited electronic technology which has allowed
detonation of IEDs from a distance. This has decreased the likelihood of apprehending

the criminal since the individual does not have to be at the scene for detonation to occur.

Trends and Statistics on Improvised Explosive Devices

According to Burke (2007), explosives account for over 70% of the devices used
in terrorist attacks. Records up until the year 2000 showed that pipe bombs represented
31% of them (Burke 2000). Pipe bombs are common as they are effective, easily
assembled and hidden, and the materials can be bought in any neighborhood hardware
store without raising suspicion. Typically, all that is required is a length of pipe, end caps,
explosive powder, and a fuse or triggering mechanism. Purchase of most of these
materials is not regulated by law and criminals frequently escape detection while
obtaining their supplies (National Research Council 1998).

The FBI Bomb Data Center General Information Bulletin 97-1 (1997) showed
that from 1987 — 1994 there was a steady increase in the number of bombing incidents in
the US. This was followed by a decrease from 1995 -— 1997. Except for a slight increase
the next year (F BI Bomb Data Center General Information Bulletin 98-1), Alcohol,
Tobacco, Firearms and Explosives (ATF) data demonstrated a continuation in the

downward trend from 2000 — 2003 (ATF Explosive Incidents Reports 2000 — 2003).

Summarizing the reports from the FBI and ATF, bombing was the leading domestic
terrorist event from 1980 — 2005 (FBI report 2002 —— 2005). Civilians or private property
were the main targets for the majority of these years, and the devices were usually hidden
in mailboxes. Black or smokeless powders were used in most pipe bomb instances, with
metal pipes being the primary container. The reports also revealed that in most cases the

events occurred either at dusk or dawn, and the main perpetrators were young adults.

Characteristics of Smokeless Powder

Smokeless powders have a variety of functions, thus they are manufactured in an
assortment of shapes and sizes to achieve optimal performance (NRC 1998). Physical
appearances range from disks and flattened balls to squares and tubes. The powders are
usually blended with stabilizers and glazed with graphite to increase the shelf life and
decrease sensitivity to friction, static electricity, and heat. Approximately 10 million
pounds of smokeless powder are sold annually in the US, of which about 70% is sold to
manufacturers of ammunition, with the remainder sold to private citizens (NRC 1998).

Smokeless powder is available in three categories: single-base, double-base, and
triple-base. The energetic material found in single-base powder is nitrocellulose.
Combining this with nitroglycerin produces the energetic material for double-base
powders. Triple-base powders include a mixture of these and nitroguanidine. The powder
becomes more potent as the base number increases, making triple-base powders the most
dangerous and the least available to the general public. Single-based smokeless powders,
therefore, are most commonly used in terrorist activity, because they are the least

regulated and most available (NRC 1998). Nitrocellulose also has non-explosive uses,

and is found in lacquers, varnishes, and some printing products. Cellulose itself is a
polymer made up of units of the sugar glucose, and nitrocellulose is produced by
replacing up to three hydroxyl groups with a nitrogen atom, into each sugar monomer.
The concentration of nitrogen in cellulose trinitrate is about 14.15% (Y inon and Zitrin
1993). The nitrogen content in single-base smokeless powders ranges between 12.6% and
13.3% (NRC 1998). The percentage determines the powder’s sensitivity (ease with which
it can be ignited), and as it increases so does the sensitivity, potency, and the heat
released during the explosion.

The amount of energy liberated as heat when the powder burns is termed the heat
of combustion. When a hydrocarbon (in this case, sugar) combusts, carbon dioxide,
water, and heat are produced as it reacts with oxygen. Because the hydroxyl groups in the
sugar have been replaced with nitrogen, burning is not as efficient and therefore the
amount of heat decreases as the percentage of nitrogen atoms increases (Table 1). The
reverse is seen however, when an explosion takes place, as the percentage of nitrogen
dictates the sensitivity and potency of the reaction. An explosion occurs as a result of the
rapid generation and release of pressure and heat; rapidity being the key element. The

more rapid the reaction, the faster the pressure builds, and thus the more heat released.

Table 1: Percent nitrogen (N) contained in nitrocellulose versus the amount of heat

 

 

 

 

 

 

 

released
N content (%)
12.60 13.15 14.0
Heat of combustion (kcal/kg) 2414 2345 2237
Heat of explosion (kcal/kg) 934.5 1016 1140

 

 

 

Adapted from: (Yinon and Zitrin 1993). Kcal represents kilocalories; kg
represents kilograms.

The energy of the gas is dispersed via a shock wave that travels from the blast
center to the surrounding space. Shock waves occur for all intensities of explosions, and
create an overpressure that may be strong enough to inflict damage; the greater the
overpressure the more damage that can ensue (Burke 2000). An overpressure between 0.5
and 1.0 pounds per square inch (psi) has enough force to shatter windows or knock down
an individual; 5 psi is capable of rupturing the eardrum, and 15 psi causes lung damage.
When the overpressure reaches 35 psi or more, the threshold for fatalities has been
reached. A fifty percent rate of fatalities has been seen for overpressures at 50 psi and a
95% rate at 65 psi.

Low explosives such as smokeless powder change their physical state (solid to
gas) at a slow rate which is called deflagration. The material burns through thermal
conductivity, where the hot layer heats and ignites the cooler layer adjacent to it. The
pressure of an explosion as a result of deflagration, however, can reach up to 50,000 psi
(Burke 2000). High explosives such as trinitrotoluene (TNT), change states almost
immediately, and are said to detonate. The burning is very rapid, propagates via shock

waves, and is able to inflict high amounts of damage.

Nuclear and Mitochondrial DNA: the T wo Human Genomes

DNA has proven to be a powerful tool for identifying both perpetrators and
victims as a result of terrorist activity. DNA is found in two places in the cell: the nucleus
and the mitochondria. The human nuclear genome has approximately 3 billion base pairs
(bp) (National Human Genome Research Institute), and is inherited from both parents.

Nuclear DNA provides high powers of genetic discrimination among individuals and can

be sourced from any cell that contains a nucleus. The most robust genetic'results for
identification are usually obtained when nuclear DNA is of high quality and quantity. A
limitation to the usefulness of nuclear DNA arises when DNA is degraded or is present in
very low amounts.

Mitochondrial DNA (mtDNA) is found in the mitochondrion, a small energy-
producing organelle within the cell. MtDNA is circular, smaller in size than the nuclear
genome with only about 16,569 bp, maternally inherited, and may number in the
thousands of copies per cell (reviewed by Holland and Parsons, 1999). Because of the
matn'lineal inheritance, the genome offers a lower power of discrimination among
individuals as all maternal relatives carry the same mitochondrial genomic sequence.
However, if a mtDNA reference sample is not available for a suspect, any maternal
relative can be used to obtain the profile. The same can be done when identifyingvvictims
of mass disasters or war. Because of its high copy number and extra layer of protection
within the mitochondrion (F oran 2006), mtDNA can provide robust results when nuclear

DNA is degraded.

DNA Profiles: Powers of Discrimination and Frequencies

Current human forensic identification is based on the examination of short tandem
repeats (STRs): regions of nuclear DNA that are made up of variable lengths of a
repeated DNA sequence usually 2 to 6 bp in length (Butler 2005). The number of repeats
at a particular locus varies among individuals. A combination of sizes, or alleles, from
different STR loci is used to produce an STR profile. Thirteen such loci have been chosen

by the FBI as the standard, and commercial kits such as the PowerPlex® 16 System by

Promega and Identifiler® by Applied Biosystems (AB1) are currently used by forensic
laboratories in the US. to carry out these analyses.

Instances occur, however, when a DNA sample cannot be assayed due to
degradation. One solution to this problem has been the development of miniSTRs. These
are smaller in size than the traditional 13 STRs; a feat accomplished by including less of
the DNA sequences that flank the repeat region. The smaller size of the target DNA
region (amplicon) increases the chance that amplification is successful, even if
degradation exists. However, while miniSTRs may be advantageous for analyzing
degraded samples, a major disadvantage is that fewer loci can be analyzed at one time
which reduces the power of discrimination.

MtDNA variation is mainly observed within the two hyper-variable regions——
HVI and HV2, and is uncovered by sequencing the DNA base by base. Polymorphisms
within a profile are compared to a database to determine the profile frequency by dividing
the number of times the profile appears by the total number of profiles in the database.
The power of discrimination is not as strong as that of nuclear STRs for two reasons:
matrilineal relatives share the same mtDNA profile and the number of profiles in the

database is small.

Touch Samples and Trace DNA

Objects that have been touched can provide a small amount of retrievable DNA
(trace DNA) from cells that have been left behind. Epithelial cells have proven to be a
convenient source of DNA as approximately 400,000 of them are shed daily by humans

(Raven and Johnson 1986). Herber and Herold (1998) showed that DNA could be

extracted and typed from human dandruff with a single particle producing an average of
4.7 ng of DNA. Van Oorschot and Jones (1997) generated STR profiles from swabs taken
from the palm of the hands and objects that were touched, and retrieved 2 — 150 ng of
DNA, with the lower amounts being recovered from hands that were dry or previously
washed. Also examined were objects that were touched by multiple individuals, which
showed that the DNA of the last person to touch an object was usually present, and that
of the other handlers was present in varying amounts. It was interesting to note that the
strongest profile did not always belong to the last individual that handled the object,
demonstrating how diverse epithelial shedding can be.

Alessandrini et al. (2003) examined DNA retrieval and genetic profiling from
fingerprints. They focused on washed and unwashed fingers pressed onto various
substrates (wood, glass, and metal) for 30 seconds. Quantification results showed that the
amount of DNA recovered varied both among donors and within multiple trials of the
same donor; indicating that a donor deemed to be a good epithelial shedder in one
experiment did not necessarily maintain that status in another. More DNA was recovered
from unwashed hands, but unlike Van Oorschot and Jones (1997) the average quantity
ranged from less than 100 pg to slightly greater than 300 pg.

Balogh et al. (2003) focused on the relationship between the length of time a
substrate was touched and the ability to recover, sequence, and type DNA. Handling time
had no effect on the quality of STR profiles obtained, as a substrate that was touched for
2 seconds yielded results as good as a substrate touched for 60 seconds. Sequencing of

mtDNA was possible for all the samples analyzed.

Secondary transfer—the passing of epithelial cells belonging to another person to
an object—has been a concern in trace DNA analysis since it is not known to what extent
extraneous DNA may be recovered. A study by Ladd et al. (1999) indicated that
secondary transfer may be negligible, as a full secondary profile was not detectable in
most of the analyses. Lowe et al. (2002) showed that secondary transfer can occur under
optimum laboratory conditions and a controlled environment, but suggested that the
results may not be reproducible when circumstances are not as meticulous. Full
secondary profiles were only obtainable in a few cases, but for the most part, mixed
profiles were generated. Proff et al. (2006) demonstrated that secondary transfer was
possible when using a non-human vector (latent fingerprint brush), that was used at
different crime scenes. While transfer did occur, it was only seen when a large surface
area was dusted with a brush that was used at several crime scenes. The results from
these studies indicate that although secondary transfer is a possibility, its effects may be

negligible when dealing with touch samples.

DNA Isolation from Touched Objects

Biological materials for DNA testing are often collected by rubbing a swab across
the surface of evidence. The double swab technique, developed by Sweet et al. (1997), is
a standard procedure used in the field of forensics to retrieve cells. The technique entails
using a wet swab to both hydrate and pick up cells, then a dry swab to soak up any
remaining material. The rehydration effect aids in loosening dried materials deposited
onto the surface, so that they are easier to pick up when the dry swab is applied. Pang and

Cheung (2007) conducted an experiment to ascertain which swab in the technique

produced a higher quantity of DNA, since both swabs were capable of retrieving the
biological material. It was shown that both swabs were equally effective in cell recovery.
STR analysis proved that profiles were obtainable from both wet and dry swabs, though
there were instances where a profile was obtained from the dry swab while none was

generated from the corresponding wet swab.

DNA Amplification and Quantification

Quantifying DNA has become a critical step in forensic analysis as STR analysis
requires specific amounts of starting material for optimal performance. Quantification
techniques have become more technologically advanced over the years, allowing for
quick, efficient testing with more accurate results. A critical step in DNA quantification
is the use of the polymerase chain reaction (PCR) to amplify a specific target sequence.
The amplification process entails numerous cycles of DNA replication, using a
polymerase to generate the new strands of DNA. As the amplicon is replicated, it is used
as the template in the following cycle, thus doubling the amount of DNA. The quantity of
DNA continues to grow exponentially until resources are depleted. A reaction that
initially started with a few copies of DNA produces billions of copies upon completion.

Quantitative (real-time) PCR (qPCR) has made the PCR technique more
convenient as it simultaneously amplifies and quantifies the target DNA in real time.
DNA levels are monitored after each cycle, and the efficiency of amplification is
calculated using a computer program. The premise of the method is the same as regular
PCR, except that a dye-labeled oligonucleotide—a short piece of DNA complimentary to

the target sequence, or probe—is used for detection of double-stranded DNA. If a target

10

sequence is successfully replicated, there is increased fluorescence from the reporter dye
on the probe, thus greater fluorescence signifies more DNA amplification (ABI Protocol
2005). The ability to quantify more than one sample in the same reaction (multiplexing)
through the use of different probes is another advantage of the qPCR technique.
Commercial kits for quantifying nuclear DNA include the QuantifilerTM Human DNA
Quantification Kit, produced by ABI. The Quantifiler assay is sensitive enough to be
used for degraded and low copy number (LCN) DNA, and contains an internal positive
control (IPC) template and oligonucleotide that amplifies along with the sample DNA.
The control aids in identifying problems that may hinder sample analysis, such as
inhibitors.

Independent research laboratories have also developed quantification assays.
Timken et a1. (2005) quantified nuclear and mtDNA in a duplex reaction. The nuclear
target DNA was THOI , one of the 13 standard STR loci, with a sequence ranging
approximately 170 — 180 bp. The locus was chosen because the amplicon size was mid-
range among the target sequences of the 13 loci, and would have given a more accurate
quantification value for larger amplicons. The mitochondrial target was a 69 base pair
region of the NADH dehydrogenase subunit 1 (N D1) gene. The quantification method
worked well with both high quality and degraded samples, the latter of which was
compared to QuantifilerTM. Results showed that the Quantifiler assay, which targets a 62
base pair region, detected significantly more DNA than the Timken et a1. (2005) assay.
Subsequent STR analysis on the degraded samples revealed that Quantifiler
overestimated the quantity of DNA for larger amplicons, as a large portion of the longer

templates failed to amplify.

ll

Previous Studies of DNA Isolation from Pipe Bombs

New approaches are being used to pinpoint suspects by recovering and analyzing
DNA that may have been left on pieces of evidence. Studies focusing on recovering DNA
from deflagrated pipe bombs and their carrying containers have been carried out at
Michigan State University. Esslinger et al. (2004) explored obtaining DNA profiles from
exploded pipe bombs after having volunteers handle the pipes. One full profile was
recovered, while 4 of 20 bombs gave partial results that were consistent with the donor’s
known profile. Alleles from an additional 8 bombs were obtained, but were not called
because they were below the fluorescence threshold of the study. Obtaining a successful
profile correlated with the concentration of DNA recovered, the level of pipe
fragmentation, and the number of bomb pieces collected. The type of pipe material, PVC
or steel, was not a critical factor in obtaining a profile. Gehring (2004) focused on
sequencing mtDNA from deflagrated pipe bombs, and was able to correctly assign 18 of
-38 bombs to a single donor and 7 to a subset of donors. Twelve of the bombs were not
assignable and 1 was incorrectly assigned. Unlike the Esslinger et a1. (2004) study, the
Gehring (2004) research was conducted blind. Although better success was achieved by
Gehring, trends similar to Esslinger (2004) were observed where lower amounts of DNA
were recovered as the amount of pipe fragmentation increased. Kremer (2008) combined
miniSTR analysis of nuclear DNA with mtDNA sequencing to identify bomb handlers,
which proved to be even more informative. Results showed that more than half of the
bombs could be assigned to a single donor if both genomes were used. In a subsequent
study, Hoffmann (2008) focused on recovering DNA (and ultimately miniSTRs) from

IED carrying containers and correctly assigned all eight backpacks to their donors, again

12

under blind conditions. The key to the success was independently testing different areas

of a backpack and building a consensus DNA profile.

Aim of the Current Study

While swabbing has been successfully used to recover DNA from pipe bomb
fragments, small bomb pieces have proven to be very tedious and time-consuming to
process. It begs the question, therefore, of whether there is an easier and more efficient
way to recover DNA from bomb pieces. Thus, a novel soaking technique was developed
in an attempt to improve the speed with which DNA can be retrieved and the amount that
can be recovered. The goal was to compare the double swab technique and the soaking
technique, examining the quantity and quality of the recovered DNA. Two hypotheses
were tested: (1.) the more pieces of pipe collected, the more DNA would be recovered;
and (2.) the soaking technique would recover more DNA than the swabbing technique
because the pieces of pipe would be submerged in a buffer and more pieces would be
used for analysis. Pipe bombs were handled by volunteers, deflagrated, and collected and
analyzed. The pipe fragments for the soaking technique were immersed into a cell-lysing
buffer that covered their entire surface area. The recovered DNA from each technique

was quantified and STR analysis was performed.

13

Materials and Methods

Pipe Bomb Preparation

F cot-long pipe nipples, one inch in diameter, along with matching end caps were
purchased at a local hardware store; half of the pipes were PVC and half galvanized steel.
Two end caps were allocated to each pipe and a ‘/4 inch hole was drilled into the center of
one of them. The pipes and caps were sterilized by soaking in a 10% bleach solution for
one hour, followed by rinsing with deionized water and UV irradiation on flip sides for 5
minutes each (approximately 7.5 J/cmz). The materials were wiped with ELIMINase® as
per the manufacturer’s instructions, and rinsed a second time with deionized water. PVC
caps without the drilled holes were glued onto the PVC pipes using PVC cement (Figure

1). Each pipe was stored in a separate brown paper bag until deflagration.

Figure 1: Steel and PVC pipe bombs

 

Steel and PVC pipe bomb nipples with attached end caps.
Also shown is a length of fuse, used to ignite the bombs.

Pipe bombs, their corresponding detonation devices (Figure 2), and carrying
containers (either a backpack or cardboard box), the latter two of which were part of a
separate study, were assigned a number. The detonation device was made up of a cellular
phone or two-way radio, a circuit board, a battery, a clamp for holding the pipe bomb, a
piece of wire, Velcro, and a piece of wood, that were cleaned of extraneous DNA. The
carrying containers were autoclaved for an hour, followed by exposure to UV radiation
for 5 minutes on flip sides. Ten pipes were deflagrated as is, ten were assigned to
backpacks, and eighteen were assigned to both a carrying container and detonation device
(Table 2). Volunteers were asked to draw a number and a letter prior to handling the
components of a specific pipe, container, and device and the numbers were recorded out
of view of the analysts to conduct bomb assignation blindly. Approval for the use of
human subjects was granted by the Michigan State University Committee on Research

Involving Human Subjects.

Figure 2: An assembled detonation devicg with a two-way radio

 

Components of a detonation device including a two-way radio (as opposed to a
cellular phone), metal clamp, battery, wire, circuit board, and piece of wood. Each
component was attached to the piece of wood using Velcro, so that they could be
easily removed, handled and replaced by the volunteer. The pipe bomb was fitted
into the clamp just prior to deflagration.

15

Bombs, detonation devices, and cardboard boxes were handled for approximately
30 seconds, while backpacks were used by volunteers for one week. The pipe bomb
designated as the positive control was handled for about a minute once a day for three
days, while the negative control underwent no handling. Following handling, the bombs
were stored in separate brown paper bags until deflagration. A control buccal swab was
collected from each volunteer and protective gear (gloves, masks, sleeves and lab coats)

were worn by the analysts while dealing with the various components.

Table 2: Pipe bombs with corresponding carm’ng container and/ or detonation device

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Pipe # Pipe material Container Detonation device

3 Steel Backpack -

4 Steel Backpack -

5 Steel Backpack -

6 Steel Backpack -

7 Steel Backpack -

8 PVC Backpack -

9 PVC Backpack -

10 PVC Backpack -

11 (pos) PVC Backpack -

12 (neg) PVC Backpack -
l3 (neg) Steel Cardboard box Yes
14 Steel Backpack Yes
15 Steel Cardboard box Yes
16 Steel Backpack Yes
17 Steel Cardboard box Yes
1 8 Steel Backpack Yes
19 Steel Cardboard box Yes
20 Steel Backpack Yes
21 Steel Cardboard box Yes
22 PVC Backpack Yes
23 PVC Cardboard box Yes
24 PVC Backpack Yes
25 PVC Cardboard box Yes
26 PVC Backpack Yes
27 PVC Cardboard box Yes
28 PVC Backpack Yes
29 PVC Cardboard box Yes
30 PVC Backpack Yes

31 Steel - -

 

 

 

 

 

 

16

Table 2 continued
32 Steel
33 Steel
34 Steel
35 Steel

36 PVC

37 PVC

38 PVC

39 PVC

40 PVC
The number of each pipe bomb, the material it was made from, along with its
corresponding carrying container and detonation device is shown. The symbol (-) denotes
that a particular component was not designated. Pipe bomb 11 was the positive control
and pipe bombs 12 and 13 were negative controls.

 

Pipe Bomb Deflagration

Twenty-eight bombs were initially deflagrated at a local fire fighter training
facility, with the aid of the Michigan State Police bomb squad. An additional ten bombs
were deflagrated in the same manner at a later time. The process took place in a
windowless, brick room with a metal door as the only access. Inside was a ventilated

metal box into which the bombs were placed (Figure 3).

l7

Figure 3: Metal box used to contain deflagrated pipe pieces

 

The metal box was supplied by the Michigan State Police bomb squad. Its
purpose was to reduce the scatter of the pipe fragments upon deflagration,
so that they could be easily collected. Ventilation holes were cut into the
sides and lid so that the pressure from the blast could be released.

The bombs were filled with 1.5 oz of Green Dot Smokeless Shotshell Powder.
Drilled PVC end caps were glued to the pipe and the steel end caps were screwed on.
Bombs were deflagrated using an approximate 45 second fuse, which was inserted into
the hole in the end cap, clipped on to its detonation device using the clamp, and placed
into its corresponding container (Figure 4). Bombs that were not assigned a carrying
container or detonation device were placed on the bottom of the metal box. Once
deflagrated, the pieces of pipe, the container, and the components of the detonation
device were collected and placed into their respective paper bags. The base of the metal
box was either swept or vacuumed between deflagrations to remove remaining fragments,
and the floor of the room was swept to prevent cross contamination. Fragments found

after the collection and clean-up of a bomb were discarded. Gloves, masks, and sleeves

were worn throughout the process by the bomb squad personnel and the analysts.

Fi ure 4: Pi e bomb with detonation device and car ' container

 

 

Fuses were inserted into the pipes through the end cap holes and the bombs were
clipped onto the detonation devices using the metal clamps (A). The bombs and

detonation devices were placed into the carrying containers and put into the metal
box to be deflagrated (B).

Processing Pipe Fragments
Buffer, plastic pouches, microcentrifuge tubes, and swabs were UV irradiated for

10 minutes prior to processing the pipe bomb fragments. The soaking technique was used
on PVC pipe bombs 8 — 12 and 22 — 30, and small steel fiagrnents from bombs 31 — 35.
Fragments were soaked in a plastic pouch, constructed by heat-sealing the top and bottom
of a of Seal-A-Meal® bag (Figure 5). Twenty mL of digestion buffer along with 25 uL of
proteinase K (20 mg/mL) were added and the bags were sealed. Pouches were agitated on
a shaker for 2 hours and incubated. Soaked steel bomb fragments 3 — 21 were incubated

overnight at 56°C while steel bomb fragments 31 — 35 were not.

Figure 5: Plastic pouch containing PVC fragments for soaking

 

The PVC fragments of pipe bomb #10 being prepared for soaking. The
plastic pouch was constructed by heat-sealing the top and bottom of the
bag (arrows). The sides were pre-sealed by the manufacturer.

Large steel bomb fragments and PVC fragments from pipe bombs 36 — 40 were
swabbed using the double swab technique (Sweet et a1. 1997). One hundred and fifty uL
of digestion buffer was used to moisten the first swab, which was rubbed across the
galvanized covered surfaces of the steel fragments and the outer surfaces of the PVC
fragments, in a circular motion. A dry swab then followed and both were placed into a
1.5 mL microcentrifuge tube along with 500 uL of digestion buffer and 4 uL of
proteinase K. The tubes were vortexed for 10 seconds and incubated overnight at 56°C.
Reagent blanks were also initiated. Following DNA isolation, the mass of the bomb

fragments was recorded.

Optimization of Vivascience Ultrafiltration Devices

Vivascience Vivaspin ultrafiltration devices were optimized for DNA use by

testing filters that retained 5,000, 10,000, 30,000, and 100,000 molecular weight (MW)

20

material. Microcentrifuge tubes, 50 mL polypropylene centrifuge tubes, plastic caps, and
buffer were UV irradiated for 10 minutes. The ultrafiltration devices were UV irradiated
for 20 minutes, with the filters being detached and flipped over after 10 minutes.
Volunteers handled 3 plastic caps for about 1 minute, and placed the caps into a 50 mL
polypropylene centrifuge tube. To the tube was added 20 mL of buffer along with 25 uL
of proteinase K. The solution was incubated overnight at 56°C. Buffer solutions were
transferred to each of the devices and centrifuged at 4000 x g for 20 minutes, or until the
liquid was concentrated to 500 11L.

Organic extractions were performed on the concentrated buffer solutions as well
as 500 11L of flow-through from each device. Five hundred uL of phenol was added to
the solution, vortexed, and centrifuged at 14,000 rpm for 5 minutes. The top aqueous
layer was placed into a new 1.5 mL tube, to which an equal volume of chloroform was
added. The mixture was vortexed and centrifuged at 14,000 rpm for 5 minutes. The
aqueous layer was transferred to a Microcon YM-30 spin column. One hundred
microliters of 10mM Tris/ lmM EDTA (TE) was added and the column was centrifuged
at 14,000 x g for 10 minutes. Three washes were subsequently performed by adding 300
11L of TE to the column and centrifuging at 14,000 x g for 10 minutes. The DNA was
eluted in 20 uL of TE after inverting the column into a new 1.5 mL tube, and centrifuging
at 1000 x g for 4 minutes.

MtDNA PCR analysis was conducted on all the extracts. The reaction mix
included the addition of 1 uL of: mitochondrial primers F15 and R285 (20 uM); 10X
PCR reaction buffer (USB); magnesium chloride (25 mM, USB); dNTPs (20 uM);

bovine serum albumin (BSA) (0.2 mg/ 11L); 1 unit HotStart-It® Taq DNA polymerase

21

(5u/ uL, USB); 2 11L of DNA sample; and 1.8 uL deionized water for a total of 10 uL.
Cycling parameters included denaturing at 94°C for 30 seconds, annealing at 55°C for 1
minute, and extension at 72°C for 1 minute for 38 cycles, followed by a final extension at
72°C for 5 minutes. PCR products were separated via electrophoresis on a 2% agarose gel
and subsequently prepped for mtDNA sequencing.

Recovered DNA was transferred to a Montage column, and rinsed three times
with 300 uL of deionized water, at 1000 x g for 15 minutes. The DNA was resuspended
in 30 uL of deionized water and reactions of 4 uL of Quick Start mix (Beckman Coulter),
l 11L of primer, and 5 uL of DNA (50 — 100 M) were prepared. Sequencing parameters
included denaturing at 96°C for 20 seconds, annealing at 50°C for 20 seconds, and
extension at 60°C for 4 minutes for 30 cycles. DNA was prepared for electrophoresis by
adding 2.5 11L of stop solution, which included 5 pL of 3 M NaOAC, 5 uL of 100 M
EDTA, and 2.5 11L of glycogen (Beckman Coulter). The DNA was precipitated through
addition of 30 uL of 95% ethanol and centrifuging at 14,000 x rpm for 15 minutes,
followed by two rinses in 180 pL of 70% ethanol, at 14,000 x rpm for 5 minutes. The
ethanol was removed, the DNA pellet was vacuum-dried for about 20 minutes, and
resuspended in 40 uL of Sample Loading Solution (Beckman Coulter). One drop of
mineral oil was added to each well. Sequencing was performed on a Beckman Coulter
CEQTM 8000 Genetic Analysis System. The program used was LFR-l and was carried
out at 50°C. Samples were denatured at 90°C for 120 seconds, and injected for 15 seconds
at 20 kV. Bomb and reference sequences were analyzed using BioEdit sequence

alignment software (Ibis Biosciences).

22

Pipe Bomb DNA Extraction

Centrifuge tubes and Microcon columns were UV irradiated for 10 minutes and
the Vivaspin 10,000 MW ultrafiltration devices were UV irradiated for 20 minutes with
the filters being detached and inverted after 10 minutes. The buffer from the soaked pipe
fragments was transferred to a 50 mL polypropylene tube and centrifuged at 4000 x g for
15 minutes, to pellet any debris. The liquid was transferred to the Vivaspin device and
centrifuged at 4000 x g for 20 minutes or until the liquid was concentrated to 500 11L.
The retentate was pipetted into a 1.5 mL microcentrifuge tube. Swabs were transferred to
a spin basket and centrifuged at 14,000 revolutions per minute (rpm) for 5 minutes and
the liquids combined into one 1.5 mL microcentrifuge tube. Organic extraction, as
outlined above, was performed on each buffer solution, as well as 500 uL of flow-
through from four randomly selected pipe bombs. The procedure was followed by
mtDNA PCR analysis on the pipe bomb DNA and flow-through extracts, using the

abovementioned protocol.

Quantification of Pipe Bomb DNA

F AM and HEX dyes supplied by Bio-Rad, and VIC dye purchased from Applied
Biosystems, were calibrated as per the iQTMS manufacturer’s instructions. DNA from the
pipe bombs was first quantified using a TaqMan assay (Timken et al. 2005). Reactions
were prepared in either duplicate or triplicate. Singleplex reactions included 7.5 11L of iQ
Supermix; 0.64 uL of primers and probe (Timken et al. (2005) final concentrations
maintained); 0.64 uL of BSA (0.2 ug/ uL); 2 uL of undiluted DNA or 2 — 3 uL of diluted

DNA, and deionized water to 15 uL. Reactions were analyzed on a Bio-Rad iQTMS

23

Multicolor Real-Time Detection System. This included a polymerase activation step of
95°C for 10 minutes, a denaturing step at 95°C for 15 seconds, and a dual annealing and
extension step at 60°C for 1 minute (Timken et al. (2005)). The latter two steps were
carried out for 45 cycles. Ten-fold serial dilutions of single-source male DNA ranging
from 20 ng/ pL to 0.02 ng/ 11L were prepared as the standards for the nuclear assay, and
10 ng/ uL to 0.01 ng/ uL of Promega genomic standard DNA (K562) were made for the
mtDNA assay. Initial mtDNA reactions were carried out utilizing a probe that lacked a
minor groove binder (MGB), which was critical for successful amplification. MtDNA
quantification was measured by copy number and was calculated using the estimation of
about 400 copies of mtDNA per 3.3 pg of K562 DNA. Data were analyzed using the Bio-
Rad iQTMS software.

Pipe bomb DNA was also quantified using a QuantifilerTM Human DNA
Quantification Kit, using the same dilutions of unknown DNA described above. Volumes
were reduced from 25 uL to 15 11L and consisted of 7.5 pL of reaction mix, 6.3 uL of
primer mix, and 1.2 uL of DNA. The QuantifilerTM standard was diluted per manual
instructions and ranged from 50 ng/ uL to 0.023 ng/ uL. The same cycling parameters as
the Timken et al. (2005) qPCR assay were used, except for a reduction from 45 to 40
cycles. Inhibition categories were created and were: N — none, P — partial, and F - full.
DNA samples giving a Quantifiler result of zero or showing signs of inhibition were

reanalyzed by preparing fresh dilutions.

24

ST R Analysis

STR analysis was carried out on the first 28 DNA samples (except for bombs 3
and 6) using a MiniFilerTM PCR Amplification Kit. Ten-microliter reactions contained 4
uL of MiniFilerTM Master Mix, 2 uL of MiniFilerTM primer mix, and 4 uL of DNA.
Swabbed bomb DNAs were used undiluted, while 1:20 dilutions were prepared for the
soaked DNA samples. A positive control contained 3 uL of 007 control DNA (Applied
Biosystems) and luL of TE. Thermal cycling parameters included an initial incubation at
95°C for 11 minutes followed by 32 cycles of denaturation at 94°C for 20 seconds,
annealing at 59°C for 2 minutes, and extension at 72°C for 1 minute, followed by a final
extension at 60°C for 45 minutes. Reference (buccal) samples were analyzed using a
PowerPlex® 16 System kit. Buccal DNA was diluted 1:100 and 1 uL of the diluted DNA
along with 3 uL of TE were added to the reaction.

Capillary electrophoresis was performed on an ABI PRISM® 310 Genetic
Analyzer. Reactions included 24.5 uL of formamide, 0.5 11L of GeneScanTM 500 LIZ ®
Size Standard (Applied Biosystems) and 1.5 uL of DNA or allelic ladder. The mixtures
were incubated at 95°C for3 minutes and immediately cooled on ice. One drop of
mineral oil was added to each tube and DNAs were separated using performance
optimized polymer 4 and 1X buffer containing EDTA (Applied Biosystems).
Electrophoresis included a run temperature of 60°C, an injection time of 5 seconds at 15
kV, and a run time of 28 minutes at 15 kV.

GeneMapper® ID software v3.2.1 was used to analyze the data. The MinifilerTM
analysis method was MiniFiler_GSSOO_HID_v1, the panel was MiniFiler_GS500_v1,

and the matrix was DS-33 Matrix 7-12-07. The PowerPlex analysis method was PP16SH,

25

the panel was PowerPlex_16ID3.l.0, the size standard was ILS 600 Advanced, and the
matrix was Matrix-UMS Test. Peaks over 50 relative fluorescent units (RF Us) were
recorded and volunteer profiles were initially called blindly before comparisons were
made to the reference samples. Results were categorized as: A — Alleles from volunteers
only, B — Volunteer (major profile) + additional alleles, C — Volunteer (non—major
profile) + additional alleles, D - Some, but not all alleles from volunteer, E -— No alleles
from volunteer, F — No alleles obtained. Additional symbols after the lettered category
indicated: T = incorrect alleles; - = missing alleles; * = complete allele dropout at 1
locus; ** = complete allele dropout at 2 loci; *** = complete allele dropout at 3 or more

loci.

Statistical Analysis

Nuclear and mtDNA quantities were compared to the type of pipe material
utilized, mass of the pipe fragments, and level of bomb fragmentation. Nuclear DNA
results using the Timken et al. (2005) assay were also compared to those using
Quantifiler, and further comparisons were made between the two DNA isolation
techniques. DNA quantities within 10 pg of one other using the two nuclear DNA
quantification techniques were considered ‘similar’, while those greater than 10 pg were
deemed as ‘higher’ or ‘lower’. Quantification averages were calculated inclusive or
exclusive of negative results (no DNA) generated from either low DNA quantities or
inhibition. Results were included if corresponding positive quantities were low, and
excluded if quantities were high, but showed signs of inhibition. Nuclear or mtDNA

quantities were converted from picograms (pg) to femtograms (fg) or mtDNA copies per

26

gram of pipe, to compare the two DNA isolation techniques based on the quantity of pipe
recovered. Deflagrated bombs were assigned to fragmentation categories which were: 1 —
low, 2 — medium, 3 — high, and 4 — complete.

The Pearson correlation coefficient (r) was calculated to ascertain correlative
relationships between: the Timken et al. (2005) and Quantifiler assays, the nuclear and

mtDNA yields, and the mass of the fragments and the amount of DNA recovered. The
formula was r = 2(x — X)(y — Y)/ \l[2(x — X)2][Z(y — Y)2], where x and y represented

DNA quantities, and X and Y denoted the mean of the quantities. Computation of ‘r’
generated a scale that ranged from -1 to +1, where -1 indicated that the correlation had a
perfect negative relationship, -0.5 was a moderate negative relationship, 0.0 was no
relationship, +0.5 was a moderate positive relationship, and +1 was a perfect positive
relationship.

DNA quantities from the nuclear DNA, mtDNA and Quantifiler assays were
compared using t-tests. DNA concentrations for the swab and soak techniques were
averaged separately, and the standard deviation for each technique was computed and
used to calculate the degrees of freedom. A significance level of 0.05 for a two-tailed t-
test was used together with the degrees of freedom to ascertain the critical values of the t-

test calculation.

27

Results

Optimization of Vivascience Ultrafiltration Devices

Vivaspin devices with larger molecular weight cutoffs concentrated the buffer
solution in a faster time. The Vivaspin 100,000 MW device filtered the solution in about
5 minutes, followed by the 30,000 MW device, which took about 10 minutes, the 10,000
MW device took about 20 minutes, and the 5,000 MW took over an hour. MtDNA PCR
showed that DNA was recovered from the concentrated buffer solution from all the
devices, but it was also retrieved from the flow-through of the 30,000 and 100,000 MW
devices. Subsequent mtDNA sequencing generated the haplotypes of the volunteers.

Therefore the Vivaspin 10,000 MW device was used for the remainder of this study.

Post Bomb Deflagration and DNA Isolation Observations

PVC pipe bombs underwent a higher level of fragmentation upon deflagration,
than did steel pipe bombs. While the entire PVC pipe broke into pieces, most of the
fragmentation of the steel pipes was at end caps and less with the nipple. Therefore, the
steel nipples were swabbed while the caps and other fragments were soaked.

Blackening of the swabs occurred with both types of pipe, while soaking
produced varying amounts of buffer discoloration after incubation. The buffer from
soaked PVC bomb fragments became grayish-brown, and from soaked steel fragments
incubated overnight, a red rust color. Closer inspection of the steel bomb fragments

revealed that a white crystal-like solid covered their surfaces. While oxidation occurred

28

on all the soaked steel fragments, it was more pronounced in the fragments that were
soaked overnight.

Centrifugation of the overnight soak solutions resulted in much larger pellets from
steel fragments than from PVC fragments, and included the white substance as well as
debris. After centrifugation the buffer from the steel fragments soaked overnight lost
most of its color and became almost colorless. The PVC soak buffers, however, had a
brownish hue, which was retained after the concentration step. The organic extraction
was not effective in removing all of the coloration, leaving the eluted DNA from soaked
PVC fragments brown in color. The eluted DNA from both the swabbed and soaked steel

fragments was colorless.

DNA Electropherograms from Swabbed and Soaked Bomb Fragments.

DNA from swabbed steel fragments amplified more frequently (Figure 6) than did
DNA from the soaked fragments (Figure 7), and was achieved without DNA dilution.
Positive DNA quantities were more often attained from the former than the latter,
although neither isolation technique showed signs of inhibition according to the

Quantifiler assay.

29

Figure 6: Amplification of DNA from swabbed steel fragments

 

Amplification Chart

  

WR’Basé-“Line’Su'btracted Curve Fit‘RFU"

 

 

v see
Electropherogram showing the amplification of DNA from swabbed steel fragments.
Relative fluorescence units (RF Us) are displayed on the y-axis using a linear scale. Cycle
number is denoted on the x-axis. The horizontal line (arrow) is the threshold and each
line represents one DNA sample. Nineteen of the 24 samples crossed the threshold
between cycles 32 and 35 and maintained the sigmoidal curve generated from successful
amplification.

30

Figure 7: Amplification of DNA from soaked steel fragments

 

Amplification Chart

  

PCR Base Line SubtractedZCurve it FIFU

 

CYGIB

-
Electropherogram showing the amplification of DNA from soaked steel
fi‘agments. RFUs are displayed on the y-axis using a linear scale, while cycle
number is denoted on the x-axis. The horizontal line (arrow) is the threshold and
each line represents one DNA sample. Only 2 of the 6 samples crossed the
threshold and maintained the sigmoidal curve generated from successful
amplification.

 

 

 

Standard and positive control DNAs amplified and produced expected results
during analysis of PVC DNA samples. DNA amplification was also successful with the
swabbed PVC fragments (Figure 8), and quantification values were obtained without
diluting the DNA. Soaked PVC fragments, however, had lower amplification success
(Figure 9) as amplification curves failed to cross the threshold for at least one of the

replicate reactions (Appendix A1). As a result, repeated trials had to be performed to

31

obtain positive outcomes. Results from the Quantifiler assay showed that 11 of the 12
DNA samples from the soaked PVC fragments were either partially or fully inhibited

(Figures 10 and 11, respectfully).

Fi ure 8: Am lification of DNA from swabbed PVC fra ments

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

+ I Ampiflcdion Chart
25: /
/ ,
a /
“ /
E zoo
co
2
3
o
1: / ..-’
g 150
e ,-”
s y",
:3
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8
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o 50 " /
n. f ,
14.95 , ’— ' ‘
_ _g_ _ , I?” k “
o- w -_
i l
t 10 23 3D 40
C

 

 

 

Electropherogram showing the amplification of DNA from swabbed PVC
fragments. RFUs are displayed on the y-axis using a linear scale, while cycle
number is denoted on the x-axis. The horizontal line (arrow) is the threshold and
each line represents one DNA sample. Eight of the 12 samples crossed the
threshold between cycles 32 and 35, and maintained the sigmoidal curve
generated from successful amplification.

32

Figure 9: Amplification of DNA from soaked PVC fragments before dilution

 

PCR Base Line Subtracted Curve Fit RFU

 

 

@018

 

 

Electropherogram showing the failure of the DNA samples from soaked PVC
fragments to amplify. RF Us ARE displayed on the y-axis using a linear scale, and
cycle number on the x-axis. The horizontal line (arrow) is the threshold.
Amplification of the standard DNA samples and the positive control is seen, with
each line representing one DNA sample. None of the samples crossed the
threshold.

33

 

Amplification Chart

 

10 15 20 25 30 35 40
vcle

depicting partial inhibition, with RF Us displayed on the y-axis using a
linear scale, and cycle number on the x-axis. The darker line represents an uninhibited
DNA sample as the IPC line crosses the threshold at cycle number 25. The lighter line
(arrow) represents a partially inhibited DNA sample as the IPC line crosses at a later
cycle number.

34

Fi ure 11: Am lification of uantifiler IPC showin full inhibition

 

 

 

 

@

 

4500

4000

3000

2500
2000

1 500
1000

3500

 

3500 ‘

:1

 

Amplification Chart

 

 

 

 

 

 

 

 

 

 

 

/

 

/

 

12281

/

 

 

’P-P

 

 

5

 

l0

 

15

 

v vacle

 

25

0 W

0

 

 

 

30 35 40

 

Electropherogram depicting full inhibition, with RF Us displayed on the y-axis using a
linear scale, and cycle number on the x-axis. The darker line represents an uninhibited
DNA sample as the IPC line crosses the threshold at cycle number 25. The horizontal IPC
line parallel to the threshold (arrow) represents a fully inhibited DNA sample as the line

does not cross the threshold.

DNAs fiom soaked PVC fragments amplified after 1:10 or 1:20 DNA dilution

(Figure 12). There were also instances of false positive quantification results, in which

amplification curves crossed the threshold prematurely or amplification was abnormal as

the curves did not maintain the sigmoidal shape (Figure 13). Table 3 displays a summary

of the results from the qPCR nuclear, mtDNA and STR assays.

35

Figpre 12: Amplification of DNA from soaked PVC fragments after DNA dilution

 

Amplification Chart

uwe Fit RFU

PCR Base. Line Subtracte

 

 

 

Electropherogram showing the amplification of DNA from soaked PVC
fragments after dilution. RFUs are displayed on the y-axis using a linear scale,
and cycle number is denoted on the x-axis. The horizontal line (arrow) is the
threshold and each line represents one DNA sample. Two of the triplicate samples
crossed the threshold, and maintained the sigmoidal curve generated from
successful amplification.

36

Fi ure 13: Electro hero ram de ictin erroneous am lification curve

 

E

 

 

 

3 IS

 

.3
m

 

 

A
O)

 

 

.5
&

 

..s
N

 

.5
O

 

 

PCR Base Line Subtracted Curve Fit RFU
a)

 

 

 

 

 

 

 

 

 

 

* 14.95 /7 .- aw

i / 21/ \V

r t’ "

1' fl »'

o i 10 i 20 so 40
CYCIO

Depiction of false positive amplification curves, where the line crossed the

threshold at a very early cycle or was uncharacteristically shaped. One of the lines
crossed the threshold at about cycle 18, whereas the other (arrow) crossed at about
cycle 36 despite being abnormally shaped. RFUs are displayed on the y-axis using
a linear scale and cycle number is denoted on the x-axis. Each line represented
one DNA sample.

37

Table 3: Results from gPCR nuclear, mitochondrial, and STR assays

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Bomb DNA Pipe Recovered 1‘ Level of Average amt. Femtogram 1 Average amt. of Average amt. STR
# Isolation Material Mass (g) fragmentation of DNA (pg) of DNA per DNA based on mtDNA category inhib. -
,_ Tech. gram of pipe 1 Quantifiler (pg) (copies)
3 swab steel 630.72 0.00 0.00 — - -
3 soak steel 242.96 3 0.00 0.00 — - -
4 swab steel 561.92 46.95 83.55 7.19 1240 B i‘
4 soak steel 393.01 4 0.00 0.00 - -
5 swab steel 676.18 7(191) (2.83) 2.44 230 C 1‘-
5 soak steel 21 l .50 2 0.00 0.00 - - —
6 swab steel 493.57 0.00 0.00 - — —
6 soak steel 406.43 3 0.00 0.00 — - -
7 swab steel 650.69 23.48 36.08 15.70 1540 B +-
7 soak steel 236.99 1 0.00 0.00 - — -
8 soak PVC 74.21 4 (2.58) (34.80) 62.4, inhib 5624 B i‘ *
9 soak PVC 93.6 4 (11.91) (127.22) 0, inhib 2699 Di-
10 soak PVC 91.12 4 (7.19) (78.85) 14.20 2461 Di-**
1 1 D—j—iwrok
(p03) soak PVC 69.96 4 (4.21) (60.14) lnhib 490
12
(neg) soak PVC 69.48 4 0.00 0.00 0. inhib 110
13
(neg) swab steel 620.96 (0.98) (1.57) 7.79 829
13 266.72 0.00 0.00 -
(neg) soak steel 1 -
l4 swab steel 768.32 45.95 59.81 36.90 1543 C -
14 soak steel 151.34 2 0.00 0.00 - - —
15 swab steel 625.03 8.50 13.60 19.20 1317 C i"
15 soak steel 274.97 2 0.00 0.00 — - _
16 swab steel 577.15 7.77 13.46 19.60 1433 B -
16 soak steel 267.07 3 0.00 0.00 - — -
17 swab steel 498.02 72.00 144.57 34.9 1657 B *
17 soak steel 475.25 3 0.00 0.00 - - —
l8 swab steel 598.03 2.64 4.41 10.10 1320 B *
18 soak steel 289.65 1 0.00 0.00 - - —
l9 swab steel 400.37 (6.75) (16.86) 0, inhib 3180 B ”r
19 soak steel 487.89 4 0.00 0.00 - - -
20 swab steel 680.79 29.08 42.72 29.20 9297 C *
20 soak steel 206.80 3 0.00 0.00 — — -
21 swab steel 572.22 4 (2.50) (4.37) 10.1 782 B ’i‘
21 soak steel 306.05 0.00 0.00 - — —
22 soak PVC 101.83 4 32.20 316.21 34.2 1022 D ***
23 soak PVC 70.03 4 (3.61) (51.48) 0, inhib 469 E °***
24 soak PVC 76.15 4 (5.53) (72.55) 0, inhib 966 D -*

 

 

 

 

 

 

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A.\
from

Gael

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

- Table 3 continued

. 25 soak PVC 94.04 4 20.35 216.40 41.1 1956 D t-*** P

g 20 soak PVC 83.65 4 5.73 68.50 0.00 1128 D 1* p

3 27 soak PVC 86.81 4 67.33 775.60 69.4 4145 F P

M 21 soak PVC 113.1 4 (0.38 (3.36) 0.00 4533 E1 *** N

29 soak PVC 53.36 4 (4.15) (77.81) 0.00 434 D7. P

7730 soak PVC 121.68 4 0.00 0.00 0. inhib 864 D’t- ** P

:_ 31 swab steel 598.9 0.00 0.00 0.00 8 - N

‘ 31 soak steel 218.25 2 (7.69) (35.22) 0.00 943 - N

32 swab steel 700.84 3.00 0.00 “ 0.00 1799 — N

32 soak steel 186.84 1 0.00 0.00 18.45 8 — N

33 swab steel 694.18 12.12 17.46 70.65 1394 - N

33 soak steel 193.5 1 (1.27) (6.56) 0.00 19 - N

34 swab steel 635.3 16.25 25.58 0.00 751 — N

34 soak steel 252.31 2 (1.35) (5.34) 0.00 21 - N

35 swab steel 650.11 7.66 11.78 85.30 3204 - N

35 soak steel 237.57 1 0.85 3.58 0.00 16 - N
73.82 -

36 swab PVC 46.71 4 47.10 1008.35 6.55 2085 N
83.10 -

37 swab PVC 54.87 4 53.79 980.32 161.50 5398 N
84.57 -

38 swab PVC 57.63 4 (45.28) (785.70) 0.00 2097 N
74.56 -

39 swab PVC 39.69 4 41.29 1040.31 103.5 3050 N
58.34 -

40 swab PVC 52.57 4 38.27 727.98 0.00 1718 N

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The lSt column denotes pipe bomb numbers; bomb 1 1 was the positive control (pos), and bombs 12 and 13 were the negative controls (neg). The 2nd and 3rd columns
display the DNA isolation technique used and the type ot‘pipe bomb material respectively. The 4th column shows the recovered mass of each pipe bomb post deflagration.
Two masses are displayed for pipe bombs 36 — 40: the mass ofall the pieces collected. and the mass of the swabbed pieces only (bold). Bomb fragmentation levels are

as noted: 1 4 low, 2 — medium, 3 — high, and 4 4 complete. The 6th column displays the average amount of DNA recovered from each pipe bomb in picograms (pg) using
the Timken et a1. (2005) assay. Values in parentheses signify the average quantity of DNA inclusive ofzero values (the Ct value was not reached). The 7th column displays
the amount of DNA in femtograms (fg) per gram of pipe. Column 8 gives the quantity ofnuclear DNA in picograms (pg) based on the Quantifiler assay and column 9
shows the quantity ofmtDNA in copy numbers. The symbol (-) indicates that no analysis was conducted. The 10th column shows STR results based on categories:

A — Alleles from volunteers only, B 4 Volunteer (major profile) + additional alleles, C 4 Volunteer (non—major profile) + additional alleles, D — Some, but not all alleles
from volunteer, E 4 No alleles from volunteer, F 4 No alleles obtained. The additional symbols after the lettered category indicate: 1 Z incorrect alleles; ° : missing alleles;
* = complete allele dropout at l locus; ** = complete allele dropout at 2 loci; *** = complete allele dropout at 3 or more loci. Column 1 1 shows the level ofinhibition for
each sample, based on the results of the Quantifiler IPC. Inhibition categories were: N — none, P — partial and F 4 full.

39

 

Comparison of T imken et al. (2005) and Quantifiler Assays: Nuclear DNA

The Timken et al. (2005) and Quantifiler assays had a correlation value of 0.48,
indicating that there was a moderate positive relationship. Seven of the DNA samples
from the Timken et a1. (2005) assay generated values higher (greater than 10 pg) than
Quantifiler, with quantities for samples 4, 36, 38 and 40 giving more than 30 pg greater
than the latter assay. The Quantifiler assay produced values that were higher than the
Timken et al. (2005) assay for eight of the DNA samples, three of which (35 swab, 37
and 39) were more than 60 pg greater than those of Timken et al. (2005). The remaining
23 DNAs were similar in concentration (less than 10 pg difference) between the assays.
No amplification was observed for 19 of the samples using the Quantifiler assay, 4 of
which (12, 30, 31 swab and 32 swab) produced the same results using the Timken et al.
(2005) assay. There were five instances where the Quantifiler assay produced a positive
value for one of its replicates but no amplification in another (8, 10, 27, 32 soak and 33

swab).

DNA Quantity Relative to Pipe Material, Pipe Mass Recovered, and Isolation Technique
DNA yields based on the type of pipe material and its recovered mass are
displayed in Table 3. DNA amounts in descending order are 'shown in Appendix A2 and
A3 respectively. Fifteen of the 18 steel bombs (83.3%) generated DNA, while no DNA
was recovered from bombs 3, 6 and 32 (16.7%). Assessing the steel bombs according to
isolation technique, 14 of the 18 swabbed bombs (77.8%) and 4 of the 18 soaked bombs
(22.2%) yielded DNA. Further breakdown of the soaked steel fragments show that 0 of

the 13 pipe bombs that were soaked overnight produced DNA, while 4 of the 5 bombs

40

incubated at room temperature for 2 hours generated positive results. Quantification
values were obtained for 16 of the 17 PVC bombs (94.1%). DNA was recovered from all
5 swabbed PVC bombs (100%) and 11 of the 12 soaked bombs (91.7%).

Higher, but not statistically different quantities of nuclear DNA per gram of pipe
were retrieved from swabbed steel fragments versus the soaked fragments (bombs 33 —
35). The mass of bomb recovered and DNA yields showed a modest positive correlation
of 0.29 using the Timken et al. (2005) assay, and 0.36 using Quantifiler. However, bombs
with larger masses did not necessarily generate the highest DNA quantities. As an
example, bomb 32 had a mass of 700.84 g but no DNA was recovered based on either
quantification assay, while bomb 16 had a lower mass of 577.15 g but generated 7.77 pg/
uL of DNA using Timken et al. (2005) and 19.6 pg/ 1.1L using Quantifiler. DNA
quantities from soaked steel fragments were similar to each other, all being less than 10
pg/ 11L.

Swabbed PVC fragments (36 — 40) yielded higher, but not statistically different
quantities of nuclear DNA per gram of pipe than soaked PVC fragments. Recovered
bomb masses for the swabbed fragments were among the lowest analyzed, ranging from
39.69 4 57.63 g. Masses of soaked PVC fragments, ranged from 53.36 4 121.68 g. More
DNA was generated from the higher fragment masses among soaked PVC samples,
although their DNA quantities were still less than that recovered from swabbed samples.
The mass of PVC bomb recovered and DNA yields showed a moderate negative

relationship of -0.47 using the Timken et al. (2005) assay and -0.31 using Quantifiler.

41

Recovered Nuclear DNA Quantities and Levels of Pipe Fragmentation

Fragmentation levels from 6 steel bombs fell into the low category, 5 into
medium, 3 into high, and 3 into complete fragmentation. Table 3 shows the level of pipe
fragmentation versus the quantity of DNA retrieved for each steel pipe. The average
quantity of DNA for each fragmentation category is recorded in Appendix A4. Using
DNA quantities from the Timken et al. (2005) assay, the greatest amount was recovered
from bombs in the high fragmentation category, followed by bombs in the complete
category. Bombs in the medium fragmentation category produced the third highest DNA
yields, while those in the low category generated the lowest amount of DNA.

The Quantifiler assay showed that bombs in the high fragmentation category
produced the most DNA, followed by those in the low fragmentation category (Appendix
A4). Bombs in the medium category yielded the third highest quantity of DNA, while

those that underwent complete fragmentation generated the lowest amount of DNA.

MtDNA Yields and Pipe Material, Pipe Mass and Isolation Technique

Steel fragments 3 — 7 and 13 — 21, incubated overnight, were not analyzed for
mtDNA quantity as no DNA was obtained for either the preliminary mtDNA PCR
analysis or the Timken et al. (2005) nuclear quantification assay. Apart from those
bombs, pipe bomb material did not appear to influence mtDNA retrieval as DNA was
generated for all other pipes (Table 3). Average mtDNA quantities, in descending order
of mass, and separated by pipe material and isolation technique, are shown in Appendix
A5. The correlation between the mass of pipe recovered and mtDNA yield was -0.04 for

PVC pipes and 0.12 for steel pipes. Swabbed PVC mtDNA yields ranged from 1718 —

42

5394 copies, while soaked quantities ranged from 110 4 5624 copies. Swabbed steel
fragments also tended to generate higher quantities of DNA (8 4 9297 copies) than did
the soaked fragments (8 4 943 copies; Appendix A6). Pipe bomb 32 yielded a positive
mtDNA result despite not producing any nuclear DNA in either the Timken et a1 (2005)
or Quantifiler assays.

Disparities among mtDNA triplicate yields were small (Appendix A1), but
instances occurred where two quantities were similar to each other while the third was
much higher or lower. Examples include pipe bomb 7 which generated 2170, 2036 and
413 copies, and pipe bomb 9 that produced 1600, 2291 and 4206 copies. A few of the
pipes produced a positive result for only two of the three replicates, while generating no
DNA for the third. These included bomb 24, which yielded 927 and 1004 copies, and
bomb 38 that produced 2109 and 2085 copies, with no DNA being obtained for the third
triplicate in both instances. This trend was not more prominent with either PVC or steel

pipes, nor for a particular isolation technique.

MtDNA Quantities and Levels of Pipe Fragmentation

Appendix A7 shows the average quantity of mtDNA recovered per gram of pipe
for each pipe fragmentation category. Pipes in the high fragmentation category generated
the most DNA, followed by complete pipe fragmentation, low pipe fragmentation, and
medium fragmentation. The order of the mtDNA categories were the same as the Timken
et a1. (2005) nuclear assay, except that the last two categories were reversed. Only the

high fragmentation category was the same as Quantifiler, which had the low

43

fragmentation category with the second highest yields, followed by medium

fiagmentation, and lastly complete fragmentation.

Recovered Nuclear DNA and MtDNA Quantities

Table 4 displays the rank of nuclear and mtDNA quantities, with nuclear DNA in
descending order, while Table 5 shows the rank of the DNAs in descending order of
mtDNA quantity. The correlation value between the Timken et al. (2005) assay and the
mtDNA assay was 0.33, while the value between the Quantifiler and mtDNA assays was
0.52, showing a moderate positive relationship in both instances. Overall rankings are

seen in Appendix A8, with samples producing a quantification value of zero not ranked.

Table 4: Comparison of nuclear DNA Quantities in descending order to mtDNA Quantities

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Average amt. of Average amt.
Isolation Pipe nuclear DNA of mtDNA
Rank Pipe # Technique Material (pg) (copy #)

l 1 7 swab steel 72.00 1657
2 27 soak PVC 67.33 4145
3 37 swab PVC 53.79 5398
4 36 swab PVC 47.10 2085
5 4 swab steel 46.95 1240
6 l4 swab steel 45 .95 1 543
7 38 swab PVC 45.28 2097
8 39 swab PVC 41.29 3050
9 40 swab PVC 38.27 1718
10 22 soak PVC 32.20 1022
1 1 20 swab steel 29.08 9297
12 7 swab steel 23.48 1540
13 25 soak PVC 20.35 1956
14 34 swab steel 16.25 751

15 33 swab steel 12.12 1394
16 9 soak PVC 1 1.91 2699
17 15 swab steel 8.50 1317
18 16 swab steel 7.77 1433
19 3 1 soak steel 7.69 943

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 4 continued
20 35 swab steel 7.66 3204
21 10 soak PVC 7. 1 9 2461
22 19 swab steel 6.75 3 180
23 26 soak PVC 5.73 1 128
24 24 soak PVC 5.53 966
25 l 1 soak PVC 4.21 490
26 29 soak PVC 4.15 434
27 23 soak PVC 3 .61 469
28 18 swab steel 2.64 1320
29 8 soak PVC 2.58 5624
30 21 swab steel 2.50 782
31 5 swab steel 1.91 230
32 34 soak steel 1 .35 21
33 33 soak steel 1 .27 19
34 13 swab steel 0.98 829
35 35 soak steel 0. 85 16
36 28 soak PVC 0.38 4533
37 12 soak PVC 0.00 1 10
38 30 soak PVC 0.00 864
39 3 1 swab steel 0.00 8
40 32 swab steel 0.00 1799
41 32 soak steel 0.00 8

 

 

 

 

 

 

 

Column 1 shows the rank of the nuclear DNA quantities in descending order. Pipe bomb
number is reported in column 2, while columns 3 and 4 display the isolation technique
and the type of pipe material, respectively. Average amount of nuclear DNA in
picograms (pg) is shown in column 5, while column 6 displays the average quantity of
mtDNA in copy number.

Pipes that generated high quantities of nuclear DNA did not necessarily produce
high mtDNA quantities. Pipe bomb 22 ranked 10th using the Timken et a1. (2005) nuclear
assay and 9th using Quantifiler, but 25th on the mtDNA scale. Interestingly, pipe bomb 8

provided one of the lowest quantities of nuclear DNA using Timken et al. (2005) assay

(29th), ranked 5th in nuclear DNA using the Quantifiler assay and yielded the 2nd highest

quantity of mtDNA (Appendix A8).

45

Ranking DNA quantities based on mtDNA yield, pipe bomb 20 produced the

most mtDNA but was 11th in the amount of nuclear DNA using the Timken et al. (2005)
th . . . th

assay (Table 4), and 10 usmg the Quantifiler assay. Pipe bomb 19 ranked 7 on the

mtDNA scale, but 22nd and 26th on the Timken et al. (2005), and Quantifiler assays

respectively. The largest disparity was seen with bomb 28, which ranked 4th on the

mtDNA scale, but 36th using the Timken et al. (2005) nuclear assay and produced no

results using Quantifiler despite there being no sign of inhibition.

Table 5: Comparison of mtDNA guantities in descending order to nuclear DNA Quantities

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Pipe Isolation Pipe Average amt. of Average amt. of
Rank # Technique Material mtDNA (copy #) nuclear DNA (ng

1 20 swab steel 9297 29.08
2 8 soak PVC 5624 2.58
3 37 swab PVC 5398 53.79
4 28 soak PVC 4533 0.38
5 27 soak PVC 4145 67.33
6 35 swab steel 3204 7.66
7 19 swab steel 3 180 6.75
8 39 swab PVC 3050 41 .29
9 9 soak PVC 2699 1 l .91
10 10 soak PVC 2461 7.19
l l 38 swab PVC 2097 45.28
12 36 swab PVC 2085 47 . 10
13 25 soak PVC 1956 20.35
14 32 swab steel 1799 0.00
15 40 swab PVC 1718 38.27
16 1 7 swab steel 1657 72.00
17 14 swab steel 1 543 45 .95
18 7 swab steel 1540 23 .48
19 16 swab steel 1433 7.77
20 33 swab steel 1394 12. 12
21 18 swab steel 1320 2.64
22 15 swab steel 13 17 8.50

 

 

 

 

 

 

46

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 5 continued
23 4 swab steel 1240 46.95
24 26 soak 1 PVC 1128 5.73
25 22 soak PVC 1022 32.20
26 24 soak PVC 966 5.53
27 3 1 soak steel 943 7.69
28 30 soak PVC 864 0.00
29 13 swab steel 829 0.98
30 21 swab steel 782 2.50
31 34 swab steel 751 16.25
32 l l soak PVC 490 4.21
33 23 soak . PVC 469 3.61
34 29 soak PVC 434 4.15
35 5 swab steel 230 l .91
36 12 soak PVC 1 10 0.00
37 34 soak steel 21 1.35
38 33 soak steel 19 l .27
39 35 soak steel 16 0.85
40 3 1 swab steel 8 0.00
41 32 soak steel 8 0.00

 

 

 

 

 

 

 

Column 1 shows the rank of the mtDNA quantity in decreasing order. Pipe bomb number
is reported in column 2, while columns 3 and 4 display the isolation technique and the
type of pipe material, respectively. Average amount of mtDNA in copy number is shown
in column 5. Column 6 displays the average quantity of nuclear DNA in picograms (pg).
S TR Results for Swabbed and Soaked DNA Samples

Total input DNA for STR analysis based on the Timken et al. (2005) assay were:
20 samples had less than 100 pg of DNA, 4 had 100 4 200 pg, and 2 had 200 4 300 pg of
DNA; based on Quantifiler: 19 samples had less than 100 pg of DNA, 5 had 100 4 200
pg, and 2 had 200 4 300 pg of DNA. STR results based on the number of alleles observed
are shown in Table 3 and complete results are in Appendix B. Figures 14 and 15 show
examples of electropherograms for STR categories B and C, and D and F respectively.
Seven of the 11 swabbed steel pipes fell into category B, wherein although there were

additional alleles, the volunteer constituted the major profile. Four of those 7 swabbed

pipes had incorrect alleles at one locus, 2 had at least one missing allele, and 2 had

47

complete allele drop-out at the FGA locus. The remaining 4 of the 11 swabbed pipes fell
into category ‘C’ where the volunteer alleles were recovered along with additional
alleles, but were not the major profile. Two of the 4 pipes produced incorrect alleles at
one locus, 3 of the pipes had at least one missing allele, and 1 pipe had total allele drop-
out at the FGA locus.

DNA from the soaked PVC pipe fragments fell into categories D — F, with only
one sample falling into category ‘B’ (8 soak). Eight pipes were categorized as ‘D’ (some
of the volunteer’s alleles obtained), 2 pipes as ‘E’ (no volunteer alleles obtained), and 1
as ‘F’ (no alleles obtained). The positive control (11) was categorized as ‘D’, with at least
one locus with incorrect alleles and complete allelic drop-out at three or more loci. STR
results compared to the level of inhibition showed that samples that generated the most
allelic drop-out were soaked PVC fragments, which tended to be inhibited. One of the
negative controls (12) showed sporadic contamination as only one allele was obtained,
while the other (13) produced alleles at every locus, 7 of which were consistent with one
of the analysts. Alleles were recovered at three loci for sample 30, although no

quantification results were obtained for the nuclear DNA assays.

48

Fi re 14: Electro hero rams of STR cat ories B and C

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1353171
:90 100 2 120 130
2800 '
2000 I;
1200
400
0 7 212
OL 11 13
62 1104 460
101.38 115.37 12.3.71
12
890
119.53
fi013s317l _43
90 100 ‘110 '120 l
6000 . g
4000
2000
0 0L 9 11 13
93 4259 2729 1414
98.61 107.03 115.36 123.50
()1.
1107
10133

Electropherograms showing STR categories B and C (volunteer (major profile) + other
alleles and volunteer (non-major profile) + other alleles) respectively. The number of
RFUs is displayed on the y-axis, while the size of the alleles is denoted on the x-axis. The
numbers within the boxes reflects the number of STR repeats, the number of RFUs, and
the length of the repeat (in bp), respectively. Vertical columns refer to grouped ‘bins’ into
which the alleles fall. Category B depicts the volunteer alleles (11, 12) as the major peaks
among additional alleles, and category C shows the volunteer alleles (13, 13) as the minor

 

 

 

peak among additional alleles.

49

Figure 15: Electropherograms of STR categories D and F

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

m .
150 160 170 180
600 l 8 l ‘ .l 4‘ I __
0 A
D 12
267
173.26
190 110 120 13.0
SON-h 1 i '
50W"
000--
3000.-
ZOWH F
1W0
.1
O

 

 

Electropherograms showing STR categories D and F respectively. The number of RFUs is
displayed on the y-axis, while the size of the alleles is denoted on the x-axis. The numbers within
the blue boxes reflects the number of STR repeats, the number of RFUs, and the length of the
repeat (in bp), respectively. Vertical columns refer to grouped ‘bins’ into which the alleles fall.
Category D depicts an instance where only some of the volunteer alleles (11, 12) are present, and
category F shows no alleles were generated.

50

Discussion

The purpose of the research presented here was to compare two techniques used
to recover DNA from exploded pipe bombs. Previous DNA-based studies of IEDs
incorporated the double swab technique (Sweet et al. 1997) to lift shed epithelial cells left
on the device by the assembler. However, the post-blast fragments are very small and
difficult to swab, especially in cases that involve PVC pipes. Thus, a soaking technique
was developed as a means of retrieving cells that remained on the small fragments, which
typically would not be processed by swabbing.

Various advantages were offered by each of the isolation techniques, a mutual one
being that they were both relatively easy. A major benefit of the swabbing technique was
that fewer reagents—including buffer, proteinase K, and other laboratory supplies—were
used. A minimal amount of buffer was applied to the swab before it was rubbed over the
pipe surface and less than 1 mL was used to digest the retrieved cells. The small amount
of moisture also limited the generation of rust from the steel bomb fiagments. Another
advantage was that the swab was applied directly to the surface of the fragments, and the
pressure likely aided in loosening and lifting any cells adhering to the surface. A third
benefit was that fewer steps were performed between swabbing the fragments and DNA
extraction, which decreased the chances of DNA loss.

One disadvantage of the swabbing technique was that it was tedious and time-
consuming. Each fragment needed to be carefully swabbed to ensure that the entire

surface was covered. Fragments from a steel bomb took about 20 minutes to swab,

51

allowing about 3 bombs to be processed in an hour. Swabbed PVC bombs took even
longer as there were many small fragments to be processed.

The soaking technique allowed more bombs to be set up for analysis in a shorter
period of time. The time taken to prepare the fragments and pouches for several bombs
was short, as was adding the buffer and sealing the pouches for incubation. A second
advantage was that more fragments per bomb were analyzed since the very small pieces,
not normally swabbed, could be processed in the pouch.

However, the soaking technique required large quantities of reagents. Further,
when the fragments were being shaken or incubated in the plastic pouches, the buffer
solution settled to the bottom of the pouch, leaving some of the top fragments uncovered.
Unless the pouch was frequently flipped over, some of the cells may not effectively be
lysed. Also, it was not possible to remove all of the buffer from the pouch and pipe
pieces, and generally only about 15 of the 20 mL was recovered. DNA was undoubtedly
left in the residual liquid or adhered to the bomb fragments. Greater DNA retrieval may
have been possible if a different device was used to soak the fragments. Instead of the
flexible pouch, a sturdier container that can be centrifuged may be advantageous. Not
only would the liquid be drawn to the bottom, but it would reduce the number of times
that the buffer was transferred during its cleanup. This, in turn, could reduce the amount
of liquid, and ultimately DNA, that is lost. Centrifugation would pellet the debris that
accumulated in the buffer as it was washed off the fragments. While centrifugation can be
a benefit, the downside is that DNA may be trapped and pelleted as well.

Owing to the large volume of buffer used in the soaking technique, the solution

had to be concentrated before extraction. The ultrafiltration devices used were intended

52

for protein concentration, thus needed to be tested on DNA. Different size filters that
retained proteins up to a specific molecular weight were tested before choosing the one
used in the experiment. The Vivaspin 10,000 MW device was selected because it retained
DNA while allowing for relatively fast filtering. MtDNA PCR analysis confirmed that no
DNA was entering the flow-through, as no PCR product was obtained for flow-through
samples. However, another problem was encountered during the concentration step, when
debris that did not pellet during centrifugation became concentrated along with the DNA.
As a result, the debris was eluted with the DNA in the final step of the organic extraction.
Subsequent DNA analysis of these samples proved to be challenging due to inhibition
and amplification failure.

A final drawback of the soaking technique occurred during the processing of the
steel fragments. The soak allowed for rust formation, seen in greater amounts for the
fragments that were left overnight. It became apparent that overnight incubation had an
adverse effect on the steel fragments and the final buffer quality, as a white substance,
not seen on fragments incubated at room temperature for two hours, formed on the
pieces. The substance was crystal or salt-like. It could have been generated from one or
more of the reagents in the buffer, such as the TRIS, EDTA, or SDS as it reacted with the
metal, presumably through oxidation. It is not known to what extent the substance
affected the DNA, although no PCR product was obtained for any of those DNA samples
and there were no signs of inhibition. The DNA could have been destroyed, or trapped
and pelleted with the debris. The latter is quite possible since the formerly rust-filled
solution became almost colorless after centrifugation. The rust in buffer solutions from

steel fragments incubated at room temperature was removed during Microcon

53

centrifugation, as the final extract yielded clean DNA that, according to the Quantifiler
assay, showed no signs of inhibition.

Correlation between the two nuclear quantification assays was particularly
important as it gave an indication of the extent to which the two assays were in sync. A
moderate positive relationship of +0.48, signified that there were differences in the
quantification results from each assay. The deviances between the assays might have
arisen due to pipetting error from stochastic sampling effects (the random chance that the
target DNA is sampled and amplified stemming from the small quantities of DNA
recovered), or from the number of times each DNA sample had to be assayed to obtain
positive results. This influenced quantification results as the chance of obtaining a
positive outcome increased as the number of trials increased. Samples assayed using
Timken et al. (2005) were run repeatedly until positive results were obtained, whereas
those assayed using Quantifiler were run once in duplicate, decreasing the number of
positive results obtained. Repeat runs sometimes required diluting the DNA 1:10 or 1:20,
which exacerbated stochastic effects. No positive quantification values were obtained
once the dilution was greater than 1:20.

A moderate positive correlation between the two nuclear assays begged the
question as to which was more accurate at quantifying the DNA. Subsequent correlations
to the mtDNA assay and the mass of the recovered pipe fragments indicated that
Quantifiler was more accurate, in spite of there being no amplification for almost half of
the DNAs assayed. This signified that the zero values were real and could not be
discounted. While the stochastic effects and small DNA quantities influenced

quantification results, the accuracy of Quantifiler could also be attributed to components

54

found in its reagents. The buffer from the Quantifiler kit was optimized for real-time
quantification (Quantifiler Kit User Manual), and it is possible that proprietary reagents
within the buffer and primer mix are more effective for quantification than are the
reagents found in the Bio-Rad buffer used for the Timken et al. (2005) assay, resulting in
more accurate Quantifiler results.

The only counterintuitive result seen between DNA quantity and recovered pipe
amounts was for swabbed PVC bombs, where DNA yields increased with decreasing
amounts of recovered pipe. This is contrast to the soaked samples, generated from greater
amounts of retrieved pipe overall, which demonstrated a positive relationship between
DNA quantity and pipe obtained, although DNA yields were still less than those
recovered from the swabbed fragments. Interestingly, high quantities of DNA were
consistently generated by the 5 swabbed bombs based on the Timken et al. (2005) assay,
while only three of those same bombs amplified using Quantifiler. Two of the bombs
analyzed using the latter assay (bombs 37 and 39), one of which (39) had the smallest
swabbed mass, had DNA quantities in excess of 100 pg/ 11L, values that were higher than
any other recovered DNA quantity for either assay. Bombs 37 and 39 also had two of the
highest levels of DNA recovered based on the Timken et al. (2005) assay as well, thus the
results seem real, and it appears these bombs, in spite of their high fragmentation,
actually contained large amounts of DNA, with bomb 39 causing the negative correlation
between DNA and bomb mass yields.

STR analysis aided in ascertaining the quality of DNA recovered from the pipe
fragments. MiniFiler required a total of 500 4 750 pg of input DNA for optimum

performance; hence, none of the samples were optimal. Good results, however, were

55

obtained from the swabbed pipe fragments, indicating that a lack of PCR inhibition was
important to successful STR analysis. For example, volunteer alleles were retrieved from
swabbed pipe bomb 5, which was analyzed using 3.61 pg of DNA based on Timken et al.
(2005) and 7.64 pg based on Quantifiler. Conversely, no alleles were recovered for
soaked pipe bomb 27 which was analyzed using 269.32 pg and 277.60 pg in the same
assays respectively. Pipe bomb 5 showed no sign of inhibition based on the Quantifiler
assay, while pipe bomb 27 was partially inhibited. Thus, adding 4 11L of DNA for STR
analysis versus 1.2 uL for quantification likely contributed to the failure of obtaining
alleles for bomb 27. The cleanness of the DNA sample, therefore, made a difference in
the generation of STR results. Also of note was bomb 8 which was the only soaked bomb
to be classified in category B (volunteer alleles along with additional alleles were
obtained, with the volunteer alleles being the major profile). This DNA showed partial
inhibition, where 10.32 pg of DNA was added based on the Timken et al. (2005) assay,
while 249.60 pg was added based on Quantifiler. In this instance, even though the DNA
sample contained inhibitors, the quantity of DNA added (based on Quantifiler values)
aided in producing volunteer alleles. These results help confirm that Quantifiler was the
better assay for nuclear DNA quantification.

Complete allele drop-out generally occurred at the largest locus (F GA) for
swabbed fragments, while it was more variable from the soaked fragments. Larger loci
tend to drop-out first when DNA is degraded (Whitaker et a1. 1995), which would explain
the lack of amplification success of the FGA locus from the swabbed fragments.
Although this would have also occurred for soaked fragments, there was the additional

issue of diluting those DNAs to reduce PCR inhibition, and hence an increase in

56

stochastic effects. It is therefore understandable why a higher level of allelic drop-out was
experienced with the DNA samples from the soaked fragments.

Stutter, an artifact of STR analysis, occurs as a result of slippage by the
polymerase, making the target DNA one repeat unit shorter than the true allele. Most of
the additional alleles at a locus were one repeat unit shorter than the true allele, and were
observed at various loci for each sample. It was difficult to ascertain whether those alleles
were real or were the result of stutter. Usually stutter products are about 5 4 10% of the
actual allele’s peak height. However, under LCN conditions stutter products are enhanced
and their peak heights can be greater than the 10% (Whitaker et al. 2001). Examining the
peak height intensities, therefore, did little to identify real alleles because in some
instances the intensity of an additional peak and that of an actual peak were almost the
same. Low intensity peaks were especially challenging to categorize as they could have
been called in various ways: they may have been stutter, a real allele from a volunteer, or
an allele from a minor contributor. All the scenarios demonstrate the difficulties that arise
when analyzing LCN samples. These could potentially be eliminated by either testing
different portions of the bomb to build a consensus profile from repeated peaks, or
comparing the unknown alleles to known profiles to ascertain whether a minor profile
existed.

Allele drop-in was also common in the STR results, occurring at about half the
loci for swabbed samples, and at all the loci for soaked samples. Drop-in is sporadic, and
is usually not reproducible when samples are retested. It was difficult to establish,
however, whether an allele was drop-in, a result of contamination, or the result of stutter,

as the DNAs were only tested once. Previous studies in the laboratory demonstrated the

57

benefits of repeated testing in generating a consensus profile (Hoffmann 2008), in which
only alleles that appeared more than once were called, and the chances of identifying all
correct alleles increased. A few of the alleles that were established to be drop-in after
making comparisons to the reference samples had higher peak intensities than those from
the volunteer, and were falsely deemed as the major allele. Although it was possible that
some of the drop-in was a result of contamination from the analysts, identifying an allele
as drop-in would have been easier had repeat testing been done.

The purity of the DNA recovered from each type of pipe was determined using
the Quantifiler IPC. DNA from swabbed steel and PVC fragments showed no signs of
inhibition and amplification curves were generated without difficulty. Interestingly,
samples from soaked steel fragments also showed no signs of inhibition, despite there
being rust and the white substance prior to extraction. It was likely that the higher
temperature of 56 °C for the overnight soaks sped up the rate of chemical reactions taking
place between the buffer and the steel, causing the formation of rust and the white salt.
Pelletting of the white substance and subsequent use of the Microcon columns, removed
the debris and rust from the final elute. In contrast, the reduced soak time of 2 hours and
incubation at room temperature, which retarded the rate of chemical reactions, allowed
for successful DNA retrieval and amplification of soaked steel samples. Low quantities
or absence of DNA from the soaked steel fragments could be attributed to degradation of
the nuclear DNA by Mg2+-dependent exonucleases, even though EDTA was added to the
buffer to chelate the Mg2+ ions and inactivate such enzymes. A high concentration of Fe2+
stemming from the steel pipes may have overwhelmed the EDTA, rendering it

ineffective. Two hour incubations, along with increasing the concentration of EDTA,

58

could reduce the chance of saturating the buffer with divalent cations, and allow the
EDTA to effectively chelate the Mg2+ions. Future soaks of steel fragments should be
done using various time intervals, to determine the shortest time possible for optimum
DNA recovery.

MtDNA was less challenging to amplify as results were always obtained after one
quantification attempt. Amplification success was likely due to dilution of the inhibited
DNA, which reduced the concentration of the inhibitors and increased polymerase
activity, coupled with factors such as higher copy number and better survivability of
mtDNA (F oran 2006). Three of the bombs generated mtDNA quantities for only two of
three triplicate samples, with the DNA fi'om one of those showing partial inhibition while
the others were uninhibited. Inhibitors from that single bomb may be responsible for the
amplification failure of its third triplicate, whereas the failure of the uninhibited DNA
from the other two bombs could be due to stochastic effects, or inhibitors introduced into
the sample from the PCR tube or pipette tip.

Amplicon size also likely influenced the successful amplification of mtDNA. The
mtDNA target of 69 bp was smaller than that of the Timken et al. (2005) nuclear target of
170 4 180 bp. Timken et a1. (2005) examined the amplification success of degraded DNA
as it related to amplicon size. High quality DNA was degraded to various extents and
analyzed using Quantifiler, which amplifies a 62 bp region, and the Timken et al. (2005)
assay. Subsequently, larger STR loci failed to amplify, indicating that while the two
assays were effective in quantifying amplicons of their respective sizes, they both
overestimated the quantities for longer amplicons when the DNA was degraded.

Quantifiler had a higher overestimation as its amplicon was shorter and more likely to be

59

intact and in greater amounts when the DNA was degraded. Thus, due to high copy
number, high quality and the small size of the mtDNA amplicon, amplification was more
successful.

Esslinger et al. (2004) and Gehring (2004) both found that as the level of pipe
fragmentation decreased, the amount of recovered DNA increased. This trend was not
seen in the current study as the low fragmentation category collectively produced the
least nuclear DNA based on Timken et al. (2005) quantification results, and the second
lowest quantity based on mtDNA. This fragmentation category, however, had the second
highest DNA yield based on the Quantifiler assay, although most of this could be
attributed to bomb 35 which generated a very high value of 131.21 fg per gram of pipe.
The same bomb only generated 15.36 fg per gram of pipe for the Timken et al. (2005)
assay. While the Quanitfiler value seemed to be an outlier, mtDNA quantities for bomb
35 was one of the highest obtained, thus more closely agreeing with the Quantifiler
nuclear quantity. It is likely, therefore, that the Quantifiler value was not an outlier, but
another confirmation that the assay was more reliable.

Low bomb fragmentation meant that a large portion of pipe nipple survived, along
with several small end cap pieces, providing a large surface area for processing. The
nipple and end caps were coated with zinc, which, if removed, would result in the loss of
cells. Zinc removal, however, was minimal when fragmentation was low, becoming more
frequent as the level of fragmentation increased. It is not certain, therefore, why pipes
with the most surface area did not generate the highest DNA quantities. It is possible that
large fragments retained the heat from the blast for a longer time and as a result, there

was further DNA degradation. In spite of this, cells containing quality DNA were still

60

recovered as all of the DNA from low fragmentation bombs fell into STR category B—
the volunteer alleles constituted the major profile. This indicated that, although yields
were low, the DNA was of good quality, and potentially represented a caliber of DNA
typical of large pipe fragments. Another explanation for low quantities of DNA from
category 1 fragments is linked to the amount of cells shed by a volunteer. There were
instances in the high and complete fragmentation categories, including bombs 37 and 39
detailed above, where certain pipes generated DNA quantities that were much higher than
the others, perhaps owing to a ‘good shedder’. This included steel bomb 17 in the high
fragmentation category which had a quantification value of 144.57 fg per gram of pipe,
while the other two high fragmentation steel bombs only generated 42.72, and 13.46 fg
per gram, and steel bomb 4 which had the highest value in the complete fragmentation
category with 83.55 fg per gram of pipe, versus 16.86 and 4.37 fg per gram for the other
two complete fragmentation steel bombs. Low DNA yields for the large category 1 bomb
fragments may have also resulted from few cells being deposited or cells being lefi
behind during swabbing. In this regard, there were times when the nipple became dry
during the application of the wet swab, which could result in cells re-adhering to the
surface of the pipe and not being effectively lifted by the dry swab.

Overall, two hypotheses were proposed for this study: 1) more recovered pipe
would generate more DNA, and 2) the soaking technique would recover more DNA than
the swabbing technique because it allowed all sized fragments to be utilized. The first
hypothesis was not necessarily supported, particularly when analyzing PVC pipes, as the
amount of DNA recovered depended less on the amount of pipe recovered than it did on

the DNA isolation technique used. Swabbed PVC fragments produced more DNA than

61

soaked PVC fragments, despite having the least amount of recovered pipe available. It
could be that there is a limit to the size of a fragment from which DNA can be recovered,
regardless of the technique. Smaller pipe pieces may be generated from hotter pipes,
which cause more cells to be either degraded by heat or removed by the impact of the
blast. If this is the case, even though more pieces are incorporated into the analysis during
the soaking technique, many would not contribute DNA.

The hypothesis that the soaking technique would recover more DNA than the
swabbing technique was not supported either, as more DNA was recovered from
swabbed fragments. It was thought that the soaking technique would have been more
successful for two main reasons: 1) in the case of PVC pipes, more of the recovered pipe
could be utilized, and 2) the fragments were fully immersed in the buffer. However, in
each instance there were limitations that hindered the amount of DNA that could be
retrieved. Improvements in the soaking technique, such as using a sturdier container or a
more effective cleanup method, may assist in producing greater quantities of DNA.

Although the swabbing technique was found to be more effective at DNA
recovery, its application exposed minor adjustments that could be incorporated to make it
more efficient. Swabs could be re-wetted when analyzing large surface areas to ensure
that cells are hydrated, or multiple wet swabs may be used on the same surface before the
dry swab is applied. The effectiveness (or ineffectiveness) of the soaking technique could
also be tested by swabbing the soaked fragments to determine if DNA remains. Further
optimization of the two isolation techniques could prove beneficial in the analysis of

IEDs.

62

Conclusion

The goal of this study was to compare the double swab and soaking techniques, to
ascertain which method was better at efficiently and effectively providing a higher
quantity and quality of recovered DNA. While the double swab technique was more
tedious, it was more effective at DNA retrieval as extracts from swabbed fragments were
cleaner, and generated greater quantities of DNA. Amplification success was more
frequent and better STR results were also obtained from swabbed bombs. The soaking
technique was quicker than the double swab technique, but was less effective at DNA
retrieval as the isolated DNA was more challenging to analyze. This resulted from an
inability to remove debris that co-extracted with the DNA, which caused PCR inhibition,
and because the soaked samples had to be diluted as a consequence. Thus, stochastic
effects were exacerbated.

MtDNA amplification was more successful than nuclear DNA amplification for
both isolation technique, which was attributed to high mtDNA copy number, the small
size of the amplicon, and the possibility that the mitochondrion protected the DNA during
deflagration more than did the nucleus. Correlative relationships between nuclear DNA,
mtDNA, and recovered pipe masses indicated that Quantifiler was a more accurate
nuclear DNA quantification assay, in addition to its benefit of determining PCR
inhibition levels. Finally, successful recovery of both types of DNA was independent of
pipe material, the level of pipe fragmentation, or the amount of recovered pipe used for
analysis, with the DNA isolation technique being the primary factor influencing DNA

recovery.

63

Table A1: Nuclear DNA and mtDNA quantities for each sample

Appendix A

Nuclear and MtDNA Data

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Isolation Pipe

Pipe # Technique Material Nuclear DNA Values MtDN A values
3 swab steel 0, 0 -
3 soak steel 0, 0 -
4 swab steel 23.85, 54.50, 43.95, 65.50 604, 1297, 1818
4 soak steel 0, 0 -
5 swab steel 5.10, 2.06, 0.49, 0 75, 291, 324
5 soak steel 0, 0 -
6 swab steel 0, 0 -
6 soak steel 0, 0 -
7 swab steel 14.00, 29.40, 20.55, 29.95 2170, 2036, 413
7 soak steel 0, 0 -
8 soak PVC 1.20, 9.13, 0, 0 810, 7479, 8582
9 soak PVC 47.63, 0, 0, 0 1600, 2291,4206
10 soak PVC 5.07, 23.67, 0, 0 1224, 2655, 3503
11 soak PVC 16.83, 0, 0, 0 117, 396, 958
12 soak PVC 0, 0, 0, 0 28, 55, 246
13 swab steel 1.95, 0 859, 630, 999
13 soak steel 0, 0 -
14 swab steel 48.65, 43.25 1394, 1491, 1745
14 soak steel 0, 0 -
15 swab steel 10.10, 6.90 1309, 132121321
15 soak steel 0, 0 -
l6 swab steel 13.05, 2.49 1208, 1333, 1758
16 soak steel 0, 0 -
l7 swab steel 72.00, 0 1673, 1770, 1527
17 soak steel 0, 0 -
18 swab steel 0.12, 5.15 1624, 967, 1370
18 soak steel 0, 0 -
19 swab steel 13.50, 0* 3382, 3309, 2848
19 soak steel 0, 0 -
20 swab steel 37.65, 20.50 10982, 9176, 7733
20 soak steel 0, 0 - '
21 swab steel 5.00, 0 962, 642, 742
21 soak steel 0, 0 -
22 soak PVC 42.60, 21.80, 0, 0* 777, 1016, 1273
23 soak PVC 1.85, 1.87, 10.7, 0 465, 608, 333
24 soak PVC 22.10, 0, 0, 0 927, 1004, 0

 

 

 

 

 

64

 

 

Table A1 continued

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

25 soak PVC 18.90, 21.80, 0, 0, 0* 1903, 1855, 2109

26 soak PVC 5.42, 8.61, 3.17, 0, 0, 0* 867, 1345, 1171

27 soak PVC 67.33,0 ,0* 4533, 3248, 4655

28 soak PVC 1.14, 0, 0 3406, 4315, 5849

29 soak PVC 13.67, 1.32, 1.62, 0 522, 284, 496

30 soak PVC 0, 0, 0, 0 573, 1078, 941

31 swab steel 0, 0, 0, 0, 0 6, 10, 0

31 soak steel 5.65, 8.75, 16.35, 0 1143, 1236, 450

32 swab steel 0, 0, 0, 0 947, 2788, 1661

32 soak steel 0, 0, 0, 0 7, 8, 0

33 swab steel 0.64, 18.05, 18.5, 11.3 885, 1806, 1491

33 soak steel 2.68, 1.60, 0.80, 0 17, 12, 27

34 swab steel 28.30, 10.60, 9.85 690, 713, 851

34 soak steel 1.30, 4.09, 0, 0 24, 25, 14

35 swab steel 0.28, 9.65, 13.05 1842, 2739, 5030‘

35 soak steel 1.67, 0.03, 0 9, 22, 0

36 swab PVC 16.80, 52.50, 72.00 1903, 1745, 2606

37 swab PVC 31.45, 35.45, 170.0, 19.20, 12.85 4606, 5467, 6121

38 swab PVC 7.90, 105.00, 113.00, 0, 0 2109, 2085, 0
8.30, 8.70, 59.50, 11.75, 61.00,

39 swab PVC 98.50 2873, 3030, 3248

40 swab PVC 40.95, 47.20, 62.50, 2.41 960, 2097, 2097

 

 

 

 

Column 1 shows the pipe bomb number, while columns 2 and 3 depict the isolation
technique and pipe material respectively. Column 4 displays the nuclear DNA quantities
and column 5 the mtDNA quantities. The symbol * indicates that the zero values were

recognized and included in the calculation of the DNA average.

Table A2: Average amount of DNA in descending order

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Pipe # Isolation Technique Pipe Material Agg. amt (pg)
1 7 swab steel 72.00
27 soak PVC 67.33
37 swab PVC 53.79
36 swab PVC 47.10
4 swab steel 46.95
14 swab steel 45.95
38 swab PVC 45.28
39 swab PVC 41 .29
40 swab PVC 38.27
22 soak PVC 32.20
20 swab steel 29.08
7 swab steel 23.48
25 soak PVC 20.35
34 swab steel 16.25
33 swab steel 12. 12

 

 

 

65

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table A2 continued
9 soak PVC 1 1 .91
1 5 swab steel 8.50
16 swab steel 7.77
3 1 soak steel 7.69
35 swab steel 7.66
10 soak PVC 7.19
19 swab steel 6.75
26 soak PVC 5.73
24 soak PVC 5.53
l 1 soak PVC 4.21
29 soak PVC 4. 15
23 soak PVC 3.61
l 8 swab steel 2.64
8 soak PVC 2.58
2 1 swab steel 2.50
5 swab steel 1 .91
34 soak steel 1 .35
33 soak steel 1 .27
13 swab steel 0.98
35 soak steel 0.85
28 soak PVC 0.38
3 swab steel 0.00
3 soak steel 0.00
4 soak steel 0.00
5 soak steel 0.00
6 swab steel 0.00
6 soak steel 0.00
7 soak steel 0.00
12 soak PVC 0.00
13 soak steel 0.00
14 soak steel 0.00
1 5 soak steel 0.00
16 soak steel 0.00
1 7 soak steel 0.00
18 soak steel 0.00
19 soak steel 0.00
20 soak steel 0.00
2 1 soak steel 0.00
30 soak PVC 0.00
3 l swab steel 0.00
32 swab steel 0.00
32 soak steel 0.00

 

 

 

 

 

 

Column 1 shows the pipe bomb number, while colunms 2 and 3 depict the isolation technique
and pipe material respectively. Column 4 depicts the average quantity of nuclear DNA in
picograms, and in descending order. Sample # 11 was the positive control, while samples # 12 &
13 were negative controls.

66

Table A3:

uanti of DNA recovered in descendin order b mass

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Avg. of fg per
Pipe Pipe Isolation Mass Avg. amt fg per gram Quantifiler gram of
# Material Technique (g) (pg) of pipe (pg) pipe
14 steel swab 768.32 45.95 59.81 36.90 48.03
32 steel swab 700.84 0.00 0. 00 0.00 0. 00
33 steel swab 694.18 12. 12 I 7.46 20.65 29. 75
20 steel swab 680.79 29.08 42. 72 29.20 42.89
5 steel swab 676.18 1.91 2.83 2.44 3. 61
7 steel swab 650.69 23.48 36. 08 15.70 24.13
35 steel swab 650.11 7.66 11. 78 85.30 131.21
34 steel swab 635.37 16.25 25. 58 0.00 0. 00
15 steel swab 625.03 8.50 13. 60 19.20 30. 72
13 steel swab 620.96 0.98 3. I 4 2.79 4.49
3 1 steel swab 598.90 0.00 0. 00 0.00 0. 00
18 steel swab 598.03 2.64 4.41 10.10 16.89
16 steel swab 577.15 7.77 13.46 19.60 33.96
21 steel swab 572.22 2.50 4.3 7 10.10 I 7.65
4 steel swab 561.92 46.95 83. 55 7.19 12.80
1 7 steel swab 498.02 72.00 144.5 7 34.90 70.08
19 steel swab 400.37 6.75 16.86 0.00 0. 00
34 steel soak 252.31 1 .35 5.34 0.00 0. 00
35 steel soak 237.57 0.85 3.58 0.00 0. 00
3 1 steel soak 21 8.25 7.69 35.22 0.00 0. 00
33 steel soak 193.50 1.69 1.27 0.00 0. 00
32 steel soak 1 86.84 0.00 0. 00 18.45 98. 75
30 PVC soak 121 .68 0.00 0. 00 0.00 0. 00
28 PVC soak 1 13.10 0.38 3.36 0.00 0.00
22 PVC soak 101.83 32.20 316.21 34.20 335.85
25 PVC soak 94.04 20.35 216.40 41.10 43 7.05
9 PVC soak 93.60 1 1.91 12 7.22 0.00 0. 00
10 PVC soak 91.12 7.19 78.85 14.20 155.84
27 PVC soak 86.81 67.33 775. 60 69.40 799.45
26 PVC soak 83.65 5.73 68.50 0.00 0. 00
24 PVC soak 76.15 5.53 72.55 0.00 0. 00
8 PVC soak 74.21 2.58 34.80 62.40 840.86
23 PVC soak 70.03 3.61 51.48 0.00 0. 00
1 1 PVC soak 69.96 4.21 60.14 0.00 0. 00
12 PVC soak 69.48 0.00 0. 00 0.00 0. 00
38 PVC swab 57.63 45.28 785. 70 0.00 0. 00
37 PVC swab 54.87 53.79 980.32 161 .50 2943.32
29 PVC soak 53.36 4.15 7 7.81 0.00 0. 00
40 PVC swab 52.57 38.27 72 7. 98 0.00 0. 00
36 PVC swab 46.71 47. 10 1008.35 6.55 140.23
39 PVC swab 39.69 41.29 1040.31 103.50 2607. 71

 

Column 1 shows the pipe bomb number, while columns 2 and 3 depict the pipe material and isolation

technique respectively. Column 4 depicts the mass of the recovered fragments for each bomb in descending

order. Column 5 shows the average quantity of nuclear DNA based on the Timken et al. (2005) assay,
while column 6 depicts the amount of femtograms per gram of pipe for column 5. Column 7 refers to the
average quantity of nuclear DNA based on the Quantifiler assay, while column 8 depicts the amount of

femtograms per gram of pipe for column 7. Sample # 11 was the positive control, while samples # 12 & 13

were negative controls.

67

 

Table A4: Average amount of DNA recovered from each fragmentation categogy

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A B C D E F G
Pipe # Pipe Frag. Total using Avg. of Total using Avg. of
Material Category Avg. amt column Avg. of column
(fg) ‘D’ (fg) Quantifiler ‘F’ (fg)
0g)
7 steel 1 36.08 12.95 24.13 34.41
13 steel 1 3.14 4.49
18 steel 1 4.41 16.89
32 steel 1 0.00 0.00
33 steel 1 18.73 29.75
35 steel 1 15.36 131.21
5 steel 2 2.83 28.47 3.61 16.47
14 steel 2 59.81 48.03
15 steel 2 13.60 30.72
31 steel 2 35.22 0.00
34 steel 2 30.92 0.00
16 steel 3 13.46 66.92 33.96 48.98
17 steel 3 144.57 70.08
20 steel 3 42.72 42.89
4 steel 4 83.55 34.93 12.80 10.15
19 steel 4 16.86 0.00
21 steel 4 4.37 17.65

 

 

 

 

 

 

 

Column 1 shows the pipe bomb number, while columns 2 depicts the pipe material.

 

Column 3 shows categories for the level of fragmentation: 1 4 low, 2 4 medium, 3 4 high,
and 4 4 complete. Column 4 shows the total average quantity of DNA based on the
Timken et a1. (2005) assay, where total DNA refers to the sum of the swabbed and soaked
results for each sample. Column 5 depicts the average for each fragmentation category in
column 4. Column 6 represents the total average quantity of DNA based on the
Quantifiler assay, while column 7 shows the average for each fragmentation category in
column 6.

68

Table A5: Average mtDNA quantity according to pipe material and isolation technique

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Pipe # Isolation Technique Pym Material Aveflge mtDNA (copy #)
31 soak steel 943
32 soak steel 8
33 soak steel 19
34 soak steel 21
35 soak steel 16
4 swab steel 1240

5 swab steel 230
7 swab steel 1540

13(neg) swab steel 829
14 swab steel 1543
15 swab steel 1317
16 swab steel 1433
17 swab steel 1657
1 8 swab steel 1320
19 swab steel 3 1 80
20 swab steel 9297
21 swab steel 782
31 swab steel 8
32 swab steel 1799
33 swab steel 1394
34 swab steel 751
35 swab steel 3204

8 soak PVC 5624
9 soak PVC 2699
10 soak PVC 2461

1 1(pos) soak PVC 490

lmeg) soak PVC 110
22 soak PVC 1022
23 soak PVC 469
24 soak PVC 966
25 soak PVC 1956
26 soak PVC 1128
27 soak PVC 4145
28 soak PVC 4533
29 soak PVC 434
30 soak PVC 864
36 swab PVC 2085
37 swab PVC 5398
38 swab PVC 2097
39 swab PVC 3050
40 swab PVC 1718

 

 

 

Column 1 shows the pipe bomb number, while columns 2 and 3 depict the isolation technique and
pipe material respectively. Column 4 depicts the average quantity of mtDNA in copy number.
Sample # 11 was the positive control, while samples # 12 & 13 were negative controls.

69

Table A6: Mitochondrial DNA copy number in descending order of mass

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Pipe Isolation Avg. mtDNA Copies per gram
Pipe # Material Technique Mass icopy #) of material
14 steel swab 768.32 1543 2.01
32 steel swab 700.84 1 799 2.57
33 steel swab 694. l 8 1394 2.01
20 steel swab 680.79 9297 13.66
5 steel swab 676. 18 230 0.34
7 steel swab 650.69 1540 2.3 7
35 steel swab 650.1 1 3204 4.93
34 steel swab 635.37 751 1.18
15 steel swab 625 .03 13 17 2.1 1
13 steel swab 620.96 829 1 .34
31 steel swab 598.9 8 0.01
18 steel swab 598.03 1320 2.21
16 steel swab 577.15 1433 2.48
21 steel swab 572.22 782 1.37
4 steel swab 561 .92 1240 2.21
17 steel . swab 498.02 1657 3.33
19 steel swab 400.37 3 1 80 7.94
34 steel soak 252.31 21 0.08
35 steel soak 237.57 16 0.07
31 steel soak 218.25 943 4.32
33 steel soak 193.5 19 0.10
32 steel soak 186.84 8 0.04
30 PVC soak 121 .68 864 7.10
28 PVC soak 1 13.1 4533 40.08
22 PVC soak 101.83 1022 10.04
25 PVC soak 94.04 1956 20.80
9 PVC soak 93.6 2699 28.84
10 PVC soak 91.12 2461 27.00
27 PVC soak 86.81 4145 47.75
26 PVC soak 83.65 1128 13.48
24 PVC soak 76. 15 966 12.68
8 PVC soak 74.21 5624 75.78
23 PVC soak 70.03 469 6.69
1 1 PVC soak 69.96 490 7.01
12 PVC soak 69.48 1 10 1.58
38 PVC swab 57.63 2097 36.39
37 PVC swab 54.87 5398 98.38
29 PVC soak 53.36 434 8.13
40 PVC swab 52.57 1718 32.68
36 PVC swab 46.71 2085 44.63
39 PVC swab 39.69 3050 76.85

 

 

 

 

 

 

 

Column 1 shows the pipe bomb number, while columns 2 and 3 depict the pipe material and
isolation technique respectively. Column 4 depicts the mass of the fragments for each pipe in
descending order. Column 5 shows the average quantity of mtDNA in copy number.

70

Table A7: Average number of copies recovered from each fragmentation categogy

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Pipe Fragmentation Average mtDNA Avg. per

Pipe # Material Catgory per gram (copy #) category

4 steel 4 2.21 3.84

19 steel 4 7.94

21 steel 4 1.37

16 steel 3 2.48 6.49

17 _ steel 3 3.33

20 steel 3 13.66

5 steel 2 0.34 2.01

14 steel 2 2.01

15 steel 2 2.1 l

3 1 steel 2 4.33

34 steel 2 1.27

7 steel 1 2.37 2.60

13 steel 1 1.34

18 steel 1 2.21

32 steel 1 2.61

33 steel 1 2.10

35 steel 1 4.99

 

 

 

 

 

 

Column 1 shows the pipe bomb number, while column 2 depicts the pipe material.
Column 3 shows categories for the level of fragmentation: l — low, 2 — medium,

3 4 high, and 4 — complete. Column 4 represents the average quantity of mtDNA in copy
number. Column 5 depicts the average for each fragmentation category in column 4.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Nuclear DNA
Pipe # MtDNA rank rank (Avg amt.) Quantifiler rank

20 1 1 1 10
8 2 29 5
37 3 3 1
28 4 36 -
27 5 2 4

35 (swab) 6 20 3
l9 7 22 26
39 8 8 2
9 9 16 -
10 10 21 16
38 11 7 -
36 12 4 20
25 13 13 6

32 (swab) 14 - -

 

71

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table A8 continued
40 15 9 -
17 16 1 8
14 17 6 7
7 18 12 15
16 19 18 12
33 (swab) 20 15 11
18 21 28 17
15 22 17 13
4 23 5 19
26 24 23 -
22 25 10 9
24 26 24 -
31 (soak) 27 19 -
30 28 - -
13 29 34 21
21 30 30 18
34 (swab) 31 14 -
11 32 25 -
23 33 27 -
29 34 26 -
5 35 31 22
12 36 - -
34 (soak) 37 32 -
33 (soak) 38 33 -
35 (soak) 39 35 -
31 (swab) 4O - -
32 (soak) 41 - 14

 

 

Column 1 shows the pipe bomb number along with the DNA isolation technique used,
while column 2 depicts the rank of the pipe according to mtDNA quantity. Column 3
refers to the nuclear DNA rank of the samples based on the Timken et al. (2005) assay.
Bold values depict rankings that are within 5 places of each other and italic values refer
to values that are within 10 places of each other. Column 4 refers to the nuclear DNA
rank of the samples based on the Quantifiler assay. Symbol (-) indicates that the value
was zero and was not ranked.

72

Table A9: Statistical differences of isolation technigues using t-tests

 

Avg. amt
(swab)

Avg. amt
(soakL

Quantifiler
(swab)

Quantifiler
(soak)

MtDNA
(swab)

 

Avg. amt

NO

flab)

 

Avg. amt NO

(soak) NO -

 

Quantifiler

QwabL NO NO - NO -

 

Quantifiler

(soak) ' ' NO ' '

 

 

MtDN A

(soak) - - - ' NO

 

 

 

 

 

 

 

Using the 1St column: row 2 refers to the average quantity of nuclear DNA recovered
fi'om swabbed fragments using the Timken et a1. (2005) qPCR assay; row 3 refers to the
average quantity of nuclear DNA recovered from soaked fragments using the Timken et
al. (2005) qPCR assay; Quantifiler denotes the average quantities of nuclear DNA
recovered from the swabbing and soaking techniques using the Quantifiler assay and
mtDNA denotes the average quantity of mtDNA recovered using the Timken et al. (2005)
qPCR assay. (-) denotes that no comparison was made between the two averages.

73

Appendix B

STR Data

Complete ST R results for samples 4 —— 30

Alleles highlighted in bold indicate a major peak and the symbol (-) denotes that a
peak over the threshold value (50 RF Us) was not obtained. “0L” indicates that the allele
was off ladder. The locus D281338 was not included in the PowerPlex l6 kit, and
therefore, could not be compared to the known sample. Sample 11 was the positive
control, while samples 12 and 13 were the negative controls. All soak samples were

analyzed using 1:20 dilutions

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

£61;
A] Volunteer alone

[B] Volunteer (maLor profilQ + other alleles

[C] Volunteer @n-maj or profilei+ other alleles

[D] At least I volunteer allele

1E] No volunteer

[F] No alleles

No comparison made to a known DNA sample
Sam le #
Locus 4 swab 5 swab 7 swab 13 swab (mg)
D13S317 8,11 [A] 11, 12, 13 [C] 8, 9, 11, 12 [B] 11,16
D78820 9,10 [A] 10,11 [C 8, 910,1112[B] 10,11
Amel X, Y [B] X 1A] X, Y [B] X, Y
D281338 19, 20, 22, 25 23, 25, 28 17, 20, 24 17, 19, 20, 21, 24
D2181] 27, 30 [A] 29 [D] 28, 30, 31 [C] 28, 31, 32
D16SS39 12,13 [A] 10 [A] 9,10,11 [C] 9,11,15
D18S51 13,16 [A] 16,19 JG] 15,17 [C] 15, 16,18
CSFlPO 11,12,13 [C] 11,12 [A] 10,11,12 [C] 10,11, 12
FGA 23.2 [E] 24, 25 [E] 24 [E] 16.2, 19, 22.2,
25, 27

 

 

 

 

 

 

74

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sam le#
Locus l4 swab 15 swab 16 swab 17 swab
D138317 9, 11,13 [B] 11, 12, 13 [B] 8, 11, 13,16 [B] 10, 11,12,13,
l4, 16 [B]
D78820 8, 10, 12 [B] 10, 11, 12 [C] 7, 8, 11 [B] 8, 10, 11 [B]
Amel X, Y X, Y [A] X [A] X, Y [B]
D281338 20, 22, 24, 25, 2], 22, 23 l7, 19, 20, 21 17, 19, 20, 21
26, 27
D21811 32, 32.2 [A] 28 [A] 29, 31.2, 32.2 28, 29, 30, 31
[C] [B]
D168539 9,10, 12,13 [B] 12,12.2,l3 [C] 11,12 [A] 9,1] [A]
01885] 14, 15, 16, 17, l3, 18 [D] 12 [A] 12, 15, 16, l7[B]
18 [C]
CSFlPO 9, 10, 11 [C] 10,11,12 [C] 10, ll, 13 JB] 10 [A]
FGA 23 [D] 27 [E] 25, 26, 28.2 [D] - [F]
Sam le#
Locus 18 swab l9 swab 20 swab 21 swab
D1383l7 11,13,14 [C] 9,11,12 [C] 11, 12,13 [B] 12 [A]
D78820 8, 10, 12 1B] 10, 11 12 [B] 9, 11 [A] 8, 9 10 1B]
Amel X, Y [B] X, Y [A] X, Y [A] X, Y [B]
0281338 19, 22, 23, 24, l8, 19, 24 20, 21 19, 25
25
D21811 29 [A] 29, 31 [A] 30, 30.2, 32.2 30, 30.2 [A]
[C]
D168539 10,12,13 [C] 8,12 [A] 9,12 [C] 9,11 [A]
D1885] 12, 15, 16 [B] 13,15,16.2,17, 11,12,13,15, 10,12,14,15,
18 [C] 17,18 [C] 16 [C
CSFlPO 10,11 [A] 10,11, 12 [B] 10,12 [Cj 11,12 [A]
FGA - [F] 24, 26 [E] - [F] 26 [E]
Sam le#
Locus 8 soak 9 soak 10 soak 11 soak jpofl
D13S317 8, 12 [A] 8, 12 [D] 11 [D] 8 [D]
D78820 - [F] 10 [D] 11 JE] - [F
Amel X [A] X, [A] X, Y [Q - [F]
0281338 20 [D] 17, 20 [A] — [F] 15, 18
D21811 28, 30 [A] 31, 32.2 [D] 29, 30 [A] 25 [E]
D168539 9,1] [A] 10,12.2 JE] 11,15 [C] - [F]
D1885] 15,16 [A] 13,16 [A] 15,16 [D] 15 JA
CSFlPO 11,13 [A] 11,12 [C] 10 [E] - [F]
FGA 22, 27 1E] 23 [E] - [F] - [F]

 

 

 

 

 

75

 

 

 

 

Sample #

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Locus 12 soak (neg) 22 soak 23 soak 24 soak 25 soak
D1383l7 - 11 [E] 11 [A] 0L [F] - [F]
D78820 - - [F] - [F] - [F] - [F]
Ame] - X [A] X [A] X, Y JA] X, Y [A]
D281338 19 18 0L [F] 17,19, 20 17,19, 20, 26
D21811 - - [F] 0L [F] 0L [F] 0L [F]
D168539 - 6, 12 [C] 1] [D] 8, 11 [D] - [F]
D1885] - 19 [D] - [F] 15,16 [D] 12,13 [D]
CSFIPO - - [F] - [F] 10,12 [C] 10 [D]
FGA - - [F] - [F] 22,23 [D] 23 [E]
Sample #
Locus 26 soak 27 soak 28 soak 29 soak 30 soak
D13S3l7 - [F] - [F] 8 [E] 9, 14 [D] ll, 14 [E]
D78820 8, 13 [A] - [F] - [F] 8, 9, 10, 12 [D] 11 [D]
Amel X, Y [A] - [F] X [A] X, Y [B] X [A]
D281338 20, 23 - [F] 24 17, 18,19, 21, 17
25, 23, 27
D2181] 0L [5] - [F] 27 [E] 27, 30.2 [E] - [F]
D168539 9,11,12 - [F] 11 [A] 10,1], 12.2 [C] 11 [A]
[C]
D1885] 13,15 [A] - [F] 15 [E] 12,14,16,19 - [F]
[C]
CSFlPO 10, 11 [C] - [F] - [F] 9,10,11,12 10 [E]
[C]
FGA 20 [E] - [F] - [F] 22, 25, 26, 27, - [F]
27.2 [E]

 

 

 

 

 

 

 

76

 

 

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