1
Chapter 1: Introduction
DNA is a vital and fragile genetic material used within many disciplines. With advances
in genetic sequencing and analysis technology, DNA has been utilized more frequently in the
court of law. Thus, the integrity of DNA is essential. The condition of DNA influences its
stability, thereby influencing the preservation, which determines the success of identifying
individuals, animals, plants, microorganisms, or food (Bonnet et al., 2009; Arenas et al., 2017).
There is ample research discussing DNA contamination throughout different aspects of the
collection process (Ladd et al., 1999; Pang and Cheung, 2007; Lapointe et al., 2015; Fonneløp et
al., 2016; Pickrahn et al., 2017; Basset and Castella, 2018). The studies focus on the crime scene,
collection, and extraction processes. However, there is a gap in the research concerning potential
contamination during storage—specifically storage between collection and extraction. The
research analyzing the storage process focuses on preserving DNA, but not considering potential
contamination, or cross-contamination, from samples stored in close proximity to one another.
Because of the value of DNA, it is crucial to understand the optimal preservation
methods. However, how genetic material is stored is dependent on the laboratory’s protocols,
which determine the packaging, storage conditions, and retention length of the evidence received
at the forensic facilities (Ballou et al., 2013; Latta et al., 2015; Martin, 2016). If the appropriate
storage conditions are not followed, then the integrity of genetic evidence becomes
compromised, jeopardizing the information that can be obtained from the DNA. This risk is even
more critical because of Hollywood’s unrealistic portrayal of evidence, which leads jurors to
expect genetic evidence to demonstrate immutable truth (Slabbert and Heathfield, 2018).
Therefore, refining the storage conditions will benefit the preservation and integrity of genetic
evidence, increasing the confidence of the genetic analysis.
2
A preliminary study was conducted to determine if cross-contamination can occur during
storage. The study tested the storage length and drying time for two collection methods: swabs
and Whatman cards by having one sample with DNA present stored in an evidence bag with a
second that did not have DNA. The samples were stored at room temperature throughout the
study. The longer Whatman cards were in storage, the more likely contamination occurred, and
when analyzed with Fisher’s Exact test the p-value was 0.00 when α = 0.05 level. This indicates
that storage length is statistically significant in the observation of contamination. However, the
buccal swabs had a p-value of 0.054, which is not statistically significant at the α = 0.05 level.
The results from the study indicate that the longer Whatman cards are in storage, the more likely
contamination is to occur (Ramey, 2019). The recommended storage conditions depend on
storage length and evidence type, which some facilities cannot provide (Ballou et al., 2013; Latta
et al., 2015; Martin, 2016). When stored, there is often no separation between evidence samples,
with some facilities storing different cases in close proximity to each other (Cordray, 2010;
Department of Public Safety - Texas, 2012; Ballou et al., 2013). Therefore, if the environment
influences the DNA movement, then the potential for contamination increases when evidence is
stored in close proximity.
The amount of time samples were left to dry after being exposed to wet DNA, or dry
time, and DNA contamination were compared for the buccal swabs and the Whatman cards. The
p-value for both the buccal swabs and the Whatman cards were greater than the significance
level (α = 0.05). This p-value shows that the longer samples are left to dry prior to storage does
not decrease the potential of contamination.
In order to continue to investigate DNA contamination, the environmental conditions that
were selected are those that protocols consider during the storage process: temperature and
3
humidity. Protocols provide four temperatures that evidence can be stored: frozen (at or below -
10˚C), refrigeration (between 2˚C and 8 ˚C, less than 25% humidity), room temperature (ambient
temperature), or temperature-controlled (between 15.5˚C and 24˚C, less than 60% humidity).
The optimal storage temperature is dependent on the evidence type (Ballou et al., 2013). In
contrast, humidity conditions are only referenced for refrigeration and temperature-controlled
storage scenarios.
Temperature and humidity are the primary environmental factors that could potentially
impact evidence throughout the storage process. When environmental factors are not considered,
it puts DNA integrity at risk because certain environmental factors can cause damage to DNA
(Alaeddini et al., 2010; Hall et al., 2014). Temperature has been found to be a factor in the
movement of molecules (Widen et al., 2004; de Fátima Poças et al., 2011; Maia et al., 2016;
Brandsch, 2017; Fang and Vitrac, 2017). Humidity has been shown to affect DNA’s structure,
beginning at 50% humidity (Westhof, 1988; Bonnet et al., 2009). Therefore, it is essential to
research temperature and humidity concerning the DNA integrity during storage.
Chapters Outline: Chapters 2-4 provide background research discussing different areas
for the project. Chapter 2 provides a general history of forensic genetics. The chapter also
discusses the various DNA analyses that have been used over the years and how the information
obtained from the analyses are utilized in the courtroom. Chapter 3 discusses the success rate and
collection process of DNA from a crime scene. Chapter 4 discusses the preservation of DNA by
analyzing environmental effects and storage methods. The chapter also discusses the movement
of DNA and the migration modeling of molecules.
The subsequent chapters 5-8 will discuss the various aspects of the research. Chapter 5
discusses preliminary research and hypotheses, and outlines the storage process and DNA
4
analysis. Chapter 6 discusses the results from the various DNA analyses. Chapter 7 analyses the
results by discussing the findings from the research. Finally, chapter 8 concludes the final
remarks on the research and discusses how the study will impact the field in a forensic context.
5
References:
Alaeddini R, Walsh SJ, Abbas A. 2010. Forensic Science International : Genetics Forensic
implications of genetic analyses from degraded DNA — A review. Forensic Sci Int Genet
4:148–157.
Arenas M, Pereira F, Oliveira M, Pinto N, Lopes AM, Gomes V, Carracedo A, Amorim A. 2017.
Forensic genetics and genomics: Much more than just a human affair. PLoS Genet 13:1–28.
Ballou S, Stolorow M, Taylor M, Bamberger PS, Brown L, Brown R, Burney Y, Davenport D,
DePalma L, Williams S, Jones C, Keaton R, Kiley W, Latta J, Kline M, Lanning K, LaPorte
G, Ledray LE, Nagy R, Ostrom BE, Schwind L, Stoiloff S. 2013. The biological evidence
preservation handbook : best practices for evidence handlers ; technical working group on
biological evidence preservation.
Basset P, Castella V. 2018. Lessons from a study of DNA contaminations from police services
and forensic laboratories in Switzerland. Forensic Sci Int Genet 33:147–154.
Bonnet J, Colotte M, Coudy D, Couallier V, Portier J, Morin B, Tuffet S. 2009. Chain and
conformation stability of solid-state DNA: Implications for room temperature storage.
Nucleic Acids Res 38:1531–1546.
Brandsch R. 2017. Probabilistic migration modelling focused on functional barrier efficiency and
low migration concepts in support of risk assessment. Food Addit Contam Part A 34:1743–
1766.
Cordray R. 2010. Guidelines for preservation and retention of biological evidence.
Department of Public Safety - Texas. 2012. Best practices for collection, packaging, storage,
preservation, and retrieval of biological evidence. :1–8.
Fang X, Vitrac O. 2017. Predicting diffusion coefficients of chemicals in and through packaging
materials. Crit Rev Food Sci Nutr 57:275–312.
de Fátima Poças M, Oliveria JC, Peteira JR, Brandsch R, Hogg T. 2011. Modelling migration
from paper into a food simulant. Food Control 22:303–312.
Fonneløp AE, Johannessen H, Egeland T, Gill P. 2016. Contamination during criminal
investigation: Detecting police contamination and secondary DNA transfer from evidence
bags. Forensic Sci Int Genet.
Hall A, Sims LM, Ballantyne J. 2014. Assessment of DNA damage induced by terrestrial UV
irradiation of dried bloodstains: Forensic implications. Forensic Sci Int Genet 8:24–32.
Ladd C, Adamowicz MS, Bourke MT, Scherczinger CA, Lee HC. 1999. A Systematic Analysis
of Secondary DNA Transfer. J Forensic Sci 44:14599J.
Lapointe M, Rogic A, Bourgoin S, Jolicoeur C, Séguin D. 2015. Leading-edge forensic DNA
analyses and the necessity of including crime scene investigators, police officers and
technicians in a DNA elimination database. Forensic Sci Int Genet 19:50–55.
Latta JT, Giles RE, President P, Hueneme P, Chief D, Fallon K, Police SC, Hammarberg SK,
Property MP, Unit E. 2015. International Association for Property and Evidence. Prof
Stand.
Maia J, Rodriguez-Bernaldo de Quirós A, Sendón R, Cruz JM, Seiler A, Franz R, Simoneau C,
6
Castle L, Driffield M, Mercea P, Oldring P, Tosa V, Paseiro P. 2016. Determination of key
diffusion and partition parameters and their use in migration modelling of benzophenone
from low-density polyethylene (LDPE) into differnt foodstuffs. Food Addit Contam Part A
33:715–724.
Martin c. 2016. Dna Storage Banks: The importance of preserving dna evidence to allow for
transparency and the preservation of justice.
Pang BCM, Cheung BKK. 2007. Double swab technique for collecting touched evidence. Leg
Med 9:181–184.
Pickrahn I, Kreindl G, Müller E, Dunkelmann B, Zahrer W, Cemper-Kiesslich J, Neuhuber F.
2017. Contamination incidents in the pre-analytical phase of forensic DNA analysis in
Austria—Statistics of 17 years. Forensic Sci Int Genet 31:12–18.
Ramey SL. 2019. DNA integrity in forensic samples.
Slabbert N, Heathfield LJ. 2018. Ethical, legal and social implications of forensic molecular
phenotyping in South Africa. Dev World Bioeth 18:171–181.
Westhof E. 1988. Water: an integral part of nucleic acid structure. Annu Rev Biophys Biophys
Chem 17:125–144.
Widen H, Leufven A, Nielsen T. 2004. Migration of Model Contaminants from PET Bottles:
Influence of Temperature, Food Simulant and Functional Barrier. Food Addit Contam
21:993–1006.
7
Chapter 2: Forensic Genetics
This chapter explores the history, DNA analysis methods, CODIS, and evidence in the
courtroom of forensic genetics. The history of genetics extends over three centuries, but it was
not until the second half of the 20th century that DNA would be used for forensics. Between the
start of forensic genetics and now, various methods have been used to try and obtain a genetic
profile for human identification. As the technologies advanced, DNA could be used in the
courtroom as evidence, which would soon expand beyond just human identification.
Background
Most of the advances in forensic genetics have occurred in the past two decades, with the
era of forensic DNA only beginning around 35 years ago. However, human identification has
existed since the start of the 20th century through forensic serology. In 1900, Karl Landsteiner
discovered the ABO blood groups (Li, 2018; Alessandrini et al., 2020; Erlich, 2020). This
method of identification uses the antigens polymorphisms on the red blood cells. The antigens
could aid in identification based on varying frequencies of the four blood types within a
population; the disadvantage is the power of discrimination because ABO blood groups can only
exclude individuals from identification, not confirm them (Alessandrini et al., 2020).
A decade after Landsteiner’s discovery, Edmond Locard established Locard’s Principle
of Exchange in 1910. The principle states, “every contact leaves a trace” (Rutty and EAM, 2005;
Byard et al., 2016; Li, 2018; Allwood et al., 2020). Modern forensic genetics relies on the trace
DNA found at crime scenes to aid in the investigation. Thus, Locard’s principle helped establish
this idea of traceable identification (Byard et al., 2016; Li, 2018; Mistek et al., 2019). In 1917,
Thomas Hunt Morgan published his theory of the gene (Morgan, 1917; Li, 2018). His theory
8
further examined the Mendelian law of heredity and discovered that genes are located on the
chromosomes and are the basic unit of heredity (Morgan, 1917), establishing the foundation of
forensic genetics, and the field of genetics as a whole (Li, 2018). However, serology would be
the source of human identification until the mid-1980s. In 1927, Landsteiner and Levine
discovered two antigens P (Globoside) and MNSs blood system. By the 1930s, red blood cells
were still used for human identification. During this time, Levine and Stetson discovered the LW
blood system, followed shortly by the discovery of the Rh factor by Alexander Weiner and
Landsteiner. In total, sixteen red blood cell antigens would eventually be used in identification,
providing a modest power of discrimination. By the 1960s, around 60 serological markers were
used for identification. These markers now included the white blood cell antigen
histocompatibility known as HLA (Pourazar, 2007; Alessandrini et al., 2020).
In 1953, the structure of DNA was discovered to be double-helical (Li, 2018). This
discovery was a significant advancement toward the use of DNA for identification and forensic
genetics (Reich et al., 2002; Li, 2018). In 1984, Dr. Alec Jeffreys discovered ‘DNA
fingerprinting,’ now known as DNA profiling. Jeffreys found DNA heritable patterns that
resemble a barcode, which the comparison was seen after completing southern blot analysis. The
targeted segments of the DNA separate by the agarose electrophoresis based on the size of the
amplified DNA, in which smaller DNA will travel faster through the gel, resulting in bands
(Roewer, 2013; Zahra et al., 2018; Carracedo and Prieto, 2019; Alessandrini et al., 2020; Bright
et al., 2020). This discovery began the era of forensic DNA, and the following decade’s research
focused on exploring DNA profiling (Roewer, 2013). With DNA profiling, genetic evidence
could now provide the ability to discriminate between individuals based on a likelihood ratio
([LR]; Carracedo and Prieto, 2019; Bright et al., 2020). The ratio can communicate the
9
significance of the DNA by measuring the probability of the genetic evidence belonging to a
suspect (Carracedo and Prieto, 2019). This was most important to understand when the use of
DNA was first introduced to the courts because DNA profiling was a new concept. The first case
to use DNA profiling involved an immigration issue where a boy was at risk of deportation, but
with DNA evidence, he was saved. The boy was thought to be either a nephew or unrelated to a
woman living in the United Kingdom. The conventional genetic markers for the time (ABO
blood group, Rh, HLA, etc.) indicated the two were related. However, the analysis could not
confirm if the boy was her son, and a DNA profile was able to prove their true relationship
(Jeffreys et al., 1985). Jeffreys stated, “If our first case had been forensic I believe it would have
been challenged and the process may well have been damaged in the courts” (Roewer, 2013).
This public acceptance of DNA profiling paved the way for the data to be used in forensic cases.
The DNA now being used for human identification instead of forensic serology created a
need for a place to access it. In 1995, England created the first DNA profile bank. This was soon
followed by Northern Ireland, Scotland, and New Zealand in 1996 (Carracedo and Prieto, 2019).
The databases are beneficial to law enforcement by providing an individual’s unique marker
identifiers. In 2004, Kirk Bloodsworth became the first death row inmate to be exonerated with
DNA (Junkin, 2005). However, the use of DNA databases raises concerns from the public,
despite their expectation of using genetic evidence within the courtroom. The general public has
three main concerns regarding the databases: a lack of DNA data transparency, lack of
international standardization of DNA analysis, and potential ethical oversight. Ethical concerns
increase when considering forensic DNA phenotyping (FDP) because of the potential
stigmatization of specific populations (Machado and Silva, 2019). These concerns still exist
10
today but could potentially be resolved through transparent dialogue with the general public
about the DNA extraction and analyzing process.
Until 2005, forensic genetics was still focusing on the standardization of the field.
However, the 2000s began the rapid advancements in forensic genetics that occurred between
2005 through 2015, focusing on new technologies and applications (Roewer, 2013; Butler,
2015). Databases were expanded, and by the end of this period the United States National DNA
Index System (NDIS) grew by 12 million genetic profiles. New STR kits were implemented in
Europe and the United States. Instruments pursued rapid DNA profiling (Butler, 2015).
The expansion of these new technologies allowed forensic genetics to become
sophisticated in less explored areas beginning in 2015 (Butler, 2015). Nonhuman genetic
elements like food, animals, microorganisms, and plants, which have overlapping applications in
forensics, were applied in a forensic context. Plants, microorganisms, and animals can all be
silent witnesses of crimes. The silent witness is the genetic evidence left behind that is not a
person’s DNA, such as pet hair, soil DNA, and grass. Food, microorganisms, and plants are also
applicable for bioterrorism (Arenas et al., 2017). Law enforcement now uses these newer areas to
identify wildlife, hunting, and food authentication (Amorim et al., 2020). Despite nonhuman
forensic genetics, there are still limitations due to minimal species having been identified and
established for comparison (Arenas et al., 2017).
Advancements in technologies have also allowed investigators to generate a phenotype
report based on an individual’s DNA (Kayser, 2015; Hopman and M’charek, 2020). This began
with the probability of an individual’s eye and hair color (Kayser, 2015). There are analysis
services that provide a phenotype report consisting of sex, ancestry, skin color, eye color, hair
11
color, freckle percentage, and facial reconstruction (Hopman and M’charek, 2020). Therefore, as
the methods and technologies advance, more information is obtained from genetics.
DNA Analysis
Retired
The recent advancements in the technology for DNA analysis and extractions have
caused RFLP, DQ alpha, and AmpFLP to become retired technological methods. These retired
methods were used in forensics to generate genetic profiles but became obsolete in forensic
genetics for various reasons.
RFLP
Restriction fragment length polymorphism (RFLP) was the first method to analyze a
pattern in the variation of tandem repeats, thus creating a genetic profile beginning in 1980
(Vitoševic et al., 2019; Dash et al., 2020a). Today, this method has been replaced with
polymerase chain reaction (PCR) based technologies. The process follows four steps: first, the
DNA is cut into fragments using restriction endonuclease. Next, the fragments were separated
with gel electrophoresis. Then the fragments are transferred to a nitrocellulose membrane to
conduct a southern blot and finally analyzed using radioactive probes (Vitoševic et al., 2019).
The DNA required for a successful RFLP analysis consisted of large quantities of intact DNA,
making the method not optimal for forensics (Roewer, 2013; Børsting and Morling, 2015;
Vitoševic et al., 2019; Erlich, 2020).
DQ alpha
In 1991, the DQ-alpha test was developed to examine the poly-allelic locus of the HLA-
DQA1 gene (Tilstone et al., 2006). Because this method used PCR, it did not require the same
12
quantity of DNA as RFLP, which seemed promising for forensic cases. However, the process
was labor-intensive, detected sequence variation, and the discriminatory power was not optimal
for forensic analysis (Saiki et al., 1986; Tilstone et al., 2006; Erlich, 2020). The analysis of this
method consists of eleven probe dot-blot assays (alleles: 1, 2, 3, 4, C, 1.1 [1.2, 1.3, 4], 1.3, all but
1.3, 4.1, [4.2, 4.3]), in which the intensity of the color the dot turns indicates the amount of
amplified DNA bound to the specific probe. The results were then compared to other samples;
however, the intensity of the dot colors could potentially be interpreted differently (Erlich, 2020).
AmpFLP
In the 1990s, Amplified fragment length polymorphism (AmpFLP) was developed using
PCR to generate a DNA fingerprint with dominant markers. The method was fast, easy,
replicable, and relatively cheap for the time. Compared to RFLP, the quantity of information was
higher. Preparing the DNA to generate AmpFLP markers consisted of template preparation,
restriction and ligation, and selective amplification. The AmpFLP markers allowed multiple
polymorphic bands to be analyzed in one gel lane simultaneously. Therefore, the bands of
different samples would be compared to each other (Blears et al., 1998; Mueller and
Wolfenbarger, 1999).
Current Methods
STR
Short tandem repeat (STR) is currently the primary method used in forensic genetics
(McCord et al., 2019). STRs are found throughout the entire genome, containing 2 – 7 base pairs
that repeat in tandem for a various number of times (Panneerchelvam and Norazmi, 2003;
Vitoševic et al., 2019; Dash et al., 2020a). STR analysis methodology expands on the retired
RFLP method because STR is more sensitive, and AmpFLP is prone to allelic drop-out (Roewer,
13
2013). An advantage to STRs is that they can be amplified simultaneously in single multiplex
amplification and detect a mixed sample (Butler et al., 2007). The process of generating an STR
profile follows four steps: DNA isolation, amplification, electrophoresis, and data analysis. The
profiles are then used to compare to the profiles of various samples (Turnbough et al., 2013;
McCord et al., 2019). STRs have a high mutation rate of approximately 10–3, which is a
limitation because it makes STRs less stable; however, it is more discriminatory than other
techniques (Butler et al., 2007; Roewer, 2013; Vitoševic et al., 2019). In comparison, the average
mutation rate per nucleotide site ranges between 1.6 x 10–7 to 2.3 x 10–9 (Nachman and Crowell,
2000). In forensics, the markers used are located in the non-coding region of the genome and the
first markers used were TH01, vWA, FES/FPS, and F13A1 because of their simplistic repeat
sequences (Wyner et al., 2020).
In 1992, the first Y-STRs were discovered. Now, a few forensic kits include Y-STRs
(Kayser, 2017). These types of STRs are only found in biological males, which is beneficial
when dealing with paternal relationships or mixed male/female samples (Diegoli, 2015;
Vitoševic et al., 2019). The addition of Y-STRs to the normal autosomal STRs has enhanced
DNA analysis. However, because of the low mutation rate, approximately 2 – 4 x 10–3, and the
lack of recombination, Y-STRs cannot discriminate between related men. In comparison, the
average X STR mutation rate is 1.35 x 10–3 (Diegoli, 2015; Kayser, 2017), resulting in more
mutational differences and, therefore, higher discriminatory power between individuals.
There are potential problems with STR readings: allele drop-out, drop-in, or stutter,
which can interfere with the interpretation of the genetic profile. If these problems occur, it can
be challenging to determine if the sample is a mixture of DNA, indicating some contamination
(McCord et al., 2019). Allele drop-out is when there is no allele observed at the locus or the loss
14
of one of the alleles leading to the appearance of homozygosity. This is common when there is
low template DNA. It is difficult to observe, often leading to the requirement to run a sample
multiple times to ensure homozygosity or that all alleles are accounted for. Allele drop-in is an
additional peak typically smaller than prominent peaks at a given locus, originating from
extraneous DNA from another DNA sample. With an allele stutter, the peak will not appear in its
intended position, and the new position will depend on the peak size (Balding and Buckleton,
2009; Buckleton, 2009; Taylor et al., 2014). It may be possible to determine that the sample is a
mixture based on various alleles if there are no overlapping alleles of the multiple DNA
contributors (Butler et al., 2007). However, if the contributors share an allele, this is known as
masking (Taylor et al., 2014).
Another potential problem is the two types of tri-allelic patterns at STR loci. In the first
type, the three peaks are uneven, but the sum of the smaller peaks equals the height of the most
prominent peak. The second type is divided into two patterns 1:1:1 or 2:1. In the first pattern
(1:1:1), the three peaks are equal in height. While in the second pattern (2:1), there are two
peaks, with one being double the height because it consists of two identical alleles (Yang et al.,
2020).
mtDNA
Mitochondrial DNA (mtDNA) is maternally inherited and analysis of it is a current
method in forensic genetics. In forensics, the process of generating a mtDNA analysis typically
follows Sanger sequencing and the most important step is the pre-extraction sample prep
(Holland et al., 2013). MtDNA is beneficial if the samples are highly degraded because of the
many copies per cell compared to the two copies of nuclear DNA (Vitoševic et al., 2019).
However, it is less discriminating than STRs because of the lack of recombination since there is
15
only a single marker maternally inherited and therefore used only when nuclear DNA is
unavailable (Holland et al., 2013; McCord et al., 2019; Vitoševic et al., 2019).
MPS
Massively parallel sequencing (MPS) is known by several names in forensics and used
interchangeably, such as next-generation sequencings and high throughput sequencing (Erlich et
al., 2020). Between 2005 to 2007, several systems using MPS were introduced (Bruijns et al.,
2018; Arora, 2020). The technology has a higher throughput compared to Sanger sequencing,
which is not frequently used with forensic human samples, has accurate sequencing capabilities
potential, rapid processing, and is low-cost. The different MPS systems follow the key steps:
library preparation, template DNA amplification and distribution, sequencing and imaging, base
calling, quality control, and data analysis (Berglund et al., 2011; Zascage et al., 2013; Murphy,
2018; Kumar et al., 2019; Erlich et al., 2020). There are advantages to using MPS over other
methods. Unlike STRs, MPS examines each nitrogenous base of the nucleotide of a region
(Murphy, 2018). MPS systems sequence reactions simultaneously and initiated with one DNA
molecule; because of this, MPS can be applied to analyze various genetic markers (McCord et
al., 2019; Novroski et al., 2019; Erlich et al., 2020). Despite these advantages, MPS is rarely
used in forensics and primarily limited to research studies. But the consensus among academics
is that MPS has the potential of impacting forensics (Bruijns et al., 2018; Murphy, 2018; Erlich
et al., 2020). In 2019, the NDIS Board of the FBI approved the uploading of MPS kits to DNA
databases. However, the NDIS can only store, upload, and search the required CODIS Core Loci
and NDIS accepted loci. This was seen in a European laboratories survey where four challenges
emerged when implementing MPS instruments in their labs used to analyze identity, ancestry, or
autosomal-STR markers: no reporting standards, not compatible with existing national DNA
16
databases, insufficient population data for statistical calculations, and minimal legislative
framework (Alonso et al., 2017; Butler and Willis, 2020).
Rapid DNA
As the name suggests, rapid DNA is capable of producing a DNA profile within two
hours. The sequencing is limited to generating an STR profile at a set number of loci because of
its speed. For instance, DNAscan 6C can analyze 27 loci under two hours (Dash et al., 2020a;
Erlich et al., 2020). These are typically machines, the size of a desktop printer, that are simple to
use and require no special training to operate (Murphy, 2018). The process is quick and
straightforward, following six steps that are completed through this one machine: signal
processing, fragment identification, comparison with internal lane standard, comparison with
allelic ladder, locus and sample specific analysis, and generated profile (Dash et al., 2020a;
Chong et al., 2021). However, the sample must be of high-quality DNA and from a swab (Erlich
et al., 2020). The analysis is intended for samples from a known individual due to the fast nature
of the sequencing. Therefore, rapid DNA is used to search against a DNA database of an arrested
suspect. The Rapid DNA Act of 2017 outlined guidelines for this technology, which is an
amendment to the DNA Identification Act of 1994. Rapid DNA analysis is no longer required to
be conducted in a qualified laboratory if the guidelines of when uploading or searching DNA
databases with rapid DNA can occur are followed. Samples are now required to be reference
samples and not a forensic sample (Anon, 2017; Murphy, 2018; Butler and Willis, 2020; Erlich
et al., 2020)
17
CODIS
The United States has influenced the standardized genetic markers used in the
identification of a genetic profile. As discussed earlier, STRs are short tandem repeats used to
establish the standardized markers and first used in 1991 (Zhang et al., 2020). The DNA
Identification Act of 1994 allowed the United States’ FBI to establish a national database to store
DNA profiles known as CODIS, the Combined DNA index system (Butler and Li, 2014;
Karantzali et al., 2019). In 1998, the first 13 standardized loci were introduced: D8S1179,
D21S11, D5S818, CSF1PO, D3S1358, TH01, D13S317, D16S539, TPOX, D18S51, vWA,
D7S820, and FGA. Then in 2001, the European standard set was established with 7 of the
genetic loci from the CODIS set: D8S1179, D21S11, D3S1358, TH01, D18S51, vWA, FGA. In
the last decade, there were discussions of expanding the current CODIS standard set to reduce
the number of adventitious matches. Validation studies were conducted on three PCR
amplification kits: Life Technologies’ GlobalFiler, Life Technologies, GlobalFiler Express, and
Promega Powerplex. Based on the validation data, it was concluded that the expansion should
retain the original 13 loci and added seven additional loci: D1S1656, D2S441, D2S1338,
D10S1248, D12S391, D19S433 and D22S1045 (Hares, 2015; Karantzali et al., 2019; Butler and
Willis, 2020). The FBI required the additional STR loci to be implemented into genetic labs by
January 1, 2017 (Hares, 2015; Moretti et al., 2016).
SE33 and Amelogenin were two of the loci considered; however, these loci did not
qualify for the CODIS expansion. However, both markers are beneficial for familial DNA
searches (paternity testing, missing people, etc.). SE33 is a highly polymorphic locus, located on
chromosome 6 (6q14). Studies have indicated that SE33 can exclude false matches and increase
the true positive rate to false positive rate ratio (Butler et al., 2009; Bhinder et al., 2018;
18
Karantzali et al., 2019). Unlike SE33, Amelogenin has incorrectly determined the individual’s
biological sex in many cases, despite it being required for genetic profiles of relatives of missing
persons or unidentified human remains along with the standardized CODIS loci (Butler and Li,
2014). Amelogenin has two homologous genes AMELX, located on the X chromosome, and
AMELY, located on the Y chromosome, which failure to amplify AMELY suggests the absence
of the Y chromosome (Steinlechner et al., 2002; Davis et al., 2012; Butler and Li, 2014; Ge et
al., 2014). However, CODIS has not decided to include other known advantageous alternatives
to Amelogenin.
What role does CODIS have in forensic genetics? CODIS is the national DNA database
in the United States comprised of three levels: local DNA index system (LDIS), state DNA index
system (SDIS), and national DNA index (NDIS; Butler and Li, 2014). Each system allows DNA
profiles to be exchanged and compared at the system’s designated level (Budowle et al., 1998).
Most countries have their own form of a forensic DNA database similar to CODIS. These
databases typically contain two types of profiles: reference profiles, and forensic profiles (Ge et
al., 2014; Arora, 2020). Databases are used to run reference DNA samples against the unknown
forensic profiles for possible profile matches, which does not always result in a match. For
example, it took five years before the Macedonia National DNA database resulted in a possible
match in one case report. The suspect was caught because of the account of an unrelated crime
five years after the initial case. While another case using the same database, a possible match
was already in the system when the unknown sample was run against the database (Jakovski et
al., 2017). Therefore, DNA databases need to be run regularly as new profiles are being
continuously added in order to check for unsuspected matches.
19
Courtroom and new evidence
The first use of DNA was not a forensic case, as mentioned earlier. The outcome of that
case was monumental in paving the way for the use of DNA in the courtroom in a forensic
context. The first forensic application was in 1987 in England (Visser and Hampikian, 2012;
Roewer, 2013). In 1996, the United States court system accepted the use of human mtDNA
(Lyons et al., 2014). However, despite the advancements in forensic genetics, people have still
been wrongly convicted based on genetic evidence. For example, in Australia, a boy was
convicted with DNA that had unknowingly been contaminated during the collection process
(Weathered et al., 2020). Genetic evidence can be useless if the database collection is limited,
there is a backlog of samples, profiles are not uploaded to CODIS, or data is misunderstood
(Visser and Hampikian, 2012). Yet, in the court system DNA is still the gold standard of
evidence. Therefore, it is essential to understand the public’s view of DNA (Visser and
Hampikian, 2012; Weathered et al., 2020). The “CSI Effect” is an increasing phenomenon that
occurs because jurors have an unrealistic expectation of evidence due to television and expect
DNA evidence to be entirely foolproof (Slabbert and Heathfield, 2018).
Today, DNA can be used in a forensic context beyond human applications. As briefly
discussed earlier, nonhuman elements are being applied to a forensic context. There are
differences seen in these uses compared to human DNA. For instance, different questions are
asked in wildlife DNA forensics, with some common questions: what is the species? is it wild or
captive-bred? or where is it from? These questions are then used to protect wildlife with various
laws and treaties (Moore and Frazier, 2019). Forensic genetics can then be applied in cases such
as protected species, food fraud, and poaching (Arenas et al., 2017; Amorim, 2019). In 2016, an
investigation of a bone necklace of expected whalebone (protected as an endangered species) led
20
to the conviction of a couple on a variety of accounts, including Lacey Acts violations, which
prohibits the trade of illegally acquired wildlife (Moore and Frazier, 2019). In 2009, the
mitochondria DNA of cat hair was first used in the murder trial of the State of Missouri versus
Henry L. Polk Jr., linking the suspect to the victim. At the time of the case, this specific cat had a
rare mitotype to allow the evidence to be admissible. However, now the dataset of USA cats is
sufficient for more general forensic applications (Lyons et al., 2014). Because of nonhuman
DNA, suspects can be linked to the crime. For example, pollen and fungal spores have also been
used to link a suspect to the scene from the palynological material on their shoes, which
contradicted the suspect’s statements (Allwood et al., 2020).
Probabilities and Likelihoods
When presenting the genetic evidence to a courtroom, its value is indicated with a
likelihood ratio. As mentioned earlier, the likelihood ratio communicates the probability of
genetic evidence belonging to a suspect (Caliebe et al., 2017; Carracedo and Prieto, 2019).
However, the ratio is not a probability according to the definition of probability theory, since the
likelihood ratio is not additive; instead, it measures the ‘rational belief’ of the two likelihood
hypotheses (Caliebe et al., 2017).
A recent analysis of probabilities indicated concern with the use of the likelihood ratio in
the courtroom. The current probability method is divided into five groups, beginning with 1 to 10
and ending with 10,000 or greater. This ratio range indicates the evidence provides limited
support through very strong support that the genetic material came from the suspect (Roberston
et al., 2016; Weathered et al., 2020). Based on this presentation of statistical evidence, two
concerns were observed. First, the likelihood ratio presentation to jury members was
significantly more challenging to understand than random match probability (RMPs). In the
21
study, the jury correctly interpreted 42% of the DNA evidence presented as likelihood ratios. In
comparison, 83% of the DNA evidence presented as RMPs was correctly interpreted (Weathered
et al., 2020). Second, the language and definition of a “partial profile” and “complete profile.”
The completeness of the profile does not indicate the number of loci used to create the profile.
Therefore, the likelihood ratio can theoretically be the same for both a partial and complete
profile. For example, a loci kit can use either 9 or 21 loci markers. If a profile were comprised of
9 loci, then depending on the kit, the 9 loci kit would generate a complete profile compared to a
partial profile for the 21 loci kit. Without this distinction, part of the evidence context is obscured
from the jury and can confuse them, potentially leading to problematic verdicts (Weathered et
al., 2020).
With the advances in analyzing DNA, additional probabilities have been introduced to
the criminal justice process. Forensic DNA phenotyping is a relatively new process that predicts
externally visible characteristics (Caliebe et al., 2017; Aggarwal, 2020). The method analyzes
single nucleotide polymorphisms to determine the phenotypes using posterior odds, which
contradicts the standard forensic reporting of only likelihood ratios in court. This probability for
FDP is used because the odds are independent of the population. Posterior odds are not currently
reported and used primarily by law enforcement, with few exceptions depending on the evidence
(Caliebe et al., 2017).
Unlike DNA profiles, FDP is not meant to be used as evidence because there are
limitations and ethical concerns about the FDA beyond its visible characteristic predictions
(Caliebe et al., 2017). The phenotypic report cannot estimate potential environmental factors that
can affect the genes, which can change the visible characteristics. The accuracy of the reports
decreases with profiles of mixed ancestry (Aggarwal, 2020). These limitations support the
22
expressed European concerns about the potential of FDP reports being misunderstood (Samuel
and Prainsack, 2019). Another concern is the privacy of the individual (Toom et al., 2016;
Slabbert and Heathfield, 2018; Samuel and Prainsack, 2019; Aggarwal, 2020). The regulation of
FDP is varying; for instance, the Netherlands allows the use of externally visible characteristics
but is limited to traits visible from birth that can contribute to the investigation, while South
Africa restricts the use of externally visible characteristics (Slabbert and Heathfield, 2018;
Wienroth, 2018). These points are essential to consider because of the “CSI Effect” phenomenon
since FDP is sometimes viewed as a “biological witness” (Kayser, 2015; Slabbert and
Heathfield, 2018; Machado and Granja, 2020).
Conclusion
Forensic genetics has a long history spanning several decades, with the most recent
advances being within the last decade. Understanding its history allows new questions to be
explored, and being able to understand the basics of each method allows for the integrity of the
DNA to be maintained throughout the forensic process. Even with recent advances in
technologies and methodologies, new concerns will arise from the general public. Research must
continue to focus on these current advances to address potential problems and allow scientist to
return to concepts not once considered.
23
References:
Abdel Hady RH, Thabet HZ, Ebrahem NE, Yassa HA. 2021. Thermal Effects on DNA
Degradation in Blood and Seminal Stains: Forensic View. Acad Forensic Pathol 11:7–23.
Adamowicz MS, Stasulli DM, Sobestanovich EM, Bille TW. 2014. Evaluation of methods to
improve the extraction and recovery of DNA from cotton swabs for forensic analysis. PLoS
One 9:1–18.
Aditya S, Sharma AK, Bhattacharyya CN, Chaudhuri K. 2011. Generating STR profile from
“touch DNA.” J Forensic Leg Med 18:295–298.
Aggarwal K. 2020. Forensic DNA Phenotyping: Significance in Criminal Investigations. Acad J
Forensic Sci 03:2581–4273.
Al-munim MFA, Al-rashedi NAM. 2021. Stability of DNA Quantitation State of Blood
Evidence under Different Conditions. 25:4965–4971.
Alaeddini R, Walsh SJ, Abbas A. 2010. Forensic Science International : Genetics Forensic
implications of genetic analyses from degraded DNA — A review. Forensic Sci Int Genet
4:148–157.
Alessandrini F, Onofri V, Turchi C, Buscemi L, Pesaresi M, Tagliabracci A. 2020. Past, Present
and Future in Forensic Human Identification. In: Longhi S, Monteriù A, Freddi A, Aquilanti
L, Ceravolo MG, Carnevali O, Giordano M, Moroncini G, editors. The First Outstanding 50
Years of “Universita Politecnica delle Marche”: Research Achievements in Life Sciences. .
p 81–92.
Alketbi SK, Goodwin W. 2019. The effect of time and environmental conditions on Touch DNA.
Forensic Sci Int Genet Suppl Ser 7:701–703.
Allwood JS, Fierer N, Dunn RR. 2020. The future of environmental DNA in forensic science.
Appl Environ Microbiol 86:1–9.
Alonso A, Müller P, Roewer L, Willuweit S, Budowle B, Parson W. 2017. European survey on
forensic applications of massively parallel sequencing. Forensic Sci Int Genet 29:e23–e25.
Aloraer D, Hassan NH, Albarzinji B, Goodwin W. 2015. Collection protocols for the recovery of
biological samples. Forensic Sci Int Genet Suppl Ser 5:e207–e209.
Ambers A, Wiley R, Novroski N, Budowle B. 2018. Direct PCR amplification of DNA from
human bloodstains, saliva, and touch samples collected with microFLOQ® swabs. Forensic
Sci Int Genet 32:80–87.
Amorim A. 2019. Nonhuman forensic genetics. Forensic Sci Int Genet Suppl Ser 7:44–46.
Amorim A, Pereira F, Alves C, Garcia O. 2020. Species assignment in forensics and the
challenge of hybrids. Forensic Sci Int Genet 48:101676.
Anchordoquy TJ, Molina MC. 2007. Preservation of DNA. Cell Preserv Technol 5:180–188.
Andersen K, Bird KL, Rasmussen M, Haile J, Breuning-Madsen H, Kjær KH, Orlando L, Gilbert
MTP, Willerslev E. 2012. Meta-barcoding of “dirt” DNA from soil reflects vertebrate
biodiversity. Mol Ecol 21:1966–1979.
Anon. 2017. Rapid DNA Act of 2017. 115th Congress. Available from:
https://www.congress.gov/bill/115th-congress/house-bill/510
24
Anzai-Kanto E, Hirata MH, Hirata RDC, Nunes FD, Melani RFH, Oliveira RN. 2005. Extração
de DNA de saliva humana depositada sobre a pele e sua aplicabilidade aos processos de
identificação forense. Braz Oral Res 19:216–222.
Arenas M, Pereira F, Oliveira M, Pinto N, Lopes AM, Gomes V, Carracedo A, Amorim A. 2017.
Forensic genetics and genomics: Much more than just a human affair. PLoS Genet 13:1–28.
Arnold LJ, Roberts RG, Macphee RDE, Haile JS, Brock F, Möller P, Froese DG, Tikhonov AN,
Chivas AR, Gilbert MTP, Willerslev E. 2011. Paper II - Dirt, dates and DNA: OSL and
radiocarbon chronologies of perennially frozen sediments in Siberia, and their implications
for sedimentary ancient DNA studies. Boreas 40:417–445.
Arora A. 2020. Future of Forensic and Crime Scene Science Technologies. Technol Forensic
Sci:357–370.
Asari M, Matsuura H, Isozaki S, Hoshina C, Okuda K, Tanaka H, Horioka K, Shiono H, Shimizu
K. 2018. Assessment of DNA degradation of buccal cells under humid conditions and DNA
repair by DOP-PCR using locked nucleic acids. Leg Med 35:29–33.
Ayala-Torres S, Chen Y, Svoboda T, Rosenblatt J, Van Houten B. 2000. Analysis of gene-
specific DNA damage and repair using quantitative polymerase chain reaction. Methods
22:135–147.
Baechler S. 2016. Study of criteria influencing the success rate of DNA swabs in operational
conditions: A contribution to an evidence-based approach to crime scene investigation and
triage. Forensic Sci Int Genet 20:130–139.
Balding DJ, Buckleton J. 2009. Interpreting low template DNA profiles. Forensic Sci Int Genet
4:1–10.
Ballou S, Stolorow M, Taylor M, Bamberger PS, Brown L, Brown R, Burney Y, Davenport D,
DePalma L, Williams S, Jones C, Keaton R, Kiley W, Latta J, Kline M, Lanning K, LaPorte
G, Ledray LE, Nagy R, Ostrom BE, Schwind L, Stoiloff S. 2013. The biological evidence
preservation handbook : best practices for evidence handlers ; technical working group on
biological evidence preservation.
Barash M, Reshef A, Brauner P. 2010. The use of adhesive tape for recovery of dna from crime
scene items. J Forensic Sci 55:1058–1064.
Barlev A, Sen D. 2018. DNA’s Encounter with Ultraviolet Light: An Instinct for Self-
Preservation? Acc Chem Res 51:526–533.
Basset P, Castella V. 2018. Lessons from a study of DNA contaminations from police services
and forensic laboratories in Switzerland. Forensic Sci Int Genet 33:147–154.
Berglund EC, Kiialainen A, Syvänen AC. 2011. Next-generation sequencing technologies and
applications for human genetic history and forensics. Investig Genet 2:1–15.
Bhinder M, Zahoor M, Sadia H, Qasim M, Perveen R, Anjum G, Iqbal M, Ullah N, Shehzad W,
Tariq M, AM Waryah. 2018. SE33 locus as a reliable genetic marker for forensic DNA
analysis systems. Turkish J Med Sci 48:611–614.
Blears MJ, De Grandis SA, Lee H, Trevors JT. 1998. Amplified fragment length polymorphism
(AFLP): A review of the procedure and its applications. J Ind Microbiol Biotechnol 21:99–
25
114.
Blozis J. 2014. Forensic DNA evidence collection at a crime scene: an investigator’s
commentary. In: Shewale JG, Liu RH, editors. Forensic DNA Analysis Current Practices
and Emerging Technologies. Boca Raton: CRC Press. p 3–17.
Bonnet J, Colotte M, Coudy D, Couallier V, Portier J, Morin B, Tuffet S. 2009. Chain and
conformation stability of solid-state DNA: Implications for room temperature storage.
Nucleic Acids Res 38:1531–1546.
Bonsu DOM, Higgins D, Austin JJ. 2020. Forensic touch DNA recovery from metal surfaces – A
review. Sci Justice 60:206–215.
Børsting C, Morling N. 2015. Next generation sequencing and its applications in forensic
genetics. Forensic Sci Int Genet 18:78–89.
Brandsch R. 2017. Probabilistic migration modelling focused on functional barrier efficiency and
low migration concepts in support of risk assessment. Food Addit Contam Part A 34:1743–
1766.
Bright JA, Kelly H, Kerr Z, McGovern C, Taylor D, Buckleton JS. 2020. The interpretation of
forensic DNA profiles: an historical perspective. J R Soc New Zeal 50:211–225.
Bright JA, Petricevic SF. 2004. Recovery of trace DNA and its application to DNA profiling of
shoe insoles. Forensic Sci Int 145:7–12.
Brogna R, Oldenhof H, Sieme H, Wolkers WF. 2020. Spectral fingerprinting to evaluate effects
of storage conditions on biomolecular structure of filter-dried saliva samples and recovered
DNA. Sci Rep 10:1–12.
Brownlow RJ, Dagnall KE, Ames CE. 2012. A Comparison of DNA Collection and Retrieval
from Two Swab Types (Cotton and Nylon Flocked Swab) when Processed Using Three
QIAGEN Extraction Methods. J Forensic Sci 57:713–717.
Bruijns B, Tiggelaar R, Gardeniers H. 2018. Massively parallel sequencing techniques for
forensics: A review. Electrophoresis 39:2642–2654.
Buckleton J. 2009. Validation issues around DNA typing of low level DNA. Forensic Sci Int
Genet 3:255–260.
Budowle B, Moretti TR, Niezgoda SJ, Brown BL. 1998. CODIS and PCR-Based Short Tandem
Repeat Loci: Law Enforcement Tools. Second Eur Symp Hum Identif 7388:73–88.
Burrill J, Daniel B, Frascione N. 2019. A review of trace “Touch DNA” deposits: Variability
factors and an exploration of cellular composition. Forensic Sci Int Genet 39:8–18.
Burrows AM, Kasu M, D’Amato ME. 2019. Preservation of DNA integrity in biological
material. Forensic Sci Int Genet Suppl Ser 7:416–418.
Burrows AM, Ristow PG, D’Amato ME. 2017. Preservation of DNA from saliva samples in
suboptimal conditions. Forensic Sci Int Genet Suppl Ser 6:e80–e81.
Butler E, Li R. 2014. Genetic Markers for Sex Identification in Forensic DNA Analysis. J
Forensic Investig 02.
Butler JM. 2015. The future of forensic DNA analysis. Philos Trans R Soc B Biol Sci 370.
Butler JM, Coble MD, Vallone PM. 2007. STRs vs. SNPs: Thoughts on the future of forensic
26
DNA testing. Forensic Sci Med Pathol 3:200–205.
Butler JM, Hill CR, Kline MC, Duewer DL, Sprecher CJ, McLaren RS, Rabbach DR, Krenke
BE, Storts DR. 2009. The single most polymorphic STR Locus: SE33 performance in U.S.
populations. Forensic Sci Int Genet Suppl Ser 2:23–24.
Butler JM, Willis S. 2020. Interpol review of forensic biology and forensic DNA typing 2016-
2019. Forensic Sci Int Synerg.
Byard RW, James H, Berketa J, Heath K. 2016. Locard’s Principle of Exchange, Dental
Examination and Fragments of Skin. J Forensic Sci 61:545–547.
Byrn SR, Xu W, Newman AW. 2001. Chemical reactivity in solid-state pharmaceuticals:
Formulation implications. Adv Drug Deliv Rev 48:115–136.
Cale CM, Earll ME, Latham KE, Bush GL. 2016. Could Secondary DNA Transfer Falsely Place
Someone at the Scene of a Crime? J Forensic Sci 61:196–203.
Caliebe A, Walsh S, Liu F, Kayser M, Krawczak M. 2017. Likelihood ratio and posterior odds in
forensic genetics: Two sides of the same coin. Forensic Sci Int Genet 28:203–210.
Carracedo Á, Prieto L. 2019. Beyond the CSI effect: The keys to good forensic genetics
communication. Metode 2019:31–37.
Cătălin M, Andrei A, Mitraşca O. 2011. Modern Methods of Collection and Preservation of
Biological Evidence for Human Identification by DNA Analysis. Abacus Diagnostics.
Chauhan M. 2020. Storage of saliva and blood specimen in different temperature. Int J Forensic
Med 2:21–24.
Chong KWY, Thong Z, Syn CK. 2021. Recent trends and developments in forensic DNA
extraction . WIREs Forensic Sci 3:1–23.
Clabaugh K, Silva B, Odigie K, Fourney R, Stevens J, Carmody G, Coble MD, Loreille O,
Scheible M, Kline M, Parsons TJ. 2007. Storage of DNA samples at ambient temperature
using DNA-SampleMatrix. Poster Present 18th Annu Meet Int Symp Hum Identification,
Hollywood, CA.
Colotte M, Coudy D, Tuffet S, Bonnet J. 2011. Adverse Effect of Air Exposure on the Stability
of DNA Stored at Room Temperature. Biopreserv Biobank:47–50.
Comte J, Baechler S, Gervaix J, Lock E, Milon MP, Delémont O, Castella V. 2019. Touch DNA
collection – Performance of four different swabs. Forensic Sci Int Genet 43.
Cordray R. 2010. Guidelines for preservation and retention of biological evidence.
Corradini B, Alù M, Magnanini E, Galinier ME, Silingardi E. 2019. The importance of forensic
storage support: DNA quality from 11-year-old saliva on FTA cards. Int J Legal Med
133:1743–1750.
Dadhania A, Nelson M, Caves G, Santiago R, Podini D. 2013. Evaluation of Copan
4N6FLOQSwabsTM used for crime scene evidence collection. Forensic Sci Int Genet Suppl
Ser 4:e336–e337.
Daly DJ, Murphy C, McDermott SD. 2012. The transfer of touch DNA from hands to glass,
fabric and wood. Forensic Sci Int Genet 6:41–46.
Dargay A, Roy R. 2016. Direct Y-STR amplification of body fluids deposited on commonly
27
found crime scene substrates. J Forensic Leg Med 39:50–60.
Dash HR, Shrivastava P, Das S. 2020a. Principles and Practices of DNA Analysis: A Laboratory
Manual for Forensic DNA Typing. New York, NY: Springer Protocols Handbook.
Dash HR, Shrivastava P, Das S. 2020b. Biological Samples: The Target Sources for DNA
Typing. In: Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic
DNA Typing. New York, NY: Humana. p 13–20.
Dash HR, Shrivastava P, Das S. 2020c. Collection, Transportation, and Preservation of
Biological Evidences for DNA Analysis. In: Principles and Practices of DNA Analysis: A
Laboratory Manual for Forensic DNA Typing. New York, NY: Humana. p 21–27.
Dash HR, Shrivastava P, Das S. 2020d. Reliable Use of WhatmanTM FTATM Cards for One-Step
Collection and Isolation of DNA. In: Principles and Practices of DNA Analysis: A
Laboratory Manual for Forensic DNA Typing. New York, NY: Humana. p 109–115.
Davis C, Illescas M, Tirado C, Lopez R, Budowle B, Cruz TD. 2012. A Case of Amelogenin Y-
null: A simple primer binding site mutation or unusual genetic anomaly? Leg Med 14:320–
323.
Davis DL, O’Brie EP, Bentzley CM. 2000. Analysis of the degradation of oligonucleotide
strands during the freezing/thawing processes using MALDI-MS. Anal Chem 72:5092–
5096.
Department of Public Safety - Texas. 2012. Best practices for collection, packaging, storage,
preservation, and retrieval of biological evidence. :1–8.
Dickerson RE, Drew HR, Conner BN, Wing RM, Fatini A V., Kopka ML. 1982. The Anatomy
of A-, B-, and Z-DNA. Science (80- ) 216:475–485.
Diegoli TM. 2015. Forensic typing of short tandem repeat markers on the X and Y
chromosomes. Forensic Sci Int Genet 18:140–151.
Dissing J, Søndervang A, Lund S. 2010. Exploring the limits for the survival of DNA in blood
stains. J Forensic Leg Med 17:392–396.
Dong H, Wang J, Zhang T, Ge JY, Dong YQ, Sun QF, Liu C, Li CX. 2017. Comparison of
preprocessing methods and storage times for touch DNA samples. Croat Med J 58:4–13.
Durose M. 2008. Census of Publicly Funded Forensic Crime Laboratories, 2005, Bureau of
Justice Statistics. :1–12.
Dziak R, Peneder A, Buetter A, Hageman C. 2018. Trace DNA Sampling Success from
Evidence Items Commonly Encountered in Forensic Casework. J Forensic Sci 63:835–841.
Emmons AL, DeBruyn JM, Mundorff AZ, Cobaugh KL, Cabana GS. 2017. The persistence of
human DNA in soil following surface decompositions. Sci Justice 57:341–348.
Erlich H. 2020. In the Begginning: Forensic Applications of DNA Technologies. In: Erlich H,
Stover E, White TJ, editors. Silent Witness: Forensic DNA Evidence in Criminal
Investigations and Humanitarian Disaters. Oxford Univeristy Press. p 15–33.
Erlich H, Calloway C, Lee SB. 2020. Recent Developments in Forensic DNA Technology. In:
Erlich H, Stover E, White TJ, editors. Silent Witness: Forensic DNA Evidence in Criminal
Investigations and Humanitarian Disaters. Oxford Univeristy Press. p 105–127.
28
Esslinger KJ, Siegel JA, Spillane H, Stallworth S. 2004. Using STR Analysis to Detect Human
DNA from Exploded Pipe Bomb Devices. J Forensic Sci 49:1–4.
Fabre A-L, Luis A, Colotte M, Tuffet S, Bonnet J. 2017. High DNA stability in white blood cells
and buffy coat lysates stored at ambient temperature under anoxic and anhydrous
atmosphere. PLoS One 12:e0188547.
Fang X, Vitrac O. 2017. Predicting diffusion coefficients of chemicals in and through packaging
materials. Crit Rev Food Sci Nutr 57:275–312.
de Fátima Poças M, Oliveria JC, Peteira JR, Brandsch R, Hogg T. 2011. Modelling migration
from paper into a food simulant. Food Control 22:303–312.
Fonneløp AE, Johannessen H, Egeland T, Gill P. 2016. Contamination during criminal
investigation: Detecting police contamination and secondary DNA transfer from evidence
bags. Forensic Sci Int Genet.
Frippiat C, Noel F. 2014. Efficiency of a novel forensic room-temperature DNA storage
medium. Forensic Sci Int Genet 9:81–84.
Gaillard C, Strauss F. 1998. Avoiding adsorption of DNA to polypropylene tubes and
denaturation of short DNA fragments. Tech Tips Online 3:63–65.
Garvin AM, Holzinger R, Berner F, Krebs W, Hostettler B, Lardi E, Hertli C, Quartermaine R,
Stamm C. 2013. The forensix evidence collection tube and its impact on dna preservation
and recovery. Biomed Res Int 2013.
GE Healthcare. 2010. Reliable extraction of DNA from Whatman FTA cards. Appl Note 28-
9822-22 AA.
Ge J, Sun H, Li H, Liu C, Yan J, Budowle B. 2014. Future directions of forensic DNA databases.
Croat Med J 55:163–166.
Ghosh A, Bansal M. 2003. A glossary of DNA structures from A to Z. Acta Crystallogr - Sect D
Biol Crystallogr 59:620–626.
Goray M, Eken E, Mitchell RJ, van Oorschot RAH. 2010. Secondary DNA transfer of biological
substances under varying test conditions. Forensic Sci Int Genet 4:62–67.
Gršković B, Zrnec D, Popović M, Petek MJ, Primorac D, Mršić G. 2013. Effect of ultraviolet c
radiation on biological samples. Croat Med J 54:263–271.
Gunnarsson J, Helena E, Ansell R. 2010. Success rates of a forensic tape-lift method for DNA
recovery. Probl Forensic Sci LXXXIII:243–254.
Haile J, Holdaway R, Oliver K, Bunce M, Gilbert MTP, Nielsen R, Munch K, Ho SYW, Shapiro
B, Willerslev E. 2007. Ancient DNA chronology within sediment deposits: Are
paleobiological reconstructions possible and is DNA leaching a factor? Mol Biol Evol
24:982–989.
Hakim HM, Lalung J, Khan HO, Ismail SA, Aziz MY, Ishak AR, Safuan S, Rasudin NS,
Chambers GK, Edinur HA. 2020. Evaluation of long-term storage effects on buccal cell
DNA from untreated cards for STR profiling. IOP Conf Ser Earth Environ Sci 596.
Hall A, Sims LM, Ballantyne J. 2014. Assessment of DNA damage induced by terrestrial UV
irradiation of dried bloodstains: Forensic implications. Forensic Sci Int Genet 8:24–32.
29
Hall D, Fairley M. 2004. A single approach to the recovery of DNA and firearm discharge
residue evidence. Sci Justice - J Forensic Sci Soc 44:15–19.
Hanson E., Ballantyne J. 2013. “Getting blood from a stone”: ultrasensitive forensic DNA
profiling of microscopic bio-particles recovered from “touch DNA” evidence. In: Nucleic
Acids Detection. Totowa, NJ: Humana Press. p 3–17.
Hara M, Nakanishi H, Yoneyama K, Saito K, Takada A. 2016. Effects of storage conditions on
forensic examinations of blood samples and bloodstains stored for 20 years. Leg Med
18:81–84.
Hares DR. 2015. Selection and implementation of expanded CODIS core loci in the United
States. Forensic Sci Int Genet 17:33–34.
Hauhart R, Menius K. 2014. DNA Evidence: Examining Police Officers’ Knowledge of
Handling Procedures in a Mid-Size Department. Int J Criminol Sociol 3:360–376.
Hebda LM, Doran AE, Foran DR. 2014. Collecting and analyzing DNA evidence from
fingernails: A comparative study. J Forensic Sci 59:1343–1350.
Hebsgaard MB, Arneborg J, Heyn P, Allentoft ME, Bunce M, Schweger C, Willerslev E. 2009.
‘The Farm Beneath the Sand’ – an archaeological case study on ancient ‘dirt’ DNA.
Antiquity 83:430–444.
Hedman J, Jansson L, Akel Y, Wallmark N, Gutierrez Liljestrand R, Forsberg C, Ansell R. 2020.
The double-swab technique versus single swabs for human DNA recovery from various
surfaces. Forensic Sci Int Genet 46:20–24.
Hefetz I, Einot N, Faerman M, Horowitz M, Almog J. 2019. Touch DNA: The effect of the
deposition pressure on the quality of latent fingermarks and STR profiles. Forensic Sci Int
Genet 38:105–112.
Helmus J, Bajanowski T, Poetsch M. 2016. DNA transfer—a never ending story. A study on
scenarios involving a second person as carrier. Int J Legal Med 130:121–125.
Hess S, Haas C. 2017. Recovery of Trace DNA on Clothing: A Comparison of Mini-tape Lifting
and Three Other Forensic Evidence Collection Techniques. J Forensic Sci 62:187–191.
Hogan C, Houten LB Van, Coticone S. 2018. Comparison of the Quantity and Overall Quality of
Trace DNA Evidence Collected from Substrates Found at Crime Scenes. J Forensic Identif
68.
Holland M, Melton T, Holland C. 2013. Forensic Mitochondrial DNA Analysis: Current Practice
and Future Potential. In: Shewale JG, Liu RH, editors. Forensic DNA Analysis: Current
Practices and Emerging Technologies. CRC Press. p 249–278.
Hopman R, M’charek A. 2020. Facing the unknown suspect: forensic DNA phenotyping and the
oscillation between the individual and the collective. Biosocieties 15:438–462.
Howlett SE, Castillo HS, Gioeni LJ, Robertson JM, Donfack J. 2014. Evaluation of
DNAstableTM for DNA storage at ambient temperature. Forensic Sci Int Genet.
Hytinen ME, Solomon AD, Miller MT, Cruz TD. 2017. Methods for Obtaining High-Quality
Touch DNA from a Nonporous Surface after Latent Fingerprint Collection. J Forensic
Identif 67:71–84.
30
Ip SCY, Yu EY, Li C. 2021. Blood DNA Preservation on Various Forensic Swab Devices. J
Forensic Identif 71.
Ivanova N V., Kuzmina ML. 2013. Protocols for dry DNA storage and shipment at room
temperature. Mol Ecol Resour 13:890–898.
Jakovski Z, Ajanovska RJ, Stankov A, Poposka V, Bitoljanu N, Belakaposka V. 2017. The
power of forensic DNA data bases in solving crime cases. Forensic Sci Int Genet Suppl Ser
6:e275–e276.
Janssen K, Aune M, Olsen M, Olsen GH, Berg T. 2019. Biological stain collection – Absorbing
paper is superior to cotton swabs. Forensic Sci Int Genet Suppl Ser 7:468–469.
Jeffreys A, Brookfield J, Semeonoff R. 1985. Positive identification of an immigration test-case
using human DNA fingerprints. Nature 317:818–819.
Joël J, Glanzmann B, Germann U, Cossu C. 2015. DNA extraction of forensic adhesive tapes—
A comparison of two different methods. Forensic Sci Int Genet Suppl Ser 5:e579–e581.
Junkin T. 2005. Bloodsworth:The True Story of One Man’s Triumph Over Injustice. Algonquin
Books.
Kamphausen T, Schadendorf D, Von Wurmb-Schwark N, Bajanowski T, Poetsch M. 2012. Good
shedder or bad shedder- The influence of skin diseases on forensic DNA analysis from
epithelial abrasions. Int J Legal Med 126:179–183.
Karantzali E, Rosmaraki P, Kotsakis A, Le Roux-Le Pajolec MG, Fitsialos G. 2019. The effect
of FBI CODIS Core STR Loci expansion on familial DNA database searching. Forensic Sci
Int Genet 43.
Kayser M. 2015. Forensic DNA Phenotyping: Predicting human appearance from crime scene
material for investigative purposes. Forensic Sci Int Genet 18:33–48.
Kayser M. 2017. Forensic use of Y-chromosome DNA: a general overview. Hum Genet
136:621–635.
Keller PE, Kouzes R. 2017. Water Vapour Permeation in Plastics. :29.
Kirgiz IA, Calloway C. 2017. Increased recovery of touch DNA evidence using FTA paper
compared to conventional collection methods. J Forensic Leg Med 47:9–15.
Kita T, Yamaguchi H, Yokoyama M, Tanaka T, Tanaka N. 2008. Morphological study of
fragmented DNA on touched objects. Forensic Sci Int Genet 3:32–36.
Kline MC, Duewer DL, Redman JW, Butler JM. 2005. Results from the NIST 2004 DNA
Quantitation Study. J Forensic Sci 50:1–8.
Kloosterman A, Sjerps M, Quak A. 2014. Error rates in forensic DNA analysis: Definition,
numbers, impact and communication. Forensic Sci Int Genet 12:77–85.
Kumar KR, Cowley MJ, Davis RL. 2019. Next-Generation Sequencing and Emerging
Technologies. Semin Thromb Hemost 45:661–673.
Ladd C, Adamowicz MS, Bourke MT, Scherczinger CA, Lee HC. 1999. A Systematic Analysis
of Secondary DNA Transfer. J Forensic Sci 44:14599J.
Lapointe M, Rogic A, Bourgoin S, Jolicoeur C, Séguin D. 2015. Leading-edge forensic DNA
analyses and the necessity of including crime scene investigators, police officers and
31
technicians in a DNA elimination database. Forensic Sci Int Genet 19:50–55.
Lawrence MG. 2005. The relationship between relative humidity and the dewpoint temperature
in moist air: A simple conversion and applications. Bull Am Meteorol Soc 86:225–233.
Lee HC, Ladd C. 2001. Preservation and collection of biological evidence. Croat Med J 42:225–
228.
Lee S, Crouse C, Kline M. 2013. Optimizing storage and handling of DNA extracts. In: Shewale
JG, editor. Forensic DNA Analysis: Current Practices and Emerging Technologies. CRC
Press. p 19–64.
Lee SB, Clabaugh KC, Silva B, Odigie KO, Coble MD, Loreille O, Scheible M, Fourney RM,
Stevens J, Carmody GR, Parsons TJ, Pozder A, Eisenberg AJ, Budowle B, Ahmad T, Miller
RW, Crouse CA. 2012. Assessing a novel room temperature DNA storage medium for
forensic biological samples. Forensic Sci Int Genet 6:31–40.
Li B, Wang ZW, Lin QB, Hu CY. 2017. Molecular dynamics simulation of three plastic
additives’ diffusion in polyethylene terephthalate. Food Addit Contam Part A 34:1086–
1099.
Li C. 2018. Forensic genetics. Forensic Sci Res 3:103–104.
Linacre A, Pekarek V, Swaran YC, Tobe SS. 2010. Generation of DNA profiles from fabrics
without DNA extraction. Forensic Sci Int Genet 4:137–141.
Lowe A, Murray C, Whitaker J, Tully G, Gill P. 2002. The propensity of individuals to deposit
DNA and secondary transfer of low level DNA from individuals to inert surfaces. Forensic
Sci Int 129:25–34.
Lyons LA, Grahn RA, Kun TJ, Netzel LR, Wictum EE, Halverson JL. 2014. Acceptance of
domestic cat mitochondrial DNA in a criminal proceeding. Forensic Sci Int Genet 13:61–
67.
Machado H, Granja R. 2020. Forensic Genetics in the Governance of Crime.
Machado H, Silva S. 2019. What influences public views on forensic DNA testing in the
criminal field? A scoping review of quantitative evidence. Hum Genomics 13:23.
Maia J, Rodriguez-Bernaldo de Quirós A, Sendón R, Cruz JM, Seiler A, Franz R, Simoneau C,
Castle L, Driffield M, Mercea P, Oldring P, Tosa V, Paseiro P. 2016. Determination of key
diffusion and partition parameters and their use in migration modelling of benzophenone
from low-density polyethylene (LDPE) into differnt foodstuffs. Food Addit Contam Part A
33:715–724.
Mandel P, Metais P. 1948. Les acides nucleiques du plasma sanguin chez l’homme. CR Seances
Soc Biol Fil 142:241–243.
Mapes AA, Kloosterman AD, van Marion V, de Poot CJ. 2016. Knowledge on DNA Success
Rates to Optimize the DNA Analysis Process: From Crime Scene to Laboratory. J Forensic
Sci 61:1055–1061.
Marrone A, Ballantyne J. 2010. Hydrolysis of DNA and its molecular components in the dry
state. Forensic Sci Int Genet 4:168–177.
McCartney C, Shorter L. 2020. Police retention and storage of evidence in England and Wales.
32
Int J Police Sci Manag 22:123–136.
McCord BR, Gauthier Q, Cho S, Roig MN, Gibson-Daw GC, Young B, Taglia F, Zapico SC,
Mariot RF, Lee SB, Duncan G. 2019. Forensic DNA Analysis. Anal Chem 91:673–688.
Milne E, Van Bockxmeer FM, Robertson L, Brisbane JM, Ashton LJ, Scott RJ, Armstrong BK.
2006. Buccal DNA collection: Comparison of buccal swabs with FTA cards. Cancer
Epidemiol Biomarkers Prev 15:816–819.
Mistek E, Fikiet MA, Khandasammy SR, Lednev IK. 2019. Toward Locard’s Exchange
Principle: Recent Developments in Forensic Trace Evidence Analysis. Anal Chem 91:637–
654.
Moore MK, Frazier K. 2019. Humans Are Animals, Too: Critical Commonalities and
Differences Between Human and Wildlife Forensic Genetics. J Forensic Sci 64:1603–1621.
Moretti TR, Moreno LI, Smerick JB, Pignone ML, Hizon R, Buckleton JS, Bright JA, Onorato
AJ. 2016. Population data on the expanded CODIS core STR loci for eleven populations of
significance for forensic DNA analyses in the United States. Forensic Sci Int Genet 25:175–
181.
Morgan TH. 1917. The Theory of the Gene. Am Nat 51:513–544.
Mozayani A, Fisher CP eds. 2017. Forensic Evidence Management: From the Crime Scene to the
Courtroom. CRC Press.
Mueller UG, Wolfenbarger LL. 1999. AFLP genotyping and fingerprinting. Trends Ecol Evol
14:389–394.
Murphy E. 2018. Forensic DNA typing. Annu Rev Criminol 1:497–515.
Nachman MW, Crowell SL. 2000. Estimate of the mutation rate per nucleotide in humans.
Genetics 156:297–304.
Nelson M. 2010. Making sense of DNA backlogs: myths vs. reality (Report No. NCJ 232197).
National Insitute of Justice, Office of Justice Programs, U.S. Department of Justice.
Ng HH, Ang HC, Hoe SY, Lim M-L, Tai HE, Soh RCH, Syn CK-C. 2018. Simple DNA
extraction of urine samples: Effects of storage temperature and storage time. Forensic Sci
Int 287:36–39.
Noël S, Lagace K, Rogic A, Granger D, Bourgoin S, Jolicoeur C, Séguin D. 2016. DNA transfer
during laundering may yield complete genetic profiles. Forensic Sci Int Genet 23:240–247.
Novroski NMM, Wendt FR, Woerner AE, Bus MM, Coble M, Budowle B. 2019. Expanding
beyond the current core STR loci: An exploration of 73 STR markers with increased
diversity for enhanced DNA mixture deconvolution. Forensic Sci Int Genet 38:121–129.
Oliveira TP, Nogueira TLS, Valentin ESB, Santos OCL, Carvalho EF, Silva DA. 2015.
Evaluation of collection and extraction methodologies of latent fingerprints for military
application. Forensic Sci Int Genet Suppl Ser 5:e474–e475.
van Oorschot RAH. 2012. Assessing DNA Profiling Success Rates: Need for More and Better
Collection of Relevant Data. Forensic Sci Policy Manag An Int J 3:37–41.
van Oorschot RAH, Ballantyne KN, Mitchell RJ. 2010. Forensic trace DNA: A review. Investig
Genet 1:1–17.
33
van Oorschot RAH, Jones MK. 1997. DNA fingerprints from fingerprints. Nature 387:766–767.
Ostojic L, Wurmbach E. 2017. Analysis of fingerprint samples, testing various conditions, for
forensic DNA identification. Sci Justice 57:35–40.
Pang BCM, Cheung BKK. 2007. Double swab technique for collecting touched evidence. Leg
Med 9:181–184.
Panneerchelvam S, Norazmi MN. 2003. Forensic DNA profiling and database. Malaysian J Med
Sci 10:20–26.
Paunescu D, Puddu M, Soellner JO, Stoessel PR, Grass RN. 2013. Reversible DNA
encapsulation in silica to produce ROS-resistant and heat-resistant synthetic DNA’fossils’.
Nat Protoc 8:2440–2448.
Pickrahn I, Kreindl G, Müller E, Dunkelmann B, Zahrer W, Cemper-Kiesslich J, Neuhuber F.
2017. Contamination incidents in the pre-analytical phase of forensic DNA analysis in
Austria—Statistics of 17 years. Forensic Sci Int Genet 31:12–18.
Pizzamiglio M, Mameli A, My D, Garofano L. 2004. Forensic identification of a murderer by
LCN DNA collected from the inside of the victim’s car. Int Congr Ser 1261:437–439.
Plaza DT, Mealy JL, Lane JN, Parsons MN, Bathrick AS, Slack DP. 2016. Nondestructive
Biological Evidence Collection with Alternative Swabs and Adhesive Lifters. J Forensic Sci
61:485–488.
Poetsch M, Bajanowski T, Kamphausen T. 2013. Influence of an individual’s age on the amount
and interpretability of DNA left on touched items. Int J Legal Med 127:1093–1096.
Poetsch M, Pfeifer M, Konrad H, Bajanowski T, Helmus J. 2018. Impact of several wearers on
the persistence of DNA on clothes—a study with experimental scenarios. Int J Legal Med
132:117–123.
Pourazar A. 2007. Red cell antigens: Structure and function. Asian J Transfus Sci 1:24–32.
Prasad MSS, Vardhanan YS. 2018. Evaluation of efficacy of collection techniques for human
genomic DNA MAOA-uVNTR polymorphism. Int J Sci Res Biol Sci 5:6–11.
Quinlan A. 2020. Visions of Public Safety, Justice, and Healing: The Making of the Rape Kit
Backlog in the United States. Soc Leg Stud 29:225–245.
Quinones I, Daniel B. 2012. Cell free DNA as a component of forensic evidence recovered from
touched surfaces. Forensic Sci Int Genet 6:26–30.
Quinque D, Kittler R, Kayser M, Stoneking M, Nasidze I. 2006. Evaluation of saliva as a source
of human DNA for population and association studies. Anal Biochem 353:272–277.
Raina A, Pramanik P, Dogra TD. 2004. Effect of storage conditions of seminal stains on
different textures of clothes in relation to DNA yield. Indian Congr Forensic Med Toxicol 2.
Ravanat J-L, Douki T, Cadet J. 2001. Direct and indirect effects of UV radiation on DNA and its
components. J Photochem Photobiol 63:88–102.
Raymond JJ, van Oorschot RAH, Gunn PR, Walsh SJ, Roux C. 2009. Trace evidence
characteristics of DNA: A preliminary investigation of the persistence of DNA at crime
scenes. Forensic Sci Int Genet 4:26–33.
Reich DE, Schaffner SF, Daly MJ, McVean G, Mullikin JC, Higgins JM, Richter DJ, Lander ES,
34
Altshuler D. 2002. Human genome sequence variation and the influence of gene history,
mutation and recombination. Nat Genet 32:135–142.
Richter C, Park JW, Ames BN. 1988. Normal oxidative damage to mitochondrial and nuclear
DNA is extensive. Proc Natl Acad Sci U S A 85:6465–6467.
Roberston B, Vignaux GA, Berger CEH. 2016. Explaining the Strength of Evidence. In:
Interpreting Evidence: Evaluating forensic science in the courtroom. 2nd ed. John Wiley &
Sons, Ltd. p 55–67.
Roewer L. 2013. DNA fingerprinting in forensics: Past, present, future. Investig Genet 4:1–10.
Rutty GN, EAM G. 2005. DNA/risk of contamination. In: Payne-James J, Byard RW, Corey TS,
Henderson C, editors. Encyclopedia of forensic and legal medicine. volume 2. . p 189–198.
Safarikova M, Kubena AA, Frankova V, Zima T, Kalousova M. 2021. The effects of different
storage conditions and repeated freeze/thaw cycles on the concentration, purity and integrity
of genomic DNA. Folia Biol (Czech Republic) 67:10–15.
Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA. 1986. Analysis of enzymatically
amplified β-globin and HLA-DQα DNA with allele-specific oligonucleotide probes. Nature
324:163–166.
Samuel G, Prainsack B. 2019. Forensic DNA phenotyping in Europe: views “on the ground”
from those who have a professional stake in the technology. New Genet Soc 38:119–141.
Sessa F, Salerno M, Bertozzi G, Messina G, Ricci P, Ledda C, Rapisarda V, Cantatore S,
Turillazzi E, Pomara C. 2019. Touch DNA: Impact of handling time on touch deposit and
evaluation of different recovery techniques: An experimental study. Sci Rep 9:1–9.
Shewale JG, Liu RH eds. 2013. Forensic DNA analysis: current practices and emerging
technologies. CRC Press.
Shi B, Shin YK, Hassanali AA, Singer SJ. 2015. DNA Binding to the Silica Surface. J Phys
Chem B 119:11030–11040.
Shikama K. 1965. Effect of freezing and thawing on the stability of double helix of DNA. Nature
207:529–530.
Sirker M, Schneider PM, Gomes I. 2016. A 17-month time course study of human RNA and
DNA degradation in body fluids under dry and humid environmental conditions. Int J Legal
Med 130:1431–1438.
Slabbert N, Heathfield LJ. 2018. Ethical, legal and social implications of forensic molecular
phenotyping in South Africa. Dev World Bioeth 18:171–181.
Stannett V, Williams JL. 2007. The permeability of poly(ethyl methacrylate) to gases and water
vapor. J Polym Sci Part C Polym Symp 10:45–59.
Steinlechner M, Berger B, Niederstätter H, Parson W. 2002. Rare failures in the amelogenin sex
test. Int J Legal Med 116:117–120.
Storm KJ, Ropero-Miller J, Jones S, Sikes N, Pope M, Horstmann N. 2009. The 2007 Survey of
Law Enforcement Forensic Evidence Processing. Rockville.
Strom KJ, Hickman M. 2010. Processing in Police Departments. Criminol Public Policy 9:381–
404.
35
Sweet D, Hildebrand D. 1999. Saliva from cheese bite yields DNA profile of burglar: A case
report. Int J Legal Med 112:201–203.
Sweet D, Lorente M, Lorente JA, Valenzuela A, Villanueva E. 1997. An Improved Method to
Recover Saliva from Human Skin: The Double Swab Technique. J Forensic Sci 42:14120J.
Talpaert-Borlè M. 1987. Formation, detection and repair of AP sites. Mutat Res 181:45–56.
Tan X, Ge L, Zhang T, Lu Z. 2021. Preservation of DNA for data storage. Russ Chem Rev
90:280–291.
Taylor D, Bright JA, Buckleton J. 2014. Interpreting forensic DNA profiling evidence without
specifying the number of contributors. Forensic Sci Int Genet 13:269–280.
ThermoFisher Scientific. 2021. 2021 Global Insights Survey of Forensics Labs: The impact of
SARS-CoV-2 on challenges, priorities, and opportunities.
Thomas A, Holben B, Dueño K, Snow M. 2018. Mitochondrial DNA extraction from burial soil
samples at incremental distances: a preliminary study. J Forensic Sci.
Tilstone WJ, Savage KA, Clark LA. 2006. Forensic Science: An Encyclopedia of History,
Methods, and Techniques. ABC-CLIO.
Toom V, Wienroth M, M’Charek A, Prainsack B, Williams R, Duster T, Heinemann T, Kruse C,
MacHado H, Murphy E. 2016. Approaching ethical, legal and social issues of emerging
forensic DNA phenotyping (FDP) technologies comprehensively: Reply to “Forensic DNA
phenotyping: Predicting human appearance from crime scene material for investigative
purposes” by Manfred Kayser. Forensic Sci Int Genet 22:e1–e4.
Tredoux S, Mfolozi S, Shires K. 2015. Efficiency of Buccal DNA Sampling Device in the
Mortuary. J Forensic Investig 3.
Turnbough MA, Eisenberg AJ, Schade L, Shewale JG. 2013. Training of Forensic DNA
Scientists - A Commentary. In: Shewale JG, Liu RH, editors. Forensic DNA Analysis:
Current Practices and Emerging Technologies. CRC Press. p 381–389.
U.S. Government Accountability Office. 2019. DNA EVIDENCE: DOJ Should Improve
Performance Measurement and Properly Design Controls for Nationwide Grant Program.
Vandewoestyne M, Van Hoofstat D, Franssen A, Van Nieuwerburgh F, Deforce D. 2013.
Presence and potential of cell free DNA in different types of forensic samples. Forensic Sci
Int Genet 7:316–320.
de Vargas Wolfgramm E, de Carvalho FM, da Costa Aguiar VR, De Nadai Sartori MP,
Hirschfeld-Campolongo GCR, Tsutsumida WM, Louro ID. 2009. Simplified buccal DNA
extraction with FTA® Elute Cards. Forensic Sci Int Genet 3:125–127.
Verdon TJ, Mitchell RJ, Van Oorschot RAH. 2014. Evaluation of tapelifting as a collection
method for touch DNA. Forensic Sci Int Genet 8:179–186.
Vickar T, Bache K, Daniel B, Frascione N. 2018. The use of the M-Vac® wet-vacuum system as
a method for DNA recovery. Sci Justice 58:282–286.
Visser R, Hampikian G. 2012. When DNA Won’t Work. Ida Law Rev 49:40–67.
Vitoševic K, Todorovic D, Slovic Z, Zivkovic-Zaric R, Todorovic M. 2019. Forensic genetics
and genotyping. Serbian J Exp Clin Res 20:75–86.
36
Weathered L, Wright K, Chaseling J. 2020. Dealing with DNA evidence in the courtroom: a
plain English review of current issues with identification, mixture and activity level
evidence. In: The Wrongful Conviction Law Review. . p 59–73.
Westhof E. 1988. Water: an integral part of nucleic acid structure. Annu Rev Biophys Biophys
Chem 17:125–144.
Wickenheiser RA. 2002. Trace DNA: a review, discussion of theory, and application of the
transfer of trace quantities of DNA through skin contact. J Forensic Sci 47:442–50.
Widen H, Leufven A, Nielsen T. 2004. Migration of Model Contaminants from PET Bottles:
Influence of Temperature, Food Simulant and Functional Barrier. Food Addit Contam
21:993–1006.
Wienroth M. 2018. Governing anticipatory technology practices. Forensic DNA phenotyping
and the forensic genetics community in Europe. New Genet Soc 37:137–152.
Wood I, Park S, Tooke J, Smith O, Morgan RM, Meakin GE. 2017. Efficiencies of recovery and
extraction of trace DNA from non-porous surfaces. Forensic Sci Int Genet Suppl Ser
6:e153–e155.
Wyner N, Barash M, Mcnevin D, Linacre AM. 2020. Forensic Autosomal Short Tandem Repeats
and Their Potential Association With Phenotype. 11:1–7.
Yang Q, Shen Y, Shao C, Liu Y, Xu H, Zhou Y, Liu Z, Sun K, Tang Q, Xie J. 2020. Genetic
analysis of tri-allelic patterns at the CODIS STR loci. Mol Genet Genomics 295:1263–
1268.
Zahra A, Hussain B, Jamil A, Ahmed Z, Mahboob S. 2018. Forensic STR profiling based smart
barcode, a highly efficient and cost effective human identification system. Saudi J Biol Sci
25:1720–1723.
Zascage RR, Shewale SJ, Planz J V. 2013. Deep-Sequencing Technologies and Potential
Applications in Forensic DNA Testing. In: Shewale JG, Liu RH, editors. Forensic DNA
Analysis: Current Practices and Emerging Technologies. CRC Press.
Zech WD, Malik N, Thali M. 2012. Applicability of DNA Analysis on Adhesive Tape in
Forensic Casework. J Forensic Sci 57:1036–1041.
Zhang J, Zhang J, Tao R, Jiang L, Chen L, Li X, Li C, Zhang S. 2020. A newly devised
multiplex assay of novel polymorphic non-CODIS STRs as a valuable tool for forensic
application. Forensic Sci Int Genet 48.
Zoppis S, Muciaccia B, D’Alessio A, Ziparo E, Vecchiotti C, Filippini A. 2014. DNA
fingerprinting secondary transfer from different skin areas: Morphological and genetic
studies. Forensic Sci Int Genet 11:137–143.
Zuidberg M, Bettman M, Aarts LHJ, Sjerps M, Kokshoorn B. 2019. Targeting relevant sampling
areas for human biological traces: Where to sample displaced bodies for offender DNA? Sci
Justice 59:153–161.
37
Chapter 3: Crime Scene Collection of DNA
Over the past decade, the process of collecting genetic evidence has made significant
advances. Today, more DNA can be collected at lower quantities, and a genetic profile can still
be reliably ascertained. In theory, this allows more crimes to be solved with the use of DNA.
However, the benefit of genetic evidence in forensics is more complicated than merely collecting
the DNA and must follow requirements to be effective: i) the collection technique (e.g., swab,
tape), ii) the collection surface where the DNA is located (e.g., glass, wood), iii) the source of the
DNA (e.g., blood, saliva, touch DNA), iv) and the sampling strategy to achieve a high success
rate of a genetic profile.
These collection requirements are interrelated. While each element needs to be
considered, the sampling strategy must be determined on the elements together. For instance, a
saliva sample from a glass surface collected using a swab will result in different success rates
based on degradation (Lee and Ladd, 2001; Hogan et al., 2018). The DNA source can also be the
same, but the surface type results in various DNA amounts (Lowe et al., 2002; Raymond et al.,
2009; Goray et al., 2010; Daly et al., 2012; Poetsch et al., 2013, 2018). Based on this knowledge,
consistent success rates can be achieved by selecting the proper sampling strategy.
Success Rate
The success rate is the ability to generate a genetic profile from the evidence collected at
a crime scene, which is dependent on the collection strategy (Zuidberg et al., 2019). This is
accomplished by considering the four elements: DNA source, collection methods and materials,
collection surface, and contact location (Baechler, 2016; Hess and Haas, 2017; Dziak et al.,
2018; Zuidberg et al., 2019). Without considering these elements, the success rate varies. For
38
instance, a study was done comparing three collection techniques, foam swab, blood FTA card,
and the saliva Oragene DNA (OG-500) kit. Despite the saliva collection method recovering a
significantly greater DNA yield, the technique also had the highest contamination rate (Prasad
and Vardhanan, 2018). Of the main four elements, the most critical success rate is the collection
method and location (Mapes et al., 2016; Hess and Haas, 2017; Zuidberg et al., 2019).
There are natural factors that cause concern for the recovery of DNA from a crime scene.
The potential of these factors affecting the success rate is present for all genetic evidence. These
factors are degradation, concentration, and purity of the DNA (Lee and Ladd, 2001; Cătălin et
al., 2011; Mapes et al., 2016). First, as observed with the collection from various substrates,
DNA degradation has a significant impact on the success rate and can potentially influence the
collection process (Lee and Ladd, 2001; Cătălin et al., 2011; Aloraer et al., 2015). Second, the
concentration of DNA can help determine the expected success rate. For example, touch DNA
has a low concentration of DNA; therefore, it is expected to have a low success rate (Mapes et
al., 2016). However, this expectation can only be accepted if the collection is free of human
errors. The third concern is purity during the collection process. The purity of the DNA is similar
to the concern of contamination. However, an impure sample could result from a variety of
sources such as dirt, dyes, or bacterial DNA (Lee and Ladd, 2001; Mapes et al., 2016). By
considering these natural concerns during every sampling strategy, then the potential for a better
success rate increases.
Law enforcement agencies
Investigators or police officers do the collection of evidence. Therefore, they must be
knowledgeable about the various sampling strategies because forensic DNA analysis can only
work with what is left at a crime scene. Many investigators still depend on experience from prior
39
casework to collect DNA (Baechler, 2016). The experience of the investigator can then, in turn,
influence the DNA success rate (Wood et al., 2017). Training should be completed before an
individual can collect evidence with annual refreshers to ensure the highest success rate (Storm
et al., 2009; Hauhart and Menius, 2014). Insufficient training and knowledge for the collection of
genetic evidence will increase the potential for contamination.
The protocols for various laboratories do not provide the necessary information for a
collection sampling strategy. If evidence collection practices are included in the protocol, then
the information is limited to swab samples (Cordray, 2010; Cătălin et al., 2011; Department of
Public Safety - Texas, 2012; Ballou et al., 2013). Nevertheless, with more funding, training, and
research, understanding the benefits of genetic evidence will advance.
The current collection strategy used by law enforcement and crime scene investigators is
singular. The strategy focuses solely on the collection method primarily the double swab
technique consisting of a wet swab followed by a dry swab (Hedman et al., 2020). It would be
advantageous to train investigators and law enforcement on the proper collection strategy to
optimize DNA collection (van Oorschot, 2012; Adamowicz et al., 2014; Hauhart and Menius,
2014; Lapointe et al., 2015). This training should focus on the collection process, transport,
storage, and contamination (Cătălin et al., 2011; van Oorschot, 2012). Together these will benefit
the DNA analysis success rate.
Contamination
There is extensive research understanding the potential contamination during the
collection process. Despite this, the contamination rate is increasing, which began after next-
generation multiplex (NGM) began being implemented. NGM more likely enhanced the
accuracy of the contamination rate because of its sensitive analysis of DNA, which has increased
40
the indication of contamination (Kloosterman et al., 2014; Fonneløp et al., 2016; Pickrahn et al.,
2017; Basset and Castella, 2018). A high percentage of the contamination originates during the
collection process from individuals handling evidence or swabbing (Lapointe et al., 2015). These
contaminations could also result from an insufficient understanding of the benefits of genetic
evidence and insufficient funding (Storm et al., 2009; Blozis, 2014; Hauhart and Menius, 2014).
DNA Source
When collecting DNA, it can originate from different sources, such as touch DNA,
saliva, or blood, which can influence the collection method. Before 2001, the analysis was
limited to evidence with nucleated cells (Lee and Ladd, 2001). Now analysis includes other DNA
sources. It is essential to consider the source of genetic evidence before collecting it because this
will influence the quality, quantity, and preservation of the DNA (Cătălin et al., 2011; Hauhart
and Menius, 2014). Collection methods and materials are not always interchangeable. Therefore,
the DNA yield variability is potentially caused by the collection method rather than low DNA
levels (Barash et al., 2010; Kirgiz and Calloway, 2017). What must be known about the genetic
evidence, before choosing the collection method? It depends on the source of the DNA. When
collecting touch DNA, more information is needed to understand its biological source (skin,
sweat, etc.) in comparison to blood or saliva. Therefore, the collection methods are more likely
to be different between these sources of DNA.
Knowing the DNA source can help identify which genetic evidence might have the
highest success rate. Thus, a collection order can be established to determine the order of
importance based on the probability of DNA's presence, and its sensitivity when collecting
genetic evidence. Therefore, trace DNA (handled objects) should be collected first, followed by
41
facial bodily fluid (e.g., glassware, utensils), then blood, and lastly semen and tissue
(Wickenheiser, 2002). Touch DNA yields low levels of loci, and one study found that 50% of
samples have had 9 loci or less, thus, the reason it needs to be collected first (Baechler, 2016).
Despite the amount of DNA amplified from a pure sample, the range can differ from samples
collected from a crime scene (Cătălin et al., 2011; Tredoux et al., 2015; Baechler, 2016).
Tredoux et al. (2015) found that buccal samples significantly yield more DNA than blood
samples. The amount of DNA from contact with the surface will vary between individuals;
however, this typically refers to the transfer of skin cells.
The potential DNA source from touch DNA consisted of five sources: cell-free DNA,
anucleate corneocytes, fragmentary cells, nucleated cells, and epithelial cells (Burrill et al.,
2019). For biological fluid (i.e., blood, semen), the DNA source is known once it is identified.
However, touch DNA may consist of multiple DNA sources, and knowing the DNA source
allows for better insight into the collection (Wickenheiser, 2002; Quinones and Daniel, 2012;
Zoppis et al., 2014; Ostojic and Wurmbach, 2017). Cell-free DNA was discovered in 1948 in the
plasma and now known to be found in biological media, such as blood and urine (Mandel and
Metais, 1948; Vandewoestyne et al., 2013; Burrill et al., 2019). Thus, it is crucial to know where
the DNA originates because the biological evidence can become cell-free from degradation and
therefore increase fragmentation after transferring (Burrill et al., 2019). Many studies found that
cell-free DNA has the potential to enhance forensic casework since it can increase the DNA
yield (Kita et al., 2008; Linacre et al., 2010; Quinones and Daniel, 2012; Vandewoestyne et al.,
2013). Anucleate corneocytes are the outer layer of the epidermis cells that have keratinized that
are thought to be nuclei free. Therefore, they are not considered significant despite being the
primary cell source in the epidermis (Quinones and Daniel, 2012; Burrill et al., 2019).
42
Fragmentary cells are single-stranded DNA that includes stripped nuclei (Kita et al., 2008;
Zoppis et al., 2014; Burrill et al., 2019). However, there is a contradiction between the source of
the fragments. Zoppis et al. (2014) found the cells forming in the sebaceous gland. This supports
the importance of sebaceous as a vector. In contrast, Kita et al. (2008) found the fragments
localized in the cornified layer located within the epidermis layer (Burrill et al., 2019).
DNA Transfer
The transfer of DNA to an object through the loss of skin cells is complicated. Four
factors impact the amount of DNA transfer: i) contact force, ii) prior activities, iii) the object, iv)
the individual (Lowe et al., 2002; Pang and Cheung, 2007; Daly et al., 2012). Many studies
sought to understand why individuals shed epithelial cells differently, but data is still
inconclusive (van Oorschot and Jones, 1997; Ladd et al., 1999; Lowe et al., 2002; Quinque et al.,
2006; Pang and Cheung, 2007; Raymond et al., 2009; Goray et al., 2010; Daly et al., 2012;
Kamphausen et al., 2012; Poetsch et al., 2013). Kamphausen et al. (2012) concluded that the
health of the individual's skin might impact the rate of shedding skin cells. Poetics et al. (2013)
found by comparing children (1-10 years old) and elders (61 and older) that there was a
correlation between the quality and quantity of DNA shed by the individual. A few studies
suggest that the quantity of DNA shed depends on the contact pressure, tendency to shed, and
prior activities (Ladd et al., 1999; Lowe et al., 2002; Pang and Cheung, 2007). These cells are
shed daily and would be ubiquitous at a crime scene due to their constant loss. Other DNA
sources will be present at a crime scene based on the situation. Therefore, touch DNA is the most
commonly researched type of biological evidence, yet it remains one of the most difficult to use.
What causes the cells to transfer? On average, around 400,000 skin cells are shed every
day by an individual (Hess and Haas, 2017). This shedding process begins with cells moving
43
from the basal layer through the epidermal layer as new cells form. These older cells eventually
will form into keratinocytes and thought to leave the traces of DNA. These results indicate the
source of DNA transfer for a primary transfer will originate from the keratinocytes. This transfer
will occur from direct contact with an object (Zoppis et al., 2014; Cale et al., 2016; Helmus et al.,
2016; Ostojic and Wurmbach, 2017; Pickrahn et al., 2017). However, Zoppis et al. (2014) found
this untrue when analyzing secondary DNA transfer. A secondary transfer occurs from indirect
contact with an object (Cale et al., 2016; Helmus et al., 2016; Pickrahn et al., 2017). In this
instance, the DNA originates from the sebaceous glands that produce a substance comprised of
cellular debris (Zoppis et al., 2014; Vickar et al., 2018).
Touch DNA is typically referring to the cells transferred to an object. It is the DNA
obtained at low trace levels from cellular and cell-free DNA (Hanson and Ballantyne, 2013;
Vickar et al., 2018; Sessa et al., 2019). Unlike other sources of DNA (blood, saliva, etc.), touch
DNA presents four difficulties in collection: i) quantity deposited, ii) quality deposited, iii)
visibility, iv) and lack of a presumptive test for all surfaces (Verdon et al., 2014; Kirgiz and
Calloway, 2017; Hefetz et al., 2019). The quantity of deposited DNA is also influenced by the
individual, the substrate, and contact pressure. A study found that the quality of DNA deposited
from latent fingerprints improves with increased contact pressure between 0 kg to 2 kg (Hefetz et
al., 2019). There are three approaches when targeting touch DNA since there is a lack of
presumptive tests to indicate if DNA is present. The first approach relies on prior cases, looking
at the success rate of various sample locations for similar items. If prior data is limited, then the
approach is limited. The second approach relies on visualization, which focuses primarily on
detection methods. The final approach relies on information on the specific case. This approach
44
is limited to the information available on the case (Zuidberg et al., 2019). Due to these
difficulties, the DNA must be collected methodically to allow the best success rate.
Blood, saliva, and other DNA sources
Unlike touch DNA, other biological materials have various DNA sources. For instance,
saliva contains the same epithelial cells as touch DNA, blood contains leukocytes, and bone
contains osteocytes (Quinque et al., 2006; Dash et al., 2020b). Nevertheless, epithelial cells are
the primary source of DNA from a crime scene. The cells are found in sweat, vaginal fluid, and
saliva (Dash et al., 2020b). The state of these biological materials, other than touch DNA, can
also vary during the collection process. The biological sample will either be a liquid, dried stain,
soft tissue, or hard tissues (i.e., bone, hair) (Lee and Ladd, 2001; Dash et al., 2020b; c). The
amount of DNA expected from a sample is dependent on the collection state. Therefore,
evidence from the same biological source but in different states will not recover the same amount
of DNA. A bloodstain will typically recover 250-500 ng/cm2 of DNA, and liquid blood will
recover 20000-40000 ng/ml; or a plucked hair will recover 1-750 ng/root and a shed hair
recovers 1-10 ng/root (Lee and Ladd, 2001; Cătălin et al., 2011; Dash et al., 2020b). In hair
samples, nuclear DNA is found only in the follicle, while mitochondrial DNA is found in the hair
shaft (Dash et al., 2020b). This indicates the importance of determining which sampling strategy
to use to achieve a high success rate.
Collection Surface
Another factor in the successful recovery of DNA is the substrate. DNA can be found on
various surfaces during an investigation. Surface types are categorized as flat or ridged, and
porous or non-porous (Hedman et al., 2020). These primary surfaces consist of fabric, wood,
45
plastic, and glass. When collecting the sample, the type of DNA and the collection method will
influence the success rate depending on the substrate (Brownlow et al., 2012; Vickar et al., 2018;
Hefetz et al., 2019; Janssen et al., 2019).
If DNA is found on multiple substrates, it is essential to know the potential DNA
recovery range for various substrates to determine the sampling priority (Daly et al., 2012).
Some substrates are challenging to recover DNA, such as bricks, producing low levels of DNA.
The difficulty is potentially caused by the substrates’ porosity and coarse nature (Hogan et al.,
2018; Vickar et al., 2018). However, the quantity of DNA recovered for wood, fabric, and glass
suggests the challenge of DNA recovery for some substrates is not because of porosity (Daly et
al., 2012; Dargay and Roy, 2016; Ostojic and Wurmbach, 2017; Burrill et al., 2019). One study
found a significant difference in recovery between wood, fabric, and glass. Wood has a recovery
range of 0 ng to 169 ng. Followed by fabric with a range of 0 ng to 14.8 ng, and lastly, glass with
a range of 0 ng to 5.2 ng (Daly et al., 2012). The low recovery range for glass might be a result
of the DNA binding to the substrate. DNA is known to bind to silica, the main component found
in various glass materials. The binding of DNA to silica is a result of phosphate-silanol and
hydrophobic interactions depending on if the DNA is single or double-stranded (Shi et al., 2015).
Over a period of time, these results change. Degradation of DNA results in the success rate
becoming higher for non-porous surfaces (Hogan et al., 2018).
When analyzing the recovery of touch DNA from non-porous surfaces, the success rates
varied. Glass and plastic surfaces could generate a greater than 70% genetic profile. In contrast,
paper surfaces could not generate that level of a genetic profile. Furthermore, most metal
surfaces could not generate any genetic profile (Ostojic and Wurmbach, 2017; Wood et al., 2017;
Bonsu et al., 2020). The deposit of DNA is more likely to occur through secondary transfer
46
(which is when deposited DNA transfers to another surface) from non-porous surfaces (Goray et
al., 2010; Cale et al., 2016; Helmus et al., 2016; Kirgiz and Calloway, 2017; Pickrahn et al.,
2017; Burrill et al., 2019). Goray et al. (2010) found the transfer rate of wet biological materials
to increase by 52.3% when the second surface is also non-porous and 94.7% for porous surfaces.
This increase in the transfer is caused by a combination of pressure and friction (Goray et al.,
2010; Noël et al., 2016; Burrill et al., 2019). Thus, non-porous surfaces are an insufficient source
of DNA compared to porous surfaces before degradation. Over time, however, the non-porous
surfaces result in the highest success rate of a genetic profile, potentially due to the lack of
absorption as seen in porous substrates (Hogan et al., 2018).
Fabric color and type will also affect the recovery of DNA from an object. It is harder to
recover DNA from darker fabrics. The extraction of the DNA from theses darker fabrics might
be collecting remnants of the dye (Linacre et al., 2010; Hess and Haas, 2017). The recovery rate
of DNA from natural and synthetic fabrics depends on the dye. Natural fabrics, such as blue
jeans, have a slightly better recovery when the dye is darker. In comparison, synthetic fabrics
have a slightly better recovery when the dye is brighter (Hess and Haas, 2017).
The DNA collected from a surface can vary in quality and quantity based on the object’s
location. When collecting DNA that is not visible, it is helpful for investigators to have known
locations with high success rates on various items to maximize efficiency. However, for some
surfaces, there is no trend for DNA hot spots, such as eyeglasses (Dziak et al., 2018). This trend
may be a result when a sample is either handled or worn extensively over the entire sample,
where DNA can easily transfer. For instance, when collecting DNA from T-shirts, the preferred
sampling areas are on the inside collar. While for gloves, the preferred sampling areas will
depend on the glove material; for fabric gloves, the preferred areas are the inside surface of all
47
fingers or the thumb pad area, and for latex gloves, the entire inside surface area should be
collected (Barash et al., 2010; Dziak et al., 2018). This same logic can be applied to a victim.
Zuidberg et al. (2019) conducted a study to create a heat map for locating the offender’s DNA on
the victim’s body and found a pattern between the offender’s DNA and the victim. When the
victim is heavier than the offender, the preferred sampling areas are the legs and ankles. When
the weight is similar between the two or the offender is heavier, the preferred sampling areas are
the arms (Zuidberg et al., 2019). Therefore, the location and surface type need to be considered
together because they will influence DNA’s success rate.
Collection Method and Material
A variety of DNA collection materials and techniques exist that are utilized at crime
scenes: swab, tape, FTA card, scraping, cutting, and vacuum. It is essential to understand the
material and technique used to collect the DNA will impact the success rate. The combination of
the technique and antimicrobial agents need to be considered before collecting the DNA
evidence. For instance, one study found that 4n6FLOQ swabs are incompatible with DNA IQ
Lysis buffer while showing a high DNA recovery increase with Prepfiler (Dadhania et al., 2013).
Another study observed a high DNA recovery on glass and metal when swabbing with NaCl
0.9% solution (Oliveira et al., 2015). Joel et al. (2015) found that the extraction protocol impacts
Scenesafe FAST™ mini tape. Therefore, exactly how a sample is collected and processed is an
important step in the collection process.
Swab
Buccal swabs are the most common collection method for genetic evidence, especially
for non-porous surfaces (Adamowicz et al., 2014; Hytinen et al., 2017; Comte et al., 2019).
48
When collecting genetic evidence with a swab, the type of buccal swab needs to be considered.
A range of materials are used for swabs using different designs between the materials to collect
the DNA: foam, microfiber, flocked nylon, and cotton (Brownlow et al., 2012; Plaza et al., 2016;
Ambers et al., 2018). Differences between the materials lie in the material’s structural design and
will determine the genetic material’s absorption rate and location. These differences will impact
the success rate of collecting various genetic evidence.
There are two techniques used to collect genetic evidence at a crime scene: single and
double swab. The single swabbing technique consists of a single wet swab, while the double
swab technique consists of a wet swab followed by a dry swab (Hedman et al., 2020). Since the
double swab technique was introduced in 1997, it has become the preferred method (Sweet et al.,
1997; Hedman et al., 2020). The technique was first recommended for recovering saliva from the
skin as the method had a higher recovery rate than the classic single swab method (Sweet et al.,
1997). This recommendation did not indicate that the new method would provide a higher
recovery rate for collecting other genetic evidence sources, such as touch DNA. However,
validation to provide evidence of the technique’s effectiveness was not completed until 2020,
two decades after the technique started being employed (Hedman et al., 2020).
Nevertheless, many studies and practitioners still use the double swab technique (Bright
and Petricevic, 2004; Esslinger et al., 2004; Anzai-Kanto et al., 2005). Prior to the validation
study, other studies using the double swab technique reported that the method improved the
recovery of DNA (Sweet and Hildebrand, 1999; Pang and Cheung, 2007). The validation study
does not support these recommendations completely.
The double swab technique’s validation study results support the importance of selecting
the proper material and method when collecting DNA. Different surfaces were tested. In general,
49
the classic single swab technique is better for collecting DNA, unless the surface is possibly
complex consisting of both ridged and porous features, such as wood. The success rate of the
second swab was tested by examining a wet and a dry swab. The results indicated that when
using a second swab, a wet swab gives a higher yield. However, the second swab still provides a
lower yield than the first swab (Hedman et al., 2020). Another study detected additional DNA on
a non-porous surface when using the single swab method after an initial fingerprint adhesive lift
recovered less than half the DNA present (Hytinen et al., 2017).
Tape
Adhesive tape is a common collection method for genetic evidence (Zech et al., 2012;
Hanson and Ballantyne, 2013; Plaza et al., 2016). When collecting genetic evidence with tape,
the adhesive type needs to be considered because it can interfere with collection (Barash et al.,
2010; Joël et al., 2015; Plaza et al., 2016). During collection, the tape can be reapplied to the
surface to increase DNA potential. However, because this method is an adhesive, there is a
collection threshold that, if exceeded, decreases the mean DNA recovery percentage as the tape
decreases tackiness (Verdon et al., 2014; Kirgiz and Calloway, 2017). For instance, Scotch®
Magic™ tape’s threshold is 8 tapings, and Scenesafe FAST™ is 32 tapings (Verdon et al., 2014).
Another study found tape recovered mixed profiles in 61% of the samples. This was significantly
higher than the cutting method for both a large and small volume sample (Gunnarsson et al.,
2010). Therefore, it is crucial to understand the benefits of an adhesive before collection.
When is an adhesive tape, the preferable collection method? Adhesives are more suitable
for recovering epithelial cells (Bright and Petricevic, 2004; Hall and Fairley, 2004; Barash et al.,
2010). In comparison to swabs, adhesive tape collects more DNA from textiles. This could result
from the porous surface of most textiles; however, the tape also outperforms swabs on raincoats,
50
which is a non-porous textile, but not on flannelette because of the loose fibers (Verdon et al.,
2014; Hess and Haas, 2017). In other instances, there is no statistically significant difference
between the methods of swabbing or tape lifting from a surface, such as steering wheels (Kirgiz
and Calloway, 2017). The use of adhesive tape was outperformed by direct cutting for porous
substrates and vacuum for non-porous substrates. However, when needing to collect from a large
surface area, the adhesive method can be beneficial (Dong et al., 2017). Thus, it is essential to
consider the DNA source and the collection surface before selecting adhesive tape.
Cutting
The third most common collection method is cutting. This is a destructive method;
therefore, it should be used sparingly. Cutting evidence with touch DNA without knowing the
key places to collect can lead to contamination or DNA loss (Dong et al., 2017). The risk of a
mixture of different trace DNA increases when using this method because both sides of the
sample are processed together (Sessa et al., 2019). Studies indicate no significant difference
between cutouts or swabs for various textile samples, such as fabric gloves, footwear, or
brassiere. However, cutouts are significant for the band on beanie-style hats (Dong et al., 2017;
Dziak et al., 2018; Sessa et al., 2019). Therefore, the cutting method provides similar results to
less destructive collection methods and should be used when those methods fail or insufficient.
Newer and less frequent methods
Materials and techniques other than swabs and adhesive tape are also used to collect
biological evidence: FTA cards, scraping, and vacuum. However, these are less frequently used,
and the research of these materials is scarce compared to swabs and tape. Because these methods
are less common, the person collecting the biological evidence should be trained on the specific
sampling strategy’s proper techniques. These alternative methods could be beneficial for
51
collection when conventional methods fail or are insufficient. The vacuum method demonstrates
the potential of collecting a higher DNA yield of bricks, which is a difficult surface for the
traditional collection methods (van Oorschot et al., 2010; Vickar et al., 2018). This method was
the best for non-porous substrates compared to swabs, direct cutting, and adhesive tape (Dong et
al., 2017). Another method, known as the scraping method, demonstrates a higher DNA yield
from porous textiles than swabs and occasionally adhesive tape (Hess and Haas, 2017). In an
instance where the collection surface contains the victim’s and offender’s DNA, such as the
victim’s nails, soaking clippings in digestion buffer results in a higher DNA yield than swabbing
or scraping (Hebda et al., 2014). However, these methods are potentially less beneficial if the
cost is the primary concern (Dong et al., 2017).
FTA cards are a material that may have technical challenges if the person is unaware of
the difficulties of working with this type of sample collection method. The FTA’s composition
helps preserve the DNA, which is beneficial for evidence storage (Kirgiz and Calloway, 2017).
The FTA is made of a paper that contains an indicating circle that often changes color and will
lyse the eukaryotic cells when a sample is introduced (Milne et al., 2006; Dash et al., 2020d).
The DNA will bind to the FTA card after drying for 5 to 10 minutes (de Vargas Wolfgramm et
al., 2009). Two collection methods that can be used to add DNA to the cards. The first is a direct
application to the card where the sample must be a liquid (Milne et al., 2006; GE Healthcare,
2010; Dash et al., 2020d). The second is a scrapping method where the card is moistened, then
scraped across the collection surface (Pizzamiglio et al., 2004; Kirgiz and Calloway, 2017;
Janssen et al., 2019). The second method’s challenge is the potential of the loss of DNA on a
rough surface (Kirgiz and Calloway, 2017). Studies have found the cards are more reliable than
buccal swabs by providing a statistically significant higher DNA yield, potentially due to the
52
card's chemical composition (Milne et al., 2006; Kirgiz and Calloway, 2017; Janssen et al.,
2019).
Conclusion
With the advancements in forensic genetics, DNA as evidence is expanding because
DNA can be collected from a variety of evidence sources. Nevertheless, the information learned
from DNA is beneficial only if the genetic evidence is collected effectively and efficiently. This
is why it is crucial to determine the best sampling strategy to achieve the highest success rate
possible. DNA cannot be retroactively fixed, especially in the absence of a reference sample.
Thus, the collection process is one of the most important steps when collecting genetic evidence
during an investigation because a sampling strategy is not the same for all genetic evidence.
Investigators need to consider the four elements for the collection process to be beneficial to the
investigation. Without the knowledge of proper sampling strategies and training increases the
risk of a low success rate.
The law enforcement agencies may not have the funding to select the best sampling
strategy for specific genetic evidence. The cost of various collection materials, such as FTA
cards, can be expensive if the method’s use is less frequent. Suppose there is a cost-effective
method with a similar success rate to the best sampling strategy; in that case, the investigators
should use the cost-effective method because the goal is to recover useful, quality DNA. It would
also be more cost-effective to increase training for the collection process because a potentially
useless low DNA success rate for an investigation wastes the agency’s funding.
53
References
Abdel Hady RH, Thabet HZ, Ebrahem NE, Yassa HA. 2021. Thermal Effects on DNA
Degradation in Blood and Seminal Stains: Forensic View. Acad Forensic Pathol 11:7–23.
Adamowicz MS, Stasulli DM, Sobestanovich EM, Bille TW. 2014. Evaluation of methods to
improve the extraction and recovery of DNA from cotton swabs for forensic analysis. PLoS
One 9:1–18.
Aditya S, Sharma AK, Bhattacharyya CN, Chaudhuri K. 2011. Generating STR profile from
“touch DNA.” J Forensic Leg Med 18:295–298.
Aggarwal K. 2020. Forensic DNA Phenotyping: Significance in Criminal Investigations. Acad J
Forensic Sci 03:2581–4273.
Al-munim MFA, Al-rashedi NAM. 2021. Stability of DNA Quantitation State of Blood
Evidence under Different Conditions. 25:4965–4971.
Alaeddini R, Walsh SJ, Abbas A. 2010. Forensic Science International : Genetics Forensic
implications of genetic analyses from degraded DNA — A review. Forensic Sci Int Genet
4:148–157.
Alessandrini F, Onofri V, Turchi C, Buscemi L, Pesaresi M, Tagliabracci A. 2020. Past, Present
and Future in Forensic Human Identification. In: Longhi S, Monteriù A, Freddi A, Aquilanti
L, Ceravolo MG, Carnevali O, Giordano M, Moroncini G, editors. The First Outstanding 50
Years of “Universita Politecnica delle Marche”: Research Achievements in Life Sciences. .
p 81–92.
Alketbi SK, Goodwin W. 2019. The effect of time and environmental conditions on Touch DNA.
Forensic Sci Int Genet Suppl Ser 7:701–703.
Allwood JS, Fierer N, Dunn RR. 2020. The future of environmental DNA in forensic science.
Appl Environ Microbiol 86:1–9.
Alonso A, Müller P, Roewer L, Willuweit S, Budowle B, Parson W. 2017. European survey on
forensic applications of massively parallel sequencing. Forensic Sci Int Genet 29:e23–e25.
Aloraer D, Hassan NH, Albarzinji B, Goodwin W. 2015. Collection protocols for the recovery of
biological samples. Forensic Sci Int Genet Suppl Ser 5:e207–e209.
Ambers A, Wiley R, Novroski N, Budowle B. 2018. Direct PCR amplification of DNA from
human bloodstains, saliva, and touch samples collected with microFLOQ® swabs. Forensic
Sci Int Genet 32:80–87.
Amorim A. 2019. Nonhuman forensic genetics. Forensic Sci Int Genet Suppl Ser 7:44–46.
Amorim A, Pereira F, Alves C, Garcia O. 2020. Species assignment in forensics and the
challenge of hybrids. Forensic Sci Int Genet 48:101676.
Anchordoquy TJ, Molina MC. 2007. Preservation of DNA. Cell Preserv Technol 5:180–188.
Andersen K, Bird KL, Rasmussen M, Haile J, Breuning-Madsen H, Kjær KH, Orlando L, Gilbert
MTP, Willerslev E. 2012. Meta-barcoding of “dirt” DNA from soil reflects vertebrate
biodiversity. Mol Ecol 21:1966–1979.
Anon. 2017. Rapid DNA Act of 2017. 115th Congress. Available from:
https://www.congress.gov/bill/115th-congress/house-bill/510
54
Anzai-Kanto E, Hirata MH, Hirata RDC, Nunes FD, Melani RFH, Oliveira RN. 2005. Extração
de DNA de saliva humana depositada sobre a pele e sua aplicabilidade aos processos de
identificação forense. Braz Oral Res 19:216–222.
Arenas M, Pereira F, Oliveira M, Pinto N, Lopes AM, Gomes V, Carracedo A, Amorim A. 2017.
Forensic genetics and genomics: Much more than just a human affair. PLoS Genet 13:1–28.
Arnold LJ, Roberts RG, Macphee RDE, Haile JS, Brock F, Möller P, Froese DG, Tikhonov AN,
Chivas AR, Gilbert MTP, Willerslev E. 2011. Paper II - Dirt, dates and DNA: OSL and
radiocarbon chronologies of perennially frozen sediments in Siberia, and their implications
for sedimentary ancient DNA studies. Boreas 40:417–445.
Arora A. 2020. Future of Forensic and Crime Scene Science Technologies. Technol Forensic
Sci:357–370.
Asari M, Matsuura H, Isozaki S, Hoshina C, Okuda K, Tanaka H, Horioka K, Shiono H, Shimizu
K. 2018. Assessment of DNA degradation of buccal cells under humid conditions and DNA
repair by DOP-PCR using locked nucleic acids. Leg Med 35:29–33.
Ayala-Torres S, Chen Y, Svoboda T, Rosenblatt J, Van Houten B. 2000. Analysis of gene-
specific DNA damage and repair using quantitative polymerase chain reaction. Methods
22:135–147.
Baechler S. 2016. Study of criteria influencing the success rate of DNA swabs in operational
conditions: A contribution to an evidence-based approach to crime scene investigation and
triage. Forensic Sci Int Genet 20:130–139.
Balding DJ, Buckleton J. 2009. Interpreting low template DNA profiles. Forensic Sci Int Genet
4:1–10.
Ballou S, Stolorow M, Taylor M, Bamberger PS, Brown L, Brown R, Burney Y, Davenport D,
DePalma L, Williams S, Jones C, Keaton R, Kiley W, Latta J, Kline M, Lanning K, LaPorte
G, Ledray LE, Nagy R, Ostrom BE, Schwind L, Stoiloff S. 2013. The biological evidence
preservation handbook : best practices for evidence handlers ; technical working group on
biological evidence preservation.
Barash M, Reshef A, Brauner P. 2010. The use of adhesive tape for recovery of dna from crime
scene items. J Forensic Sci 55:1058–1064.
Barlev A, Sen D. 2018. DNA’s Encounter with Ultraviolet Light: An Instinct for Self-
Preservation? Acc Chem Res 51:526–533.
Basset P, Castella V. 2018. Lessons from a study of DNA contaminations from police services
and forensic laboratories in Switzerland. Forensic Sci Int Genet 33:147–154.
Berglund EC, Kiialainen A, Syvänen AC. 2011. Next-generation sequencing technologies and
applications for human genetic history and forensics. Investig Genet 2:1–15.
Bhinder M, Zahoor M, Sadia H, Qasim M, Perveen R, Anjum G, Iqbal M, Ullah N, Shehzad W,
Tariq M, AM Waryah. 2018. SE33 locus as a reliable genetic marker for forensic DNA
analysis systems. Turkish J Med Sci 48:611–614.
Blears MJ, De Grandis SA, Lee H, Trevors JT. 1998. Amplified fragment length polymorphism
(AFLP): A review of the procedure and its applications. J Ind Microbiol Biotechnol 21:99–
55
114.
Blozis J. 2014. Forensic DNA evidence collection at a crime scene: an investigator’s
commentary. In: Shewale JG, Liu RH, editors. Forensic DNA Analysis Current Practices
and Emerging Technologies. Boca Raton: CRC Press. p 3–17.
Bonnet J, Colotte M, Coudy D, Couallier V, Portier J, Morin B, Tuffet S. 2009. Chain and
conformation stability of solid-state DNA: Implications for room temperature storage.
Nucleic Acids Res 38:1531–1546.
Bonsu DOM, Higgins D, Austin JJ. 2020. Forensic touch DNA recovery from metal surfaces – A
review. Sci Justice 60:206–215.
Børsting C, Morling N. 2015. Next generation sequencing and its applications in forensic
genetics. Forensic Sci Int Genet 18:78–89.
Brandsch R. 2017. Probabilistic migration modelling focused on functional barrier efficiency and
low migration concepts in support of risk assessment. Food Addit Contam Part A 34:1743–
1766.
Bright JA, Kelly H, Kerr Z, McGovern C, Taylor D, Buckleton JS. 2020. The interpretation of
forensic DNA profiles: an historical perspective. J R Soc New Zeal 50:211–225.
Bright JA, Petricevic SF. 2004. Recovery of trace DNA and its application to DNA profiling of
shoe insoles. Forensic Sci Int 145:7–12.
Brogna R, Oldenhof H, Sieme H, Wolkers WF. 2020. Spectral fingerprinting to evaluate effects
of storage conditions on biomolecular structure of filter-dried saliva samples and recovered
DNA. Sci Rep 10:1–12.
Brownlow RJ, Dagnall KE, Ames CE. 2012. A Comparison of DNA Collection and Retrieval
from Two Swab Types (Cotton and Nylon Flocked Swab) when Processed Using Three
QIAGEN Extraction Methods. J Forensic Sci 57:713–717.
Bruijns B, Tiggelaar R, Gardeniers H. 2018. Massively parallel sequencing techniques for
forensics: A review. Electrophoresis 39:2642–2654.
Buckleton J. 2009. Validation issues around DNA typing of low level DNA. Forensic Sci Int
Genet 3:255–260.
Budowle B, Moretti TR, Niezgoda SJ, Brown BL. 1998. CODIS and PCR-Based Short Tandem
Repeat Loci: Law Enforcement Tools. Second Eur Symp Hum Identif 7388:73–88.
Burrill J, Daniel B, Frascione N. 2019. A review of trace “Touch DNA” deposits: Variability
factors and an exploration of cellular composition. Forensic Sci Int Genet 39:8–18.
Burrows AM, Kasu M, D’Amato ME. 2019. Preservation of DNA integrity in biological
material. Forensic Sci Int Genet Suppl Ser 7:416–418.
Burrows AM, Ristow PG, D’Amato ME. 2017. Preservation of DNA from saliva samples in
suboptimal conditions. Forensic Sci Int Genet Suppl Ser 6:e80–e81.
Butler E, Li R. 2014. Genetic Markers for Sex Identification in Forensic DNA Analysis. J
Forensic Investig 02.
Butler JM. 2015. The future of forensic DNA analysis. Philos Trans R Soc B Biol Sci 370.
Butler JM, Coble MD, Vallone PM. 2007. STRs vs. SNPs: Thoughts on the future of forensic
56
DNA testing. Forensic Sci Med Pathol 3:200–205.
Butler JM, Hill CR, Kline MC, Duewer DL, Sprecher CJ, McLaren RS, Rabbach DR, Krenke
BE, Storts DR. 2009. The single most polymorphic STR Locus: SE33 performance in U.S.
populations. Forensic Sci Int Genet Suppl Ser 2:23–24.
Butler JM, Willis S. 2020. Interpol review of forensic biology and forensic DNA typing 2016-
2019. Forensic Sci Int Synerg.
Byard RW, James H, Berketa J, Heath K. 2016. Locard’s Principle of Exchange, Dental
Examination and Fragments of Skin. J Forensic Sci 61:545–547.
Byrn SR, Xu W, Newman AW. 2001. Chemical reactivity in solid-state pharmaceuticals:
Formulation implications. Adv Drug Deliv Rev 48:115–136.
Cale CM, Earll ME, Latham KE, Bush GL. 2016. Could Secondary DNA Transfer Falsely Place
Someone at the Scene of a Crime? J Forensic Sci 61:196–203.
Caliebe A, Walsh S, Liu F, Kayser M, Krawczak M. 2017. Likelihood ratio and posterior odds in
forensic genetics: Two sides of the same coin. Forensic Sci Int Genet 28:203–210.
Carracedo Á, Prieto L. 2019. Beyond the CSI effect: The keys to good forensic genetics
communication. Metode 2019:31–37.
Cătălin M, Andrei A, Mitraşca O. 2011. Modern Methods of Collection and Preservation of
Biological Evidence for Human Identification by DNA Analysis. Abacus Diagnostics.
Chauhan M. 2020. Storage of saliva and blood specimen in different temperature. Int J Forensic
Med 2:21–24.
Chong KWY, Thong Z, Syn CK. 2021. Recent trends and developments in forensic DNA
extraction . WIREs Forensic Sci 3:1–23.
Clabaugh K, Silva B, Odigie K, Fourney R, Stevens J, Carmody G, Coble MD, Loreille O,
Scheible M, Kline M, Parsons TJ. 2007. Storage of DNA samples at ambient temperature
using DNA-SampleMatrix. Poster Present 18th Annu Meet Int Symp Hum Identification,
Hollywood, CA.
Colotte M, Coudy D, Tuffet S, Bonnet J. 2011. Adverse Effect of Air Exposure on the Stability
of DNA Stored at Room Temperature. Biopreserv Biobank:47–50.
Comte J, Baechler S, Gervaix J, Lock E, Milon MP, Delémont O, Castella V. 2019. Touch DNA
collection – Performance of four different swabs. Forensic Sci Int Genet 43.
Cordray R. 2010. Guidelines for preservation and retention of biological evidence.
Corradini B, Alù M, Magnanini E, Galinier ME, Silingardi E. 2019. The importance of forensic
storage support: DNA quality from 11-year-old saliva on FTA cards. Int J Legal Med
133:1743–1750.
Dadhania A, Nelson M, Caves G, Santiago R, Podini D. 2013. Evaluation of Copan
4N6FLOQSwabsTM used for crime scene evidence collection. Forensic Sci Int Genet Suppl
Ser 4:e336–e337.
Daly DJ, Murphy C, McDermott SD. 2012. The transfer of touch DNA from hands to glass,
fabric and wood. Forensic Sci Int Genet 6:41–46.
Dargay A, Roy R. 2016. Direct Y-STR amplification of body fluids deposited on commonly
57
found crime scene substrates. J Forensic Leg Med 39:50–60.
Dash HR, Shrivastava P, Das S. 2020a. Principles and Practices of DNA Analysis: A Laboratory
Manual for Forensic DNA Typing. New York, NY: Springer Protocols Handbook.
Dash HR, Shrivastava P, Das S. 2020b. Biological Samples: The Target Sources for DNA
Typing. In: Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic
DNA Typing. New York, NY: Humana. p 13–20.
Dash HR, Shrivastava P, Das S. 2020c. Collection, Transportation, and Preservation of
Biological Evidences for DNA Analysis. In: Principles and Practices of DNA Analysis: A
Laboratory Manual for Forensic DNA Typing. New York, NY: Humana. p 21–27.
Dash HR, Shrivastava P, Das S. 2020d. Reliable Use of WhatmanTM FTATM Cards for One-Step
Collection and Isolation of DNA. In: Principles and Practices of DNA Analysis: A
Laboratory Manual for Forensic DNA Typing. New York, NY: Humana. p 109–115.
Davis C, Illescas M, Tirado C, Lopez R, Budowle B, Cruz TD. 2012. A Case of Amelogenin Y-
null: A simple primer binding site mutation or unusual genetic anomaly? Leg Med 14:320–
323.
Davis DL, O’Brie EP, Bentzley CM. 2000. Analysis of the degradation of oligonucleotide
strands during the freezing/thawing processes using MALDI-MS. Anal Chem 72:5092–
5096.
Department of Public Safety - Texas. 2012. Best practices for collection, packaging, storage,
preservation, and retrieval of biological evidence. :1–8.
Dickerson RE, Drew HR, Conner BN, Wing RM, Fatini A V., Kopka ML. 1982. The Anatomy
of A-, B-, and Z-DNA. Science (80- ) 216:475–485.
Diegoli TM. 2015. Forensic typing of short tandem repeat markers on the X and Y
chromosomes. Forensic Sci Int Genet 18:140–151.
Dissing J, Søndervang A, Lund S. 2010. Exploring the limits for the survival of DNA in blood
stains. J Forensic Leg Med 17:392–396.
Dong H, Wang J, Zhang T, Ge JY, Dong YQ, Sun QF, Liu C, Li CX. 2017. Comparison of
preprocessing methods and storage times for touch DNA samples. Croat Med J 58:4–13.
Durose M. 2008. Census of Publicly Funded Forensic Crime Laboratories, 2005, Bureau of
Justice Statistics. :1–12.
Dziak R, Peneder A, Buetter A, Hageman C. 2018. Trace DNA Sampling Success from
Evidence Items Commonly Encountered in Forensic Casework. J Forensic Sci 63:835–841.
Emmons AL, DeBruyn JM, Mundorff AZ, Cobaugh KL, Cabana GS. 2017. The persistence of
human DNA in soil following surface decompositions. Sci Justice 57:341–348.
Erlich H. 2020. In the Begginning: Forensic Applications of DNA Technologies. In: Erlich H,
Stover E, White TJ, editors. Silent Witness: Forensic DNA Evidence in Criminal
Investigations and Humanitarian Disaters. Oxford Univeristy Press. p 15–33.
Erlich H, Calloway C, Lee SB. 2020. Recent Developments in Forensic DNA Technology. In:
Erlich H, Stover E, White TJ, editors. Silent Witness: Forensic DNA Evidence in Criminal
Investigations and Humanitarian Disaters. Oxford Univeristy Press. p 105–127.
58
Esslinger KJ, Siegel JA, Spillane H, Stallworth S. 2004. Using STR Analysis to Detect Human
DNA from Exploded Pipe Bomb Devices. J Forensic Sci 49:1–4.
Fabre A-L, Luis A, Colotte M, Tuffet S, Bonnet J. 2017. High DNA stability in white blood cells
and buffy coat lysates stored at ambient temperature under anoxic and anhydrous
atmosphere. PLoS One 12:e0188547.
Fang X, Vitrac O. 2017. Predicting diffusion coefficients of chemicals in and through packaging
materials. Crit Rev Food Sci Nutr 57:275–312.
de Fátima Poças M, Oliveria JC, Peteira JR, Brandsch R, Hogg T. 2011. Modelling migration
from paper into a food simulant. Food Control 22:303–312.
Fonneløp AE, Johannessen H, Egeland T, Gill P. 2016. Contamination during criminal
investigation: Detecting police contamination and secondary DNA transfer from evidence
bags. Forensic Sci Int Genet.
Frippiat C, Noel F. 2014. Efficiency of a novel forensic room-temperature DNA storage
medium. Forensic Sci Int Genet 9:81–84.
Gaillard C, Strauss F. 1998. Avoiding adsorption of DNA to polypropylene tubes and
denaturation of short DNA fragments. Tech Tips Online 3:63–65.
Garvin AM, Holzinger R, Berner F, Krebs W, Hostettler B, Lardi E, Hertli C, Quartermaine R,
Stamm C. 2013. The forensix evidence collection tube and its impact on dna preservation
and recovery. Biomed Res Int 2013.
GE Healthcare. 2010. Reliable extraction of DNA from Whatman FTA cards. Appl Note 28-
9822-22 AA.
Ge J, Sun H, Li H, Liu C, Yan J, Budowle B. 2014. Future directions of forensic DNA databases.
Croat Med J 55:163–166.
Ghosh A, Bansal M. 2003. A glossary of DNA structures from A to Z. Acta Crystallogr - Sect D
Biol Crystallogr 59:620–626.
Goray M, Eken E, Mitchell RJ, van Oorschot RAH. 2010. Secondary DNA transfer of biological
substances under varying test conditions. Forensic Sci Int Genet 4:62–67.
Gršković B, Zrnec D, Popović M, Petek MJ, Primorac D, Mršić G. 2013. Effect of ultraviolet c
radiation on biological samples. Croat Med J 54:263–271.
Gunnarsson J, Helena E, Ansell R. 2010. Success rates of a forensic tape-lift method for DNA
recovery. Probl Forensic Sci LXXXIII:243–254.
Haile J, Holdaway R, Oliver K, Bunce M, Gilbert MTP, Nielsen R, Munch K, Ho SYW, Shapiro
B, Willerslev E. 2007. Ancient DNA chronology within sediment deposits: Are
paleobiological reconstructions possible and is DNA leaching a factor? Mol Biol Evol
24:982–989.
Hakim HM, Lalung J, Khan HO, Ismail SA, Aziz MY, Ishak AR, Safuan S, Rasudin NS,
Chambers GK, Edinur HA. 2020. Evaluation of long-term storage effects on buccal cell
DNA from untreated cards for STR profiling. IOP Conf Ser Earth Environ Sci 596.
Hall A, Sims LM, Ballantyne J. 2014. Assessment of DNA damage induced by terrestrial UV
irradiation of dried bloodstains: Forensic implications. Forensic Sci Int Genet 8:24–32.
59
Hall D, Fairley M. 2004. A single approach to the recovery of DNA and firearm discharge
residue evidence. Sci Justice - J Forensic Sci Soc 44:15–19.
Hanson E., Ballantyne J. 2013. “Getting blood from a stone”: ultrasensitive forensic DNA
profiling of microscopic bio-particles recovered from “touch DNA” evidence. In: Nucleic
Acids Detection. Totowa, NJ: Humana Press. p 3–17.
Hara M, Nakanishi H, Yoneyama K, Saito K, Takada A. 2016. Effects of storage conditions on
forensic examinations of blood samples and bloodstains stored for 20 years. Leg Med
18:81–84.
Hares DR. 2015. Selection and implementation of expanded CODIS core loci in the United
States. Forensic Sci Int Genet 17:33–34.
Hauhart R, Menius K. 2014. DNA Evidence: Examining Police Officers’ Knowledge of
Handling Procedures in a Mid-Size Department. Int J Criminol Sociol 3:360–376.
Hebda LM, Doran AE, Foran DR. 2014. Collecting and analyzing DNA evidence from
fingernails: A comparative study. J Forensic Sci 59:1343–1350.
Hebsgaard MB, Arneborg J, Heyn P, Allentoft ME, Bunce M, Schweger C, Willerslev E. 2009.
‘The Farm Beneath the Sand’ – an archaeological case study on ancient ‘dirt’ DNA.
Antiquity 83:430–444.
Hedman J, Jansson L, Akel Y, Wallmark N, Gutierrez Liljestrand R, Forsberg C, Ansell R. 2020.
The double-swab technique versus single swabs for human DNA recovery from various
surfaces. Forensic Sci Int Genet 46:20–24.
Hefetz I, Einot N, Faerman M, Horowitz M, Almog J. 2019. Touch DNA: The effect of the
deposition pressure on the quality of latent fingermarks and STR profiles. Forensic Sci Int
Genet 38:105–112.
Helmus J, Bajanowski T, Poetsch M. 2016. DNA transfer—a never ending story. A study on
scenarios involving a second person as carrier. Int J Legal Med 130:121–125.
Hess S, Haas C. 2017. Recovery of Trace DNA on Clothing: A Comparison of Mini-tape Lifting
and Three Other Forensic Evidence Collection Techniques. J Forensic Sci 62:187–191.
Hogan C, Houten LB Van, Coticone S. 2018. Comparison of the Quantity and Overall Quality of
Trace DNA Evidence Collected from Substrates Found at Crime Scenes. J Forensic Identif
68.
Holland M, Melton T, Holland C. 2013. Forensic Mitochondrial DNA Analysis: Current Practice
and Future Potential. In: Shewale JG, Liu RH, editors. Forensic DNA Analysis: Current
Practices and Emerging Technologies. CRC Press. p 249–278.
Hopman R, M’charek A. 2020. Facing the unknown suspect: forensic DNA phenotyping and the
oscillation between the individual and the collective. Biosocieties 15:438–462.
Howlett SE, Castillo HS, Gioeni LJ, Robertson JM, Donfack J. 2014. Evaluation of
DNAstableTM for DNA storage at ambient temperature. Forensic Sci Int Genet.
Hytinen ME, Solomon AD, Miller MT, Cruz TD. 2017. Methods for Obtaining High-Quality
Touch DNA from a Nonporous Surface after Latent Fingerprint Collection. J Forensic
Identif 67:71–84.
60
Ip SCY, Yu EY, Li C. 2021. Blood DNA Preservation on Various Forensic Swab Devices. J
Forensic Identif 71.
Ivanova N V., Kuzmina ML. 2013. Protocols for dry DNA storage and shipment at room
temperature. Mol Ecol Resour 13:890–898.
Jakovski Z, Ajanovska RJ, Stankov A, Poposka V, Bitoljanu N, Belakaposka V. 2017. The
power of forensic DNA data bases in solving crime cases. Forensic Sci Int Genet Suppl Ser
6:e275–e276.
Janssen K, Aune M, Olsen M, Olsen GH, Berg T. 2019. Biological stain collection – Absorbing
paper is superior to cotton swabs. Forensic Sci Int Genet Suppl Ser 7:468–469.
Jeffreys A, Brookfield J, Semeonoff R. 1985. Positive identification of an immigration test-case
using human DNA fingerprints. Nature 317:818–819.
Joël J, Glanzmann B, Germann U, Cossu C. 2015. DNA extraction of forensic adhesive tapes—
A comparison of two different methods. Forensic Sci Int Genet Suppl Ser 5:e579–e581.
Junkin T. 2005. Bloodsworth:The True Story of One Man’s Triumph Over Injustice. Algonquin
Books.
Kamphausen T, Schadendorf D, Von Wurmb-Schwark N, Bajanowski T, Poetsch M. 2012. Good
shedder or bad shedder- The influence of skin diseases on forensic DNA analysis from
epithelial abrasions. Int J Legal Med 126:179–183.
Karantzali E, Rosmaraki P, Kotsakis A, Le Roux-Le Pajolec MG, Fitsialos G. 2019. The effect
of FBI CODIS Core STR Loci expansion on familial DNA database searching. Forensic Sci
Int Genet 43.
Kayser M. 2015. Forensic DNA Phenotyping: Predicting human appearance from crime scene
material for investigative purposes. Forensic Sci Int Genet 18:33–48.
Kayser M. 2017. Forensic use of Y-chromosome DNA: a general overview. Hum Genet
136:621–635.
Keller PE, Kouzes R. 2017. Water Vapour Permeation in Plastics. :29.
Kirgiz IA, Calloway C. 2017. Increased recovery of touch DNA evidence using FTA paper
compared to conventional collection methods. J Forensic Leg Med 47:9–15.
Kita T, Yamaguchi H, Yokoyama M, Tanaka T, Tanaka N. 2008. Morphological study of
fragmented DNA on touched objects. Forensic Sci Int Genet 3:32–36.
Kline MC, Duewer DL, Redman JW, Butler JM. 2005. Results from the NIST 2004 DNA
Quantitation Study. J Forensic Sci 50:1–8.
Kloosterman A, Sjerps M, Quak A. 2014. Error rates in forensic DNA analysis: Definition,
numbers, impact and communication. Forensic Sci Int Genet 12:77–85.
Kumar KR, Cowley MJ, Davis RL. 2019. Next-Generation Sequencing and Emerging
Technologies. Semin Thromb Hemost 45:661–673.
Ladd C, Adamowicz MS, Bourke MT, Scherczinger CA, Lee HC. 1999. A Systematic Analysis
of Secondary DNA Transfer. J Forensic Sci 44:14599J.
Lapointe M, Rogic A, Bourgoin S, Jolicoeur C, Séguin D. 2015. Leading-edge forensic DNA
analyses and the necessity of including crime scene investigators, police officers and
61
technicians in a DNA elimination database. Forensic Sci Int Genet 19:50–55.
Lawrence MG. 2005. The relationship between relative humidity and the dewpoint temperature
in moist air: A simple conversion and applications. Bull Am Meteorol Soc 86:225–233.
Lee HC, Ladd C. 2001. Preservation and collection of biological evidence. Croat Med J 42:225–
228.
Lee S, Crouse C, Kline M. 2013. Optimizing storage and handling of DNA extracts. In: Shewale
JG, editor. Forensic DNA Analysis: Current Practices and Emerging Technologies. CRC
Press. p 19–64.
Lee SB, Clabaugh KC, Silva B, Odigie KO, Coble MD, Loreille O, Scheible M, Fourney RM,
Stevens J, Carmody GR, Parsons TJ, Pozder A, Eisenberg AJ, Budowle B, Ahmad T, Miller
RW, Crouse CA. 2012. Assessing a novel room temperature DNA storage medium for
forensic biological samples. Forensic Sci Int Genet 6:31–40.
Li B, Wang ZW, Lin QB, Hu CY. 2017. Molecular dynamics simulation of three plastic
additives’ diffusion in polyethylene terephthalate. Food Addit Contam Part A 34:1086–
1099.
Li C. 2018. Forensic genetics. Forensic Sci Res 3:103–104.
Linacre A, Pekarek V, Swaran YC, Tobe SS. 2010. Generation of DNA profiles from fabrics
without DNA extraction. Forensic Sci Int Genet 4:137–141.
Lowe A, Murray C, Whitaker J, Tully G, Gill P. 2002. The propensity of individuals to deposit
DNA and secondary transfer of low level DNA from individuals to inert surfaces. Forensic
Sci Int 129:25–34.
Lyons LA, Grahn RA, Kun TJ, Netzel LR, Wictum EE, Halverson JL. 2014. Acceptance of
domestic cat mitochondrial DNA in a criminal proceeding. Forensic Sci Int Genet 13:61–
67.
Machado H, Granja R. 2020. Forensic Genetics in the Governance of Crime.
Machado H, Silva S. 2019. What influences public views on forensic DNA testing in the
criminal field? A scoping review of quantitative evidence. Hum Genomics 13:23.
Maia J, Rodriguez-Bernaldo de Quirós A, Sendón R, Cruz JM, Seiler A, Franz R, Simoneau C,
Castle L, Driffield M, Mercea P, Oldring P, Tosa V, Paseiro P. 2016. Determination of key
diffusion and partition parameters and their use in migration modelling of benzophenone
from low-density polyethylene (LDPE) into differnt foodstuffs. Food Addit Contam Part A
33:715–724.
Mandel P, Metais P. 1948. Les acides nucleiques du plasma sanguin chez l’homme. CR Seances
Soc Biol Fil 142:241–243.
Mapes AA, Kloosterman AD, van Marion V, de Poot CJ. 2016. Knowledge on DNA Success
Rates to Optimize the DNA Analysis Process: From Crime Scene to Laboratory. J Forensic
Sci 61:1055–1061.
Marrone A, Ballantyne J. 2010. Hydrolysis of DNA and its molecular components in the dry
state. Forensic Sci Int Genet 4:168–177.
McCartney C, Shorter L. 2020. Police retention and storage of evidence in England and Wales.
62
Int J Police Sci Manag 22:123–136.
McCord BR, Gauthier Q, Cho S, Roig MN, Gibson-Daw GC, Young B, Taglia F, Zapico SC,
Mariot RF, Lee SB, Duncan G. 2019. Forensic DNA Analysis. Anal Chem 91:673–688.
Milne E, Van Bockxmeer FM, Robertson L, Brisbane JM, Ashton LJ, Scott RJ, Armstrong BK.
2006. Buccal DNA collection: Comparison of buccal swabs with FTA cards. Cancer
Epidemiol Biomarkers Prev 15:816–819.
Mistek E, Fikiet MA, Khandasammy SR, Lednev IK. 2019. Toward Locard’s Exchange
Principle: Recent Developments in Forensic Trace Evidence Analysis. Anal Chem 91:637–
654.
Moore MK, Frazier K. 2019. Humans Are Animals, Too: Critical Commonalities and
Differences Between Human and Wildlife Forensic Genetics. J Forensic Sci 64:1603–1621.
Moretti TR, Moreno LI, Smerick JB, Pignone ML, Hizon R, Buckleton JS, Bright JA, Onorato
AJ. 2016. Population data on the expanded CODIS core STR loci for eleven populations of
significance for forensic DNA analyses in the United States. Forensic Sci Int Genet 25:175–
181.
Morgan TH. 1917. The Theory of the Gene. Am Nat 51:513–544.
Mozayani A, Fisher CP eds. 2017. Forensic Evidence Management: From the Crime Scene to the
Courtroom. CRC Press.
Mueller UG, Wolfenbarger LL. 1999. AFLP genotyping and fingerprinting. Trends Ecol Evol
14:389–394.
Murphy E. 2018. Forensic DNA typing. Annu Rev Criminol 1:497–515.
Nachman MW, Crowell SL. 2000. Estimate of the mutation rate per nucleotide in humans.
Genetics 156:297–304.
Nelson M. 2010. Making sense of DNA backlogs: myths vs. reality (Report No. NCJ 232197).
National Insitute of Justice, Office of Justice Programs, U.S. Department of Justice.
Ng HH, Ang HC, Hoe SY, Lim M-L, Tai HE, Soh RCH, Syn CK-C. 2018. Simple DNA
extraction of urine samples: Effects of storage temperature and storage time. Forensic Sci
Int 287:36–39.
Noël S, Lagace K, Rogic A, Granger D, Bourgoin S, Jolicoeur C, Séguin D. 2016. DNA transfer
during laundering may yield complete genetic profiles. Forensic Sci Int Genet 23:240–247.
Novroski NMM, Wendt FR, Woerner AE, Bus MM, Coble M, Budowle B. 2019. Expanding
beyond the current core STR loci: An exploration of 73 STR markers with increased
diversity for enhanced DNA mixture deconvolution. Forensic Sci Int Genet 38:121–129.
Oliveira TP, Nogueira TLS, Valentin ESB, Santos OCL, Carvalho EF, Silva DA. 2015.
Evaluation of collection and extraction methodologies of latent fingerprints for military
application. Forensic Sci Int Genet Suppl Ser 5:e474–e475.
van Oorschot RAH. 2012. Assessing DNA Profiling Success Rates: Need for More and Better
Collection of Relevant Data. Forensic Sci Policy Manag An Int J 3:37–41.
van Oorschot RAH, Ballantyne KN, Mitchell RJ. 2010. Forensic trace DNA: A review. Investig
Genet 1:1–17.
63
van Oorschot RAH, Jones MK. 1997. DNA fingerprints from fingerprints. Nature 387:766–767.
Ostojic L, Wurmbach E. 2017. Analysis of fingerprint samples, testing various conditions, for
forensic DNA identification. Sci Justice 57:35–40.
Pang BCM, Cheung BKK. 2007. Double swab technique for collecting touched evidence. Leg
Med 9:181–184.
Panneerchelvam S, Norazmi MN. 2003. Forensic DNA profiling and database. Malaysian J Med
Sci 10:20–26.
Paunescu D, Puddu M, Soellner JO, Stoessel PR, Grass RN. 2013. Reversible DNA
encapsulation in silica to produce ROS-resistant and heat-resistant synthetic DNA’fossils’.
Nat Protoc 8:2440–2448.
Pickrahn I, Kreindl G, Müller E, Dunkelmann B, Zahrer W, Cemper-Kiesslich J, Neuhuber F.
2017. Contamination incidents in the pre-analytical phase of forensic DNA analysis in
Austria—Statistics of 17 years. Forensic Sci Int Genet 31:12–18.
Pizzamiglio M, Mameli A, My D, Garofano L. 2004. Forensic identification of a murderer by
LCN DNA collected from the inside of the victim’s car. Int Congr Ser 1261:437–439.
Plaza DT, Mealy JL, Lane JN, Parsons MN, Bathrick AS, Slack DP. 2016. Nondestructive
Biological Evidence Collection with Alternative Swabs and Adhesive Lifters. J Forensic Sci
61:485–488.
Poetsch M, Bajanowski T, Kamphausen T. 2013. Influence of an individual’s age on the amount
and interpretability of DNA left on touched items. Int J Legal Med 127:1093–1096.
Poetsch M, Pfeifer M, Konrad H, Bajanowski T, Helmus J. 2018. Impact of several wearers on
the persistence of DNA on clothes—a study with experimental scenarios. Int J Legal Med
132:117–123.
Pourazar A. 2007. Red cell antigens: Structure and function. Asian J Transfus Sci 1:24–32.
Prasad MSS, Vardhanan YS. 2018. Evaluation of efficacy of collection techniques for human
genomic DNA MAOA-uVNTR polymorphism. Int J Sci Res Biol Sci 5:6–11.
Quinlan A. 2020. Visions of Public Safety, Justice, and Healing: The Making of the Rape Kit
Backlog in the United States. Soc Leg Stud 29:225–245.
Quinones I, Daniel B. 2012. Cell free DNA as a component of forensic evidence recovered from
touched surfaces. Forensic Sci Int Genet 6:26–30.
Quinque D, Kittler R, Kayser M, Stoneking M, Nasidze I. 2006. Evaluation of saliva as a source
of human DNA for population and association studies. Anal Biochem 353:272–277.
Raina A, Pramanik P, Dogra TD. 2004. Effect of storage conditions of seminal stains on
different textures of clothes in relation to DNA yield. Indian Congr Forensic Med Toxicol 2.
Ravanat J-L, Douki T, Cadet J. 2001. Direct and indirect effects of UV radiation on DNA and its
components. J Photochem Photobiol 63:88–102.
Raymond JJ, van Oorschot RAH, Gunn PR, Walsh SJ, Roux C. 2009. Trace evidence
characteristics of DNA: A preliminary investigation of the persistence of DNA at crime
scenes. Forensic Sci Int Genet 4:26–33.
Reich DE, Schaffner SF, Daly MJ, McVean G, Mullikin JC, Higgins JM, Richter DJ, Lander ES,
64
Altshuler D. 2002. Human genome sequence variation and the influence of gene history,
mutation and recombination. Nat Genet 32:135–142.
Richter C, Park JW, Ames BN. 1988. Normal oxidative damage to mitochondrial and nuclear
DNA is extensive. Proc Natl Acad Sci U S A 85:6465–6467.
Roberston B, Vignaux GA, Berger CEH. 2016. Explaining the Strength of Evidence. In:
Interpreting Evidence: Evaluating forensic science in the courtroom. 2nd ed. John Wiley &
Sons, Ltd. p 55–67.
Roewer L. 2013. DNA fingerprinting in forensics: Past, present, future. Investig Genet 4:1–10.
Rutty GN, EAM G. 2005. DNA/risk of contamination. In: Payne-James J, Byard RW, Corey TS,
Henderson C, editors. Encyclopedia of forensic and legal medicine. volume 2. . p 189–198.
Safarikova M, Kubena AA, Frankova V, Zima T, Kalousova M. 2021. The effects of different
storage conditions and repeated freeze/thaw cycles on the concentration, purity and integrity
of genomic DNA. Folia Biol (Czech Republic) 67:10–15.
Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA. 1986. Analysis of enzymatically
amplified β-globin and HLA-DQα DNA with allele-specific oligonucleotide probes. Nature
324:163–166.
Samuel G, Prainsack B. 2019. Forensic DNA phenotyping in Europe: views “on the ground”
from those who have a professional stake in the technology. New Genet Soc 38:119–141.
Sessa F, Salerno M, Bertozzi G, Messina G, Ricci P, Ledda C, Rapisarda V, Cantatore S,
Turillazzi E, Pomara C. 2019. Touch DNA: Impact of handling time on touch deposit and
evaluation of different recovery techniques: An experimental study. Sci Rep 9:1–9.
Shewale JG, Liu RH eds. 2013. Forensic DNA analysis: current practices and emerging
technologies. CRC Press.
Shi B, Shin YK, Hassanali AA, Singer SJ. 2015. DNA Binding to the Silica Surface. J Phys
Chem B 119:11030–11040.
Shikama K. 1965. Effect of freezing and thawing on the stability of double helix of DNA. Nature
207:529–530.
Sirker M, Schneider PM, Gomes I. 2016. A 17-month time course study of human RNA and
DNA degradation in body fluids under dry and humid environmental conditions. Int J Legal
Med 130:1431–1438.
Slabbert N, Heathfield LJ. 2018. Ethical, legal and social implications of forensic molecular
phenotyping in South Africa. Dev World Bioeth 18:171–181.
Stannett V, Williams JL. 2007. The permeability of poly(ethyl methacrylate) to gases and water
vapor. J Polym Sci Part C Polym Symp 10:45–59.
Steinlechner M, Berger B, Niederstätter H, Parson W. 2002. Rare failures in the amelogenin sex
test. Int J Legal Med 116:117–120.
Storm KJ, Ropero-Miller J, Jones S, Sikes N, Pope M, Horstmann N. 2009. The 2007 Survey of
Law Enforcement Forensic Evidence Processing. Rockville.
Strom KJ, Hickman M. 2010. Processing in Police Departments. Criminol Public Policy 9:381–
404.
65
Sweet D, Hildebrand D. 1999. Saliva from cheese bite yields DNA profile of burglar: A case
report. Int J Legal Med 112:201–203.
Sweet D, Lorente M, Lorente JA, Valenzuela A, Villanueva E. 1997. An Improved Method to
Recover Saliva from Human Skin: The Double Swab Technique. J Forensic Sci 42:14120J.
Talpaert-Borlè M. 1987. Formation, detection and repair of AP sites. Mutat Res 181:45–56.
Tan X, Ge L, Zhang T, Lu Z. 2021. Preservation of DNA for data storage. Russ Chem Rev
90:280–291.
Taylor D, Bright JA, Buckleton J. 2014. Interpreting forensic DNA profiling evidence without
specifying the number of contributors. Forensic Sci Int Genet 13:269–280.
ThermoFisher Scientific. 2021. 2021 Global Insights Survey of Forensics Labs: The impact of
SARS-CoV-2 on challenges, priorities, and opportunities.
Thomas A, Holben B, Dueño K, Snow M. 2018. Mitochondrial DNA extraction from burial soil
samples at incremental distances: a preliminary study. J Forensic Sci.
Tilstone WJ, Savage KA, Clark LA. 2006. Forensic Science: An Encyclopedia of History,
Methods, and Techniques. ABC-CLIO.
Toom V, Wienroth M, M’Charek A, Prainsack B, Williams R, Duster T, Heinemann T, Kruse C,
MacHado H, Murphy E. 2016. Approaching ethical, legal and social issues of emerging
forensic DNA phenotyping (FDP) technologies comprehensively: Reply to “Forensic DNA
phenotyping: Predicting human appearance from crime scene material for investigative
purposes” by Manfred Kayser. Forensic Sci Int Genet 22:e1–e4.
Tredoux S, Mfolozi S, Shires K. 2015. Efficiency of Buccal DNA Sampling Device in the
Mortuary. J Forensic Investig 3.
Turnbough MA, Eisenberg AJ, Schade L, Shewale JG. 2013. Training of Forensic DNA
Scientists - A Commentary. In: Shewale JG, Liu RH, editors. Forensic DNA Analysis:
Current Practices and Emerging Technologies. CRC Press. p 381–389.
U.S. Government Accountability Office. 2019. DNA EVIDENCE: DOJ Should Improve
Performance Measurement and Properly Design Controls for Nationwide Grant Program.
Vandewoestyne M, Van Hoofstat D, Franssen A, Van Nieuwerburgh F, Deforce D. 2013.
Presence and potential of cell free DNA in different types of forensic samples. Forensic Sci
Int Genet 7:316–320.
de Vargas Wolfgramm E, de Carvalho FM, da Costa Aguiar VR, De Nadai Sartori MP,
Hirschfeld-Campolongo GCR, Tsutsumida WM, Louro ID. 2009. Simplified buccal DNA
extraction with FTA® Elute Cards. Forensic Sci Int Genet 3:125–127.
Verdon TJ, Mitchell RJ, Van Oorschot RAH. 2014. Evaluation of tapelifting as a collection
method for touch DNA. Forensic Sci Int Genet 8:179–186.
Vickar T, Bache K, Daniel B, Frascione N. 2018. The use of the M-Vac® wet-vacuum system as
a method for DNA recovery. Sci Justice 58:282–286.
Visser R, Hampikian G. 2012. When DNA Won’t Work. Ida Law Rev 49:40–67.
Vitoševic K, Todorovic D, Slovic Z, Zivkovic-Zaric R, Todorovic M. 2019. Forensic genetics
and genotyping. Serbian J Exp Clin Res 20:75–86.
66
Weathered L, Wright K, Chaseling J. 2020. Dealing with DNA evidence in the courtroom: a
plain English review of current issues with identification, mixture and activity level
evidence. In: The Wrongful Conviction Law Review. . p 59–73.
Westhof E. 1988. Water: an integral part of nucleic acid structure. Annu Rev Biophys Biophys
Chem 17:125–144.
Wickenheiser RA. 2002. Trace DNA: a review, discussion of theory, and application of the
transfer of trace quantities of DNA through skin contact. J Forensic Sci 47:442–50.
Widen H, Leufven A, Nielsen T. 2004. Migration of Model Contaminants from PET Bottles:
Influence of Temperature, Food Simulant and Functional Barrier. Food Addit Contam
21:993–1006.
Wienroth M. 2018. Governing anticipatory technology practices. Forensic DNA phenotyping
and the forensic genetics community in Europe. New Genet Soc 37:137–152.
Wood I, Park S, Tooke J, Smith O, Morgan RM, Meakin GE. 2017. Efficiencies of recovery and
extraction of trace DNA from non-porous surfaces. Forensic Sci Int Genet Suppl Ser
6:e153–e155.
Wyner N, Barash M, Mcnevin D, Linacre AM. 2020. Forensic Autosomal Short Tandem Repeats
and Their Potential Association With Phenotype. 11:1–7.
Yang Q, Shen Y, Shao C, Liu Y, Xu H, Zhou Y, Liu Z, Sun K, Tang Q, Xie J. 2020. Genetic
analysis of tri-allelic patterns at the CODIS STR loci. Mol Genet Genomics 295:1263–
1268.
Zahra A, Hussain B, Jamil A, Ahmed Z, Mahboob S. 2018. Forensic STR profiling based smart
barcode, a highly efficient and cost effective human identification system. Saudi J Biol Sci
25:1720–1723.
Zascage RR, Shewale SJ, Planz J V. 2013. Deep-Sequencing Technologies and Potential
Applications in Forensic DNA Testing. In: Shewale JG, Liu RH, editors. Forensic DNA
Analysis: Current Practices and Emerging Technologies. CRC Press.
Zech WD, Malik N, Thali M. 2012. Applicability of DNA Analysis on Adhesive Tape in
Forensic Casework. J Forensic Sci 57:1036–1041.
Zhang J, Zhang J, Tao R, Jiang L, Chen L, Li X, Li C, Zhang S. 2020. A newly devised
multiplex assay of novel polymorphic non-CODIS STRs as a valuable tool for forensic
application. Forensic Sci Int Genet 48.
Zoppis S, Muciaccia B, D’Alessio A, Ziparo E, Vecchiotti C, Filippini A. 2014. DNA
fingerprinting secondary transfer from different skin areas: Morphological and genetic
studies. Forensic Sci Int Genet 11:137–143.
Zuidberg M, Bettman M, Aarts LHJ, Sjerps M, Kokshoorn B. 2019. Targeting relevant sampling
areas for human biological traces: Where to sample displaced bodies for offender DNA? Sci
Justice 59:153–161.
67
Chapter 4: DNA Preservation and Movement
Compared to other aspects of forensic genetics, the protocols for DNA preservation have
gone through minimal changes since their inception. The protocols consistently cover two
aspects: storage length and temperature. However, the storage length and temperature are in
relation to each other. Protocols are created to increase evidence integrity; however, they can
vary and that can cause inconsistencies. The preservation of genetic evidence is crucial in
maintaining the integrity of the DNA because it can be a vital piece of information in a court of
law. Therefore, it is critical to use the proper preservation methods because natural factors
potentially affect the success rate of DNA recovery. This chapter explores the environmental
effects and storage methods to understand the best preservation for DNA. This is then related to
DNA leaching and the theoretical movement of DNA through packaging based on migration
modeling.
Environmental factors can cause damage to the DNA, and this degradation is often what
is discussed with the quality and quantity of forensic DNA, which can impact the integrity of the
DNA. Yet, damage can occur beyond degradation that can impact the integrity of the DNA.
These damages include hydrolytic reaction, oxidation, and radiation.
Hydrolytic Reaction
Also known as hydrolysis, hydrolytic reactions encompass two reactions: deamination
and base loss from the 2’-deoxyribose backbone. First, hydrolytic deamination occurs where
cytosine transforms to uracil, adenine transforms to hypoxanthine (6-oxy purine), and guanine
transforms to xanthine (2-oxy-6-oxy purine). However, hydrolytic deamination primarily occurs
to the cytosines. Secondly, hydrolytic base loss primarily occurs through depurination with the
glycosidic base-sugar bond as the main target (Alaeddini et al., 2010; Marrone and Ballantyne,
68
2010). Therefore, the underlying cause of hydrolysis is the loss of amino groups and the effects
change the coding potential on the DNA template which causes PCR miscoding lesions
(Alaeddini et al., 2010). A study analyzed the process of hydrolysis on DNA in both hydrated
and dried states, which found there to be the same systematic process between both DNA states.
However, the dried state of DNA offers more protection to the effects of hydrolysis (Marrone
and Ballantyne, 2010). In addition, both water and heat can cause this type of DNA damage to
occur (Bonnet et al., 2009; Asari et al., 2018; Chauhan, 2020).
Oxidation
Reactive oxygen species result in the oxidation of DNA, which causes degradation. One
of these species includes hydroxyl radicals. The presence of water can cause oxidation to occur
because of the hydrogen and oxygen that forms the molecule (Bonnet et al., 2009; Tan et al.,
2021). In addition, high temperatures can also cause oxidation to cause DNA damage (Bonnet et
al., 2009; Asari et al., 2018; Chauhan, 2020). In the absence of reactive oxygen species, the
thermal stability of DNA is higher at room temperature (Paunescu et al., 2013; Tan et al., 2021).
The underlying cause of oxidation is the bacterial metabolism and radiation (Richter et al., 1988;
Alaeddini et al., 2010). This then effects the base or sugar fragmentation of the DNA template
which then causes PCR failure (Alaeddini et al., 2010). Oxidation will also contribute to chain
breaks, base modification, and the formation of abasic sites which is a region in DNA where
purine and pyrimidine bases are absent leaving just the DNA backbone (Talpaert-Borlè, 1987;
Bonnet et al., 2009; Tan et al., 2021).
Radiation
Solar radiation produces three types of ultraviolet rays that transmit towards the earth’s
surface: UVA, UVB, and UVC. The least energetic rays are UVA rays that range from 320 to
69
400 nanometers that transmit 95% of the ultraviolet rays that transmit to the earth’s surface. The
other five percent is transmitted by UVB rays that range from 290 to 320 nanometers. The UVC
rays that range from 100 to 290 nanometers do not reach the earth’s surface (Hall et al., 2014).
Both UVB and UVC can be absorbed by DNA, which leads to damages (Gršković et al., 2013).
However, UV rays may not be the primary cause of DNA damage as water absorbs UVA and
UVB rays which can produce reactive oxygen species (Hall et al., 2014).The DNA damage seen
with exposure to UV rays include: base modification, strand breaks, photoproducts, oxidative
damages, cross-linking, and dimer formation (Alaeddini et al., 2010; Hall et al., 2014; Tan et al.,
2021). Allele drop-out only occurred after exposure to the equivalent of 795 days of UVB rays
(Hall et al., 2014). The damage is primarily found in the repetitive sequences of DNA (Barlev
and Sen, 2018). Yet, the use of radiation can be beneficial. As UVC rays can be used to reverse
the formation of cyclobutadipyrimidine by splitting the forming cyclobutene rings (Ravanat et
al., 2001). The exposure of high doses of UVC rays have also cause a rapid DNA degradation
after exposure, making this form of radiation for laboratories to use for decontamination
purposes. However, UVC more easily degraded longer fragmented DNA than short fragments
(Gršković et al., 2013).
Storage Environments: Effects
Time was thought to affect the integrity of DNA; however, research found that the
greatest effect on the integrity of DNA is environmental elements (Alketbi and Goodwin, 2019).
Therefore, it is important to use the appropriate storage methods for genetic evidence because of
the known effects various environmental elements can have on the DNA. Research has
demonstrated that genetic degradation will occur from environmental factors, which includes
70
water and temperature (Lee et al., 2012; Sirker et al., 2016; Asari et al., 2018; Hakim et al., 2020;
Ip et al., 2021).
Water
Humidity refers to the concentration of water vapor in the air. The level of moisture held
in the air is dependent on the temperature; this is known as relative humidity (Lawrence, 2005).
How does humidity potentially damage genetic evidence? Water is known to cause DNA
damage, and since humidity is a percentage of water in a gaseous state, it introduces the risk of
hydrolytic damage, oxidation, and molecular mobility (Bonnet et al., 2009; Tan et al., 2021).
Thus, it is important to dry genetic evidence before placing it into storage when possible. For
example, at 50 percent relative humidity, two things begin to occur: first, the rehydration of
DNA if exposed to air, and second, the start of denaturation of DNA (Bonnet et al., 2009;
Colotte et al., 2011; Tan et al., 2021). In one study, three environmental conditions were tested:
25°C with relative humidity less than 30 percent, and 25°C and 40°C with a relative humidity
greater than 80 percent. The study found that high levels of humidity accelerate the degradation
of DNA, and after 6 months, the degradation ratio 129:41-bp (129 base pair DNA fragment to 41
base pair DNA fragment) had significantly decreased at 25°C with 80 percent RH (Asari et al.,
2018). Another study found DNA degradation after 90 days in storage with a 50 percent relative
humidity (Brogna et al., 2020). The length of the DNA fragments and the sample type will affect
the survival rate of DNA in humid environmental conditions. For instance, blood stains are not
significantly impacted by humidity up to 93 percent relative humidity possibly due to the DNA
still being encapsulated in the nucleus (Dissing et al., 2010). The use of a stabilizing agent, that
protects the cellular structure, can prolong DNA integrity even in high humidity; however, this is
dependent on the agent used (Lee et al., 2012; Shewale and Liu, 2013).
71
Under inadequate environmental conditions, the presence of water will allow the growth
of microbials, which interferes with the extraction and the stability of DNA. Humid conditions
have been shown to be the primary cause for the growth of microbials, which prevents little to no
human DNA from surviving because of the microbial exoenzyme activity interacting with water
(Dissing et al., 2010; Sirker et al., 2016; Al-munim and Al-rashedi, 2021). In one study,
microbial growth began as early as a month and a half with the environmental conditions of
35°C at 100 percent relative humidity (Dissing et al., 2010).
Additionally, environmental conditions affect DNA prior to collection. These effects
should be applied to the current understandings of DNA storage. A study found that high levels
of humidity have different effects depending on whether the collection surface is porous or
nonporous, and more DNA was collected from glass and stainless steel than porous surfaces in
high humidity (Alketbi and Goodwin, 2019). Forensic evidence can be collected using various
materials, which are then placed into a collection container. For example, there are swabs with
plastic collection tubes and others that require a collection cardboard box. If the swabs are not
dried completely, then the cardboard box could lead absorption or leaking of the sample. The
study also found that moisture may increase the DNA transfer rate (Alketbi and Goodwin, 2019).
Thus, it is important to ensure genetic evidence is dried.
Temperature
Studies analyzing various environments have shown that temperature plays an essential
role in the degradation of DNA. The rate that degradation occurs is dependent on the
temperature; for instance, DNA degrades faster at room temperature compared to refrigeration or
freezer, and begins to degrade immediately in extremely hot temperatures (Frippiat and Noel,
2014; Hara et al., 2016; Ng et al., 2018; Abdel Hady et al., 2021; Al-munim and Al-rashedi,
72
2021). The temperature range of -20°C to 37°C produce the highest recovered concentration of
DNA after exposure to these temperatures for twenty-four hours (Raina et al., 2004; Abdel Hady
et al., 2021). However, the type of sample may determine how temperature affects the DNA
because various studies have come to different conclusions on the effects of high temperatures.
For instance, higher temperatures has been found to cause chain-breaking oxidative and
hydrolytic reaction to degrade the DNA (Bonnet et al., 2009; Asari et al., 2018; Chauhan, 2020).
Yet, another study found higher temperatures have less of an effect on the quality of the DNA
(Abdel Hady et al., 2021).
Despite the freezer degrading DNA slower than other temperatures, the freeze-thaw cycle
does have an impact on the integrity of DNA (Corradini et al., 2019). Exposure to multiple
freeze-thaw cycles can reduce the DNA integrity, which might be a result of the formation of ice
crystals during freezing (Lee et al., 2013; Tan et al., 2021). There are significant changes in the
quantity of DNA during some of the freeze-thaw cycles, however, the percentage was within an
acceptable range of ± 5% (Safarikova et al., 2021). There are conflicting conclusions on the rate
at which a sample should be frozen. A more recent study found rapid freezing is less harmful
than slower freezing of DNA (Anchordoquy and Molina, 2007; Fabre et al., 2017; Tan et al.,
2021). Another study found the freezer to be an acceptable storage method for long term storage
despite the freeze-thaw cycle. Rather, liquid nitrogen is unsuitable for long term storage as it
causes DNA to clump (Safarikova et al., 2021).
Storage Methods
Throughout the investigation process forensic evidence will be stored for various reasons.
The first step in the preservation of genetic evidence begins with the collection process. This is
73
typically done by crime scene investigators or police officers (Pickrahn et al., 2017). These
individuals are not always trained and many investigators still depend on experience from prior
casework to collect DNA (Baechler, 2016). A 2017 study found 67.1% of the contaminated
samples originated from the investigators; while another noticed an increase of contamination
over the years (Kloosterman et al., 2014; Pickrahn et al., 2017). After collection, evidence is
often shipped to a forensic facility through various methods, such as UPS, FedEx, or USPS.
During transport, packages can be exposed to extreme temperatures or repeated freeze-thaw
cycle (Shikama, 1965; Davis et al., 2000; Howlett et al., 2014). Therefore, it is vital to ensure the
integrity of genetic evidence throughout the transport and storage process. Optimal storage
conditions prevent the exposure to water and oxygen (Bonnet et al., 2009; Shewale and Liu,
2013). Currently, there are two standard protocols that the majority of laboratories follow
pertaining to time and temperature that are discussed further below. However, the protocols do
not provide the necessary information for the collection of genetic evidence. For instance,
protocols state evidence should be dried prior to packaging, but no further information is
provided than that simple fact. Nevertheless, the protocols do provide adequate information on
the packaging and preservation of genetic evidence (Cordray, 2010; Cătălin et al., 2011;
Department of Public Safety - Texas, 2012; Ballou et al., 2013).
When evidence arrives to a laboratory, the evidence goes through internal laboratory
processing procedures. Upon receiving the evidence there are a series of notes that must be
recorded as a laboratory receipt of evidence. These notes include: type of examination request,
inventory sheet, signs of contamination, and if the packages were properly packaged. Then the
laboratory will conduct an internal processing procedure, which is for the laboratories records
(Lee and Ladd, 2001). After these steps are completed, the evidence will move into storage.
74
Time
Forensic biological evidence is stored during various phases of the investigation process.
The storage conditions are dependent on the stage of the investigation. Short term storage, which
is sometimes rereferred to as temporary storage, pertains to any biological evidence storage
under a designated length of time. According to the National Institute of Justice and the National
Institute of Standards and Technology evidence may be stored in short term conditions for less
than 72 hours. The environmental guidelines for evidence stored in temporary storage will differ
from long term storage. While long term storage pertains to any biological evidence storage
beyond that of the short term storage. The National Institute of Justice recommends long term
storage conditions when storing biological evidence for more than 72 hours (Ballou et al., 2013).
Despite these guidelines, due to the backlog of evidence extractions, evidence is held in storage
until the evidence is extracted.
A backlog in the crime laboratory caused by low funding, low staffing, or evidence
quantity, leads evidence to be held longer in storage. Evidence is potentially considered
backlogged at varying rates based on the type of evidence. For instance, sexual assault kits are
generally considered backlogged between 30 and 90 days after being placed into storage.
However, there is no standard time frame for when evidence begins to be considered backlogged
(Nelson, 2010; Quinlan, 2020). In 1997, 70% of the laboratories reported backlog, which then
increased to 80% by 2000. By 2005, the average backlog request per lab increased from 86 to
152 requests by the end of the year for DNA analysis alone (Durose, 2008; Strom and Hickman,
2010). In the United States between 2011 and 2017 there has been an increase in the number of
backlogged DNA requests at the end of each year (U.S. Government Accountability Office,
2019). A global audit of 148 forensic labs, between December 2020 and January 2021, was
75
conducted. The audit indicated there was a 34% net reduction of the 2020 backlogged samples.
While, the audit predicts a 33% net increase of backlogged samples for 2021(ThermoFisher
Scientific, 2021). Therefore, more samples will be in storage for longer.
Another type of long term storage pertains to evidence retention of DNA from the
extracted evidence. It is important to retain the evidence after DNA extraction because another
extraction or analysis may be required. The retention of evidence will more likely held in police
custody, while the forensic genetics laboratories will retain the extracted samples. Currently, in
England and Wales the retention period is dependent of the national policy and the agencies
retain the evidence. For example, the NPCC V2.1 policies suggests police and forensic services
retain evidence of serious crime for six years; while Forensics 21 policy suggests forensic
services should retain serious crimes for seven years (McCartney and Shorter, 2020). In Ohio, if
a conviction occurs for a SB 77 crime, then the evidence must be retained for either thirty years
or the latest period the convicted is in custody (Cordray, 2010). In Texas, the retention for
unsolved cases is no less than forty years or until the applicable statute of limitations expires. For
convicted cases, the evidence should be retained for as long as the convicted is in custody
(Department of Public Safety - Texas, 2012). Therefore, the evidence retention length is
dependent on the local government’s laws.
Temperature
Currently, there are four temperatures evidence can be stored at: frozen (at or below
-10°C), refrigerated (between 2°C and 8°C, less than 25% humidity), room temperature (ambient
temperature), or temperature controlled (between 15.5°C and 24°C, less than 60% humidity). The
optimal storage temperature is dependent on the evidence type and the length of storage (Ballou
76
et al., 2013). It is important to maintain the temperature and humidity levels based on the storage
environment and to monitor the levels weekly.
The most common temperature for DNA extracts requires many freezers and generators,
which are rather expensive to maintain (Howlett et al., 2014; Corradini et al., 2019; Tan et al.,
2021). According to the National Institute of Justice and the National Institute of Standards and
Technology the freezer is best suited for wet type samples. However, liquid blood is the only
type of evidence that should never be frozen. Both feces and urine are best stored in the freezer
for any length of time. While it is best to freeze wet bloody samples for short term storage and
liquid DNA extracts for long term storage. In addition, it is acceptable to freeze bones for short
term storage (Ballou et al., 2013; Dash et al., 2020c).
According to the National Institute of Justice and the National Institute of Standards and
Technology, refrigeration is not frequently recommended for long term storage. Refrigeration is
more suitable for short term storage for many types of evidence including liquid blood and wet
swabs. While it is considered acceptable to store wet bloody items in refrigeration, urine samples
can only be stored at this temperature for less than 24 hours. In contrast, for long term
refrigeration is best for only liquid blood, however, it is considered acceptable to store liquid
DNA extracts in this environment (Ballou et al., 2013; Dash et al., 2020c).
Room temperature storage is the most cost-effective. Therefore, it is often the method
chosen despite being an optimal storage temperature for a select few evidence types. Three types
of evidence are considered acceptable for room temperature storage: dry biological stained items,
bones, and hair. While buccal swabs can be stored at room temperature, it should be for less than
24 hours. However, only the hair samples are acceptable for long term storage at room
temperature (Ballou et al., 2013; Dash et al., 2020c).
77
According to the National Institute of Justice and the National Institute of Standards and
Technology, temperature-controlled storage is the best environment for most types of evidence
for any length of time. This includes dry biological stained items, hair, dried swabs, buccal
swabs, and vaginal smears. Liquid blood and wet bloody items are able to be stored in
temperature-controlled environment for less than 24 hours. Finally, it is acceptable to store dried
DNA extracts in long term temperature controlled storage (Ballou et al., 2013; Dash et al.,
2020c).
Shelving
The type of shelving used for evidence storage is dependent on both the temperature and
length of storage. A forensic facility has different storage areas based on those variations. The
ideal shelving should be nonporous to prevent contamination. For general storage areas, high-
density shelving can be used, which is beneficial when the facility has limited space as this
shelving system is often mobile allowing the shelves to become compact by eliminating excess
aisle space. High-security vaults are typically reserved for storing money, weapons, or drugs
(Mozayani and Fisher, 2017). The storage equipment will vary from that of long term storage.
For instance, evidence can be temporarily stored in metal lockers while the samples dry.
Temporary storage can include manufactured or repurposed lockers, or under rooms and closets,
commercial, residential or under-the-counter refrigerators and freezers. While long term storage
includes larger shelving and rooms, such as walk-in commercial refrigerators and freezers
(Ballou et al., 2013).
Solution preservation
A DNA preserving agent may be added to a sample, depending on the type of evidence.
In some instances, the preserving agent can preserve DNA better in the normal storage and the
78
optimal agent is dependent on the storage environment. Preserving agents must be void of trace
metals because it causes oxidative damage to the DNA. If stored at -80°C, then DNA must be
isolated from ethanol and then transferred to an aqueous buffer (Anchordoquy and Molina,
2007). SampleMatrix® was tested during the shipment of genetic samples, and was found to not
maintain DNA as well as PTFE containers that are stored at 4°C. However, the cost of the PTFE
containers in large numbers are not cost effective (Clabaugh et al., 2007; Shewale and Liu,
2013). DNAstableTM has been found to be effective in preserving extracted DNA at room
temperature. However, this has been shown to be more effective on low quantity (<20 ng) DNA
samples (Howlett et al., 2014). Trehalose is another preserving agent that was found to improve
the recovery of dried low-quantity DNA at room temperature. However, the presence of
trehalose did not have a significant impact on DNA preservation when stored at -80°C (Shewale
and Liu, 2013). Biomatrica® was found provide better preservation than trehalose (Ivanova and
Kuzmina, 2013). One study tested the preservation of saliva and blood in the presence of a
buffer, which was found to preserve the integrity of the DNA for both sample types. However,
the study analyzed the preservation for four years for the saliva samples and four months for the
blood samples. Therefore, it is unknown if the buffer is able to preserve the beyond the four
months (Burrows et al., 2017, 2019). Since preserving agents are not equal in the preserving of
DNA, the agent used should be selected based on environmental conditions, storage material,
sample type, and if the sample can be dried.
Storage Material
Certain types of container materials are known to better preserve DNA samples during
storage. When packaging collected evidence samples the National Institute of Standards and
Technology recommends using evidence bags, boxes, or envelopes for general evidence
79
packaging, which will then be sealed with adhesive tape. These materials allow for easy storage
and organization. It is preferred that the containers are breathable to prevent condensation and
allow oxygen to prevent bacteria growth (Ballou et al., 2013; Mozayani and Fisher, 2017).
However, through the exposure to air dried DNA can regain water, which will lead to moisture
within the container (Colotte et al., 2011). Plastic packaging is it to be avoided for both inner and
outer packaging (Cordray, 2010; Department of Public Safety - Texas, 2012; Ballou et al., 2013).
However, many forensic swabs available today come with a plastic collection tube, but some of
the collection tubes have breathable ventilation portion near the swab tip. This ventilation allows
excessive moisture to diffuse out of the tube (Aditya et al., 2011; Garvin et al., 2013; Ip et al.,
2021). Some of the collection tubes are constructed of polypropylene, which is a type of plastic,
that is found to interfere with the preservation of DNA because it binds with DNA for an
unknown reason (Gaillard and Strauss, 1998; Kline et al., 2005; Lee et al., 2012). This binding
could cause the DNA to stick to the sides of a plastic tube, and thus, lead to a decrease in the
obtainable DNA from an extracted sample. The DNA bound to the tube would potentially need
to be reextracted.
DNA Movement and Leaching
DNA leaching is the movement of DNA, typically through soil. Different studies have
observed leaching under certain environmental conditions within the soil that migrates radially
out from its source (Haile et al., 2007; Andersen et al., 2012; Emmons et al., 2017; Thomas et al.,
2018). The texture and structure of the soil influences DNA leaching. Research has not detected
leaching in frozen sediments. Nor has DNA been detected migrating beyond the clay strata,
which research theorizes to be caused by the soil structure of the clay (Hebsgaard et al., 2009;
80
Arnold et al., 2011; Andersen et al., 2012). Nevertheless, DNA has been detected between 10 cm
to 16 cm away from its originating source (Andersen et al., 2012; Thomas et al., 2018).
However, it is still not fully understood how the DNA is moving through the soil.
Currently, there is little understood of the external movement of DNA. However, when
DNA is in a solid state, which is when most water has been removed, it is known to decrease
molecular mobility with only small movement possible (Bonnet et al., 2009; Colotte et al.,
2011). Despite the DNA being in a solid state, the environment can reactivate the DNA mobility.
Water and heat enhance the mobility of amorphous solids (Byrn et al., 2001; Bonnet et al.,
2009). As DNA is dehydrated it changes its form because water is important for the nucleic acid
structure. Therefore, DNA’s structure changes with the level of water present. The B-form
structure is a right-handed double helix that consists of ten base pairs per turn that are
perpendicular to the helix axis, which is the most similar structure to the original DNA model.
While the A-form structure consists of eleven base pairs per turn that are displaced and inclined
to the helix axis (Dickerson et al., 1982; Ghosh and Bansal, 2003). There is twice as many water
molecules per nucleotide in the B-form compared to the A-form. As water levels in DNA
reduces below 70% relative humidity, the structure changes to its A-form (Westhof, 1988;
Bonnet et al., 2009). If a sample is not dried completely, then the effects of water, as discussed
above, is detectable in closed vials if the sample volume is significantly smaller than the
container volume (Ayala-Torres et al., 2000; Bonnet et al., 2009).
Despite the limited understanding on the movement of DNA, migration modeling offers
the opportunity to examine known theories of the movement of substances through materials. If
the model is applied with the knowledge of DNA leaching, then this could lead to theories of the
movement of DNA in storage. The elements encompassed in the movement of the models
81
include time, temperature, and vapor pressure (Widen et al., 2004; de Fátima Poças et al., 2011;
Maia et al., 2016; Brandsch, 2017; Fang and Vitrac, 2017; Li et al., 2017). According to Fickian
Diffusion, which is an ideal diffusion, diffusion depends on five things: the polymer, molecule
size, temperature, pressure, and concentration (Keller and Kouzes, 2017). Materials have
different permeation rates, for instance metal has the lowest rate and polypropylene has a
medium permeation rate. As temperature rises the permeability increases approximately five
percent per degree in Celsius (Stannett and Williams, 2007; Keller and Kouzes, 2017). For
instance, using the moisture vapor transmission rate (MVTR), which measures the rate water
vapor passes through material, the rate for a 0.58 mm thick piece of polyvinyl toluene (PVT) in
100% humidity was 2.65 g*m-2*d-1 at 30°C, 4.4 g*m-2*d-1 at 40°C, and 7.19 g*m-2*d-1 at 50°C.
Therefore, permeation will occur at a faster rate in higher temperatures. To keep water from
premating, the MVTR must be below 10-6 g*m-2*d-1 and packaging greater than 0.25 mm thick
(Keller and Kouzes, 2017). Therefore, it is important to maintain low humidity and temperature
levels when storing evidence samples to reduce the various permeability rates.
Conclusion
When preserving DNA there are several aspects that need to be considered before placing
genetic evidence into storage. The storage environment is the most important element to consider
when preserving DNA, as the environment can significantly impact the DNA because many of
the effects of the environmental factors coincide. Ensuring that genetic evidence is dried
completely may be the most important element in preserving DNA as water causes DNA
damage, microbial growth, and the rehydration at a relative humidity of fifty percent. The
preservation of DNA must begin during the collection process since the materials used to collect
82
the DNA will influence the preservation of the sample. Since evidence can be in storage for an
extended period of time it is important that the preservation methods are available in detail for
law enforcement. In addition, the knowledge of migration models provides insight into how
DNA potentially moves and how molecules can move through packaging material. Thus, the
rehydration of DNA potentially allows it to permeate through layers of packaging.
83
References:
Abdel Hady RH, Thabet HZ, Ebrahem NE, Yassa HA. 2021. Thermal Effects on DNA
Degradation in Blood and Seminal Stains: Forensic View. Acad Forensic Pathol 11:7–23.
Adamowicz MS, Stasulli DM, Sobestanovich EM, Bille TW. 2014. Evaluation of methods to
improve the extraction and recovery of DNA from cotton swabs for forensic analysis. PLoS
One 9:1–18.
Aditya S, Sharma AK, Bhattacharyya CN, Chaudhuri K. 2011. Generating STR profile from
“touch DNA.” J Forensic Leg Med 18:295–298.
Aggarwal K. 2020. Forensic DNA Phenotyping: Significance in Criminal Investigations. Acad J
Forensic Sci 03:2581–4273.
Al-munim MFA, Al-rashedi NAM. 2021. Stability of DNA Quantitation State of Blood
Evidence under Different Conditions. 25:4965–4971.
Alaeddini R, Walsh SJ, Abbas A. 2010. Forensic Science International : Genetics Forensic
implications of genetic analyses from degraded DNA — A review. Forensic Sci Int Genet
4:148–157.
Alessandrini F, Onofri V, Turchi C, Buscemi L, Pesaresi M, Tagliabracci A. 2020. Past, Present
and Future in Forensic Human Identification. In: Longhi S, Monteriù A, Freddi A, Aquilanti
L, Ceravolo MG, Carnevali O, Giordano M, Moroncini G, editors. The First Outstanding 50
Years of “Universita Politecnica delle Marche”: Research Achievements in Life Sciences. .
p 81–92.
Alketbi SK, Goodwin W. 2019. The effect of time and environmental conditions on Touch DNA.
Forensic Sci Int Genet Suppl Ser 7:701–703.
Allwood JS, Fierer N, Dunn RR. 2020. The future of environmental DNA in forensic science.
Appl Environ Microbiol 86:1–9.
Alonso A, Müller P, Roewer L, Willuweit S, Budowle B, Parson W. 2017. European survey on
forensic applications of massively parallel sequencing. Forensic Sci Int Genet 29:e23–e25.
Aloraer D, Hassan NH, Albarzinji B, Goodwin W. 2015. Collection protocols for the recovery of
biological samples. Forensic Sci Int Genet Suppl Ser 5:e207–e209.
Ambers A, Wiley R, Novroski N, Budowle B. 2018. Direct PCR amplification of DNA from
human bloodstains, saliva, and touch samples collected with microFLOQ® swabs. Forensic
Sci Int Genet 32:80–87.
Amorim A. 2019. Nonhuman forensic genetics. Forensic Sci Int Genet Suppl Ser 7:44–46.
Amorim A, Pereira F, Alves C, Garcia O. 2020. Species assignment in forensics and the
challenge of hybrids. Forensic Sci Int Genet 48:101676.
Anchordoquy TJ, Molina MC. 2007. Preservation of DNA. Cell Preserv Technol 5:180–188.
Andersen K, Bird KL, Rasmussen M, Haile J, Breuning-Madsen H, Kjær KH, Orlando L, Gilbert
MTP, Willerslev E. 2012. Meta-barcoding of “dirt” DNA from soil reflects vertebrate
biodiversity. Mol Ecol 21:1966–1979.
Anon. 2017. Rapid DNA Act of 2017. 115th Congress. Available from:
https://www.congress.gov/bill/115th-congress/house-bill/510
84
Anzai-Kanto E, Hirata MH, Hirata RDC, Nunes FD, Melani RFH, Oliveira RN. 2005. Extração
de DNA de saliva humana depositada sobre a pele e sua aplicabilidade aos processos de
identificação forense. Braz Oral Res 19:216–222.
Arenas M, Pereira F, Oliveira M, Pinto N, Lopes AM, Gomes V, Carracedo A, Amorim A. 2017.
Forensic genetics and genomics: Much more than just a human affair. PLoS Genet 13:1–28.
Arnold LJ, Roberts RG, Macphee RDE, Haile JS, Brock F, Möller P, Froese DG, Tikhonov AN,
Chivas AR, Gilbert MTP, Willerslev E. 2011. Paper II - Dirt, dates and DNA: OSL and
radiocarbon chronologies of perennially frozen sediments in Siberia, and their implications
for sedimentary ancient DNA studies. Boreas 40:417–445.
Arora A. 2020. Future of Forensic and Crime Scene Science Technologies. Technol Forensic
Sci:357–370.
Asari M, Matsuura H, Isozaki S, Hoshina C, Okuda K, Tanaka H, Horioka K, Shiono H, Shimizu
K. 2018. Assessment of DNA degradation of buccal cells under humid conditions and DNA
repair by DOP-PCR using locked nucleic acids. Leg Med 35:29–33.
Ayala-Torres S, Chen Y, Svoboda T, Rosenblatt J, Van Houten B. 2000. Analysis of gene-
specific DNA damage and repair using quantitative polymerase chain reaction. Methods
22:135–147.
Baechler S. 2016. Study of criteria influencing the success rate of DNA swabs in operational
conditions: A contribution to an evidence-based approach to crime scene investigation and
triage. Forensic Sci Int Genet 20:130–139.
Balding DJ, Buckleton J. 2009. Interpreting low template DNA profiles. Forensic Sci Int Genet
4:1–10.
Ballou S, Stolorow M, Taylor M, Bamberger PS, Brown L, Brown R, Burney Y, Davenport D,
DePalma L, Williams S, Jones C, Keaton R, Kiley W, Latta J, Kline M, Lanning K, LaPorte
G, Ledray LE, Nagy R, Ostrom BE, Schwind L, Stoiloff S. 2013. The biological evidence
preservation handbook : best practices for evidence handlers ; technical working group on
biological evidence preservation.
Barash M, Reshef A, Brauner P. 2010. The use of adhesive tape for recovery of dna from crime
scene items. J Forensic Sci 55:1058–1064.
Barlev A, Sen D. 2018. DNA’s Encounter with Ultraviolet Light: An Instinct for Self-
Preservation? Acc Chem Res 51:526–533.
Basset P, Castella V. 2018. Lessons from a study of DNA contaminations from police services
and forensic laboratories in Switzerland. Forensic Sci Int Genet 33:147–154.
Berglund EC, Kiialainen A, Syvänen AC. 2011. Next-generation sequencing technologies and
applications for human genetic history and forensics. Investig Genet 2:1–15.
Bhinder M, Zahoor M, Sadia H, Qasim M, Perveen R, Anjum G, Iqbal M, Ullah N, Shehzad W,
Tariq M, AM Waryah. 2018. SE33 locus as a reliable genetic marker for forensic DNA
analysis systems. Turkish J Med Sci 48:611–614.
Blears MJ, De Grandis SA, Lee H, Trevors JT. 1998. Amplified fragment length polymorphism
(AFLP): A review of the procedure and its applications. J Ind Microbiol Biotechnol 21:99–
85
114.
Blozis J. 2014. Forensic DNA evidence collection at a crime scene: an investigator’s
commentary. In: Shewale JG, Liu RH, editors. Forensic DNA Analysis Current Practices
and Emerging Technologies. Boca Raton: CRC Press. p 3–17.
Bonnet J, Colotte M, Coudy D, Couallier V, Portier J, Morin B, Tuffet S. 2009. Chain and
conformation stability of solid-state DNA: Implications for room temperature storage.
Nucleic Acids Res 38:1531–1546.
Bonsu DOM, Higgins D, Austin JJ. 2020. Forensic touch DNA recovery from metal surfaces – A
review. Sci Justice 60:206–215.
Børsting C, Morling N. 2015. Next generation sequencing and its applications in forensic
genetics. Forensic Sci Int Genet 18:78–89.
Brandsch R. 2017. Probabilistic migration modelling focused on functional barrier efficiency and
low migration concepts in support of risk assessment. Food Addit Contam Part A 34:1743–
1766.
Bright JA, Kelly H, Kerr Z, McGovern C, Taylor D, Buckleton JS. 2020. The interpretation of
forensic DNA profiles: an historical perspective. J R Soc New Zeal 50:211–225.
Bright JA, Petricevic SF. 2004. Recovery of trace DNA and its application to DNA profiling of
shoe insoles. Forensic Sci Int 145:7–12.
Brogna R, Oldenhof H, Sieme H, Wolkers WF. 2020. Spectral fingerprinting to evaluate effects
of storage conditions on biomolecular structure of filter-dried saliva samples and recovered
DNA. Sci Rep 10:1–12.
Brownlow RJ, Dagnall KE, Ames CE. 2012. A Comparison of DNA Collection and Retrieval
from Two Swab Types (Cotton and Nylon Flocked Swab) when Processed Using Three
QIAGEN Extraction Methods. J Forensic Sci 57:713–717.
Bruijns B, Tiggelaar R, Gardeniers H. 2018. Massively parallel sequencing techniques for
forensics: A review. Electrophoresis 39:2642–2654.
Buckleton J. 2009. Validation issues around DNA typing of low level DNA. Forensic Sci Int
Genet 3:255–260.
Budowle B, Moretti TR, Niezgoda SJ, Brown BL. 1998. CODIS and PCR-Based Short Tandem
Repeat Loci: Law Enforcement Tools. Second Eur Symp Hum Identif 7388:73–88.
Burrill J, Daniel B, Frascione N. 2019. A review of trace “Touch DNA” deposits: Variability
factors and an exploration of cellular composition. Forensic Sci Int Genet 39:8–18.
Burrows AM, Kasu M, D’Amato ME. 2019. Preservation of DNA integrity in biological
material. Forensic Sci Int Genet Suppl Ser 7:416–418.
Burrows AM, Ristow PG, D’Amato ME. 2017. Preservation of DNA from saliva samples in
suboptimal conditions. Forensic Sci Int Genet Suppl Ser 6:e80–e81.
Butler E, Li R. 2014. Genetic Markers for Sex Identification in Forensic DNA Analysis. J
Forensic Investig 02.
Butler JM. 2015. The future of forensic DNA analysis. Philos Trans R Soc B Biol Sci 370.
Butler JM, Coble MD, Vallone PM. 2007. STRs vs. SNPs: Thoughts on the future of forensic
86
DNA testing. Forensic Sci Med Pathol 3:200–205.
Butler JM, Hill CR, Kline MC, Duewer DL, Sprecher CJ, McLaren RS, Rabbach DR, Krenke
BE, Storts DR. 2009. The single most polymorphic STR Locus: SE33 performance in U.S.
populations. Forensic Sci Int Genet Suppl Ser 2:23–24.
Butler JM, Willis S. 2020. Interpol review of forensic biology and forensic DNA typing 2016-
2019. Forensic Sci Int Synerg.
Byard RW, James H, Berketa J, Heath K. 2016. Locard’s Principle of Exchange, Dental
Examination and Fragments of Skin. J Forensic Sci 61:545–547.
Byrn SR, Xu W, Newman AW. 2001. Chemical reactivity in solid-state pharmaceuticals:
Formulation implications. Adv Drug Deliv Rev 48:115–136.
Cale CM, Earll ME, Latham KE, Bush GL. 2016. Could Secondary DNA Transfer Falsely Place
Someone at the Scene of a Crime? J Forensic Sci 61:196–203.
Caliebe A, Walsh S, Liu F, Kayser M, Krawczak M. 2017. Likelihood ratio and posterior odds in
forensic genetics: Two sides of the same coin. Forensic Sci Int Genet 28:203–210.
Carracedo Á, Prieto L. 2019. Beyond the CSI effect: The keys to good forensic genetics
communication. Metode 2019:31–37.
Cătălin M, Andrei A, Mitraşca O. 2011. Modern Methods of Collection and Preservation of
Biological Evidence for Human Identification by DNA Analysis. Abacus Diagnostics.
Chauhan M. 2020. Storage of saliva and blood specimen in different temperature. Int J Forensic
Med 2:21–24.
Chong KWY, Thong Z, Syn CK. 2021. Recent trends and developments in forensic DNA
extraction . WIREs Forensic Sci 3:1–23.
Clabaugh K, Silva B, Odigie K, Fourney R, Stevens J, Carmody G, Coble MD, Loreille O,
Scheible M, Kline M, Parsons TJ. 2007. Storage of DNA samples at ambient temperature
using DNA-SampleMatrix. Poster Present 18th Annu Meet Int Symp Hum Identification,
Hollywood, CA.
Colotte M, Coudy D, Tuffet S, Bonnet J. 2011. Adverse Effect of Air Exposure on the Stability
of DNA Stored at Room Temperature. Biopreserv Biobank:47–50.
Comte J, Baechler S, Gervaix J, Lock E, Milon MP, Delémont O, Castella V. 2019. Touch DNA
collection – Performance of four different swabs. Forensic Sci Int Genet 43.
Cordray R. 2010. Guidelines for preservation and retention of biological evidence.
Corradini B, Alù M, Magnanini E, Galinier ME, Silingardi E. 2019. The importance of forensic
storage support: DNA quality from 11-year-old saliva on FTA cards. Int J Legal Med
133:1743–1750.
Dadhania A, Nelson M, Caves G, Santiago R, Podini D. 2013. Evaluation of Copan
4N6FLOQSwabsTM used for crime scene evidence collection. Forensic Sci Int Genet Suppl
Ser 4:e336–e337.
Daly DJ, Murphy C, McDermott SD. 2012. The transfer of touch DNA from hands to glass,
fabric and wood. Forensic Sci Int Genet 6:41–46.
Dargay A, Roy R. 2016. Direct Y-STR amplification of body fluids deposited on commonly
87
found crime scene substrates. J Forensic Leg Med 39:50–60.
Dash HR, Shrivastava P, Das S. 2020a. Principles and Practices of DNA Analysis: A Laboratory
Manual for Forensic DNA Typing. New York, NY: Springer Protocols Handbook.
Dash HR, Shrivastava P, Das S. 2020b. Biological Samples: The Target Sources for DNA
Typing. In: Principles and Practices of DNA Analysis: A Laboratory Manual for Forensic
DNA Typing. New York, NY: Humana. p 13–20.
Dash HR, Shrivastava P, Das S. 2020c. Collection, Transportation, and Preservation of
Biological Evidences for DNA Analysis. In: Principles and Practices of DNA Analysis: A
Laboratory Manual for Forensic DNA Typing. New York, NY: Humana. p 21–27.
Dash HR, Shrivastava P, Das S. 2020d. Reliable Use of WhatmanTM FTATM Cards for One-Step
Collection and Isolation of DNA. In: Principles and Practices of DNA Analysis: A
Laboratory Manual for Forensic DNA Typing. New York, NY: Humana. p 109–115.
Davis C, Illescas M, Tirado C, Lopez R, Budowle B, Cruz TD. 2012. A Case of Amelogenin Y-
null: A simple primer binding site mutation or unusual genetic anomaly? Leg Med 14:320–
323.
Davis DL, O’Brie EP, Bentzley CM. 2000. Analysis of the degradation of oligonucleotide
strands during the freezing/thawing processes using MALDI-MS. Anal Chem 72:5092–
5096.
Department of Public Safety - Texas. 2012. Best practices for collection, packaging, storage,
preservation, and retrieval of biological evidence. :1–8.
Dickerson RE, Drew HR, Conner BN, Wing RM, Fatini A V., Kopka ML. 1982. The Anatomy
of A-, B-, and Z-DNA. Science (80- ) 216:475–485.
Diegoli TM. 2015. Forensic typing of short tandem repeat markers on the X and Y
chromosomes. Forensic Sci Int Genet 18:140–151.
Dissing J, Søndervang A, Lund S. 2010. Exploring the limits for the survival of DNA in blood
stains. J Forensic Leg Med 17:392–396.
Dong H, Wang J, Zhang T, Ge JY, Dong YQ, Sun QF, Liu C, Li CX. 2017. Comparison of
preprocessing methods and storage times for touch DNA samples. Croat Med J 58:4–13.
Durose M. 2008. Census of Publicly Funded Forensic Crime Laboratories, 2005, Bureau of
Justice Statistics. :1–12.
Dziak R, Peneder A, Buetter A, Hageman C. 2018. Trace DNA Sampling Success from
Evidence Items Commonly Encountered in Forensic Casework. J Forensic Sci 63:835–841.
Emmons AL, DeBruyn JM, Mundorff AZ, Cobaugh KL, Cabana GS. 2017. The persistence of
human DNA in soil following surface decompositions. Sci Justice 57:341–348.
Erlich H. 2020. In the Begginning: Forensic Applications of DNA Technologies. In: Erlich H,
Stover E, White TJ, editors. Silent Witness: Forensic DNA Evidence in Criminal
Investigations and Humanitarian Disaters. Oxford Univeristy Press. p 15–33.
Erlich H, Calloway C, Lee SB. 2020. Recent Developments in Forensic DNA Technology. In:
Erlich H, Stover E, White TJ, editors. Silent Witness: Forensic DNA Evidence in Criminal
Investigations and Humanitarian Disaters. Oxford Univeristy Press. p 105–127.
88
Esslinger KJ, Siegel JA, Spillane H, Stallworth S. 2004. Using STR Analysis to Detect Human
DNA from Exploded Pipe Bomb Devices. J Forensic Sci 49:1–4.
Fabre A-L, Luis A, Colotte M, Tuffet S, Bonnet J. 2017. High DNA stability in white blood cells
and buffy coat lysates stored at ambient temperature under anoxic and anhydrous
atmosphere. PLoS One 12:e0188547.
Fang X, Vitrac O. 2017. Predicting diffusion coefficients of chemicals in and through packaging
materials. Crit Rev Food Sci Nutr 57:275–312.
de Fátima Poças M, Oliveria JC, Peteira JR, Brandsch R, Hogg T. 2011. Modelling migration
from paper into a food simulant. Food Control 22:303–312.
Fonneløp AE, Johannessen H, Egeland T, Gill P. 2016. Contamination during criminal
investigation: Detecting police contamination and secondary DNA transfer from evidence
bags. Forensic Sci Int Genet.
Frippiat C, Noel F. 2014. Efficiency of a novel forensic room-temperature DNA storage
medium. Forensic Sci Int Genet 9:81–84.
Gaillard C, Strauss F. 1998. Avoiding adsorption of DNA to polypropylene tubes and
denaturation of short DNA fragments. Tech Tips Online 3:63–65.
Garvin AM, Holzinger R, Berner F, Krebs W, Hostettler B, Lardi E, Hertli C, Quartermaine R,
Stamm C. 2013. The forensix evidence collection tube and its impact on dna preservation
and recovery. Biomed Res Int 2013.
GE Healthcare. 2010. Reliable extraction of DNA from Whatman FTA cards. Appl Note 28-
9822-22 AA.
Ge J, Sun H, Li H, Liu C, Yan J, Budowle B. 2014. Future directions of forensic DNA databases.
Croat Med J 55:163–166.
Ghosh A, Bansal M. 2003. A glossary of DNA structures from A to Z. Acta Crystallogr - Sect D
Biol Crystallogr 59:620–626.
Goray M, Eken E, Mitchell RJ, van Oorschot RAH. 2010. Secondary DNA transfer of biological
substances under varying test conditions. Forensic Sci Int Genet 4:62–67.
Gršković B, Zrnec D, Popović M, Petek MJ, Primorac D, Mršić G. 2013. Effect of ultraviolet c
radiation on biological samples. Croat Med J 54:263–271.
Gunnarsson J, Helena E, Ansell R. 2010. Success rates of a forensic tape-lift method for DNA
recovery. Probl Forensic Sci LXXXIII:243–254.
Haile J, Holdaway R, Oliver K, Bunce M, Gilbert MTP, Nielsen R, Munch K, Ho SYW, Shapiro
B, Willerslev E. 2007. Ancient DNA chronology within sediment deposits: Are
paleobiological reconstructions possible and is DNA leaching a factor? Mol Biol Evol
24:982–989.
Hakim HM, Lalung J, Khan HO, Ismail SA, Aziz MY, Ishak AR, Safuan S, Rasudin NS,
Chambers GK, Edinur HA. 2020. Evaluation of long-term storage effects on buccal cell
DNA from untreated cards for STR profiling. IOP Conf Ser Earth Environ Sci 596.
Hall A, Sims LM, Ballantyne J. 2014. Assessment of DNA damage induced by terrestrial UV
irradiation of dried bloodstains: Forensic implications. Forensic Sci Int Genet 8:24–32.
89
Hall D, Fairley M. 2004. A single approach to the recovery of DNA and firearm discharge
residue evidence. Sci Justice - J Forensic Sci Soc 44:15–19.
Hanson E., Ballantyne J. 2013. “Getting blood from a stone”: ultrasensitive forensic DNA
profiling of microscopic bio-particles recovered from “touch DNA” evidence. In: Nucleic
Acids Detection. Totowa, NJ: Humana Press. p 3–17.
Hara M, Nakanishi H, Yoneyama K, Saito K, Takada A. 2016. Effects of storage conditions on
forensic examinations of blood samples and bloodstains stored for 20 years. Leg Med
18:81–84.
Hares DR. 2015. Selection and implementation of expanded CODIS core loci in the United
States. Forensic Sci Int Genet 17:33–34.
Hauhart R, Menius K. 2014. DNA Evidence: Examining Police Officers’ Knowledge of
Handling Procedures in a Mid-Size Department. Int J Criminol Sociol 3:360–376.
Hebda LM, Doran AE, Foran DR. 2014. Collecting and analyzing DNA evidence from
fingernails: A comparative study. J Forensic Sci 59:1343–1350.
Hebsgaard MB, Arneborg J, Heyn P, Allentoft ME, Bunce M, Schweger C, Willerslev E. 2009.
‘The Farm Beneath the Sand’ – an archaeological case study on ancient ‘dirt’ DNA.
Antiquity 83:430–444.
Hedman J, Jansson L, Akel Y, Wallmark N, Gutierrez Liljestrand R, Forsberg C, Ansell R. 2020.
The double-swab technique versus single swabs for human DNA recovery from various
surfaces. Forensic Sci Int Genet 46:20–24.
Hefetz I, Einot N, Faerman M, Horowitz M, Almog J. 2019. Touch DNA: The effect of the
deposition pressure on the quality of latent fingermarks and STR profiles. Forensic Sci Int
Genet 38:105–112.
Helmus J, Bajanowski T, Poetsch M. 2016. DNA transfer—a never ending story. A study on
scenarios involving a second person as carrier. Int J Legal Med 130:121–125.
Hess S, Haas C. 2017. Recovery of Trace DNA on Clothing: A Comparison of Mini-tape Lifting
and Three Other Forensic Evidence Collection Techniques. J Forensic Sci 62:187–191.
Hogan C, Houten LB Van, Coticone S. 2018. Comparison of the Quantity and Overall Quality of
Trace DNA Evidence Collected from Substrates Found at Crime Scenes. J Forensic Identif
68.
Holland M, Melton T, Holland C. 2013. Forensic Mitochondrial DNA Analysis: Current Practice
and Future Potential. In: Shewale JG, Liu RH, editors. Forensic DNA Analysis: Current
Practices and Emerging Technologies. CRC Press. p 249–278.
Hopman R, M’charek A. 2020. Facing the unknown suspect: forensic DNA phenotyping and the
oscillation between the individual and the collective. Biosocieties 15:438–462.
Howlett SE, Castillo HS, Gioeni LJ, Robertson JM, Donfack J. 2014. Evaluation of
DNAstableTM for DNA storage at ambient temperature. Forensic Sci Int Genet.
Hytinen ME, Solomon AD, Miller MT, Cruz TD. 2017. Methods for Obtaining High-Quality
Touch DNA from a Nonporous Surface after Latent Fingerprint Collection. J Forensic
Identif 67:71–84.
90
Ip SCY, Yu EY, Li C. 2021. Blood DNA Preservation on Various Forensic Swab Devices. J
Forensic Identif 71.
Ivanova N V., Kuzmina ML. 2013. Protocols for dry DNA storage and shipment at room
temperature. Mol Ecol Resour 13:890–898.
Jakovski Z, Ajanovska RJ, Stankov A, Poposka V, Bitoljanu N, Belakaposka V. 2017. The
power of forensic DNA data bases in solving crime cases. Forensic Sci Int Genet Suppl Ser
6:e275–e276.
Janssen K, Aune M, Olsen M, Olsen GH, Berg T. 2019. Biological stain collection – Absorbing
paper is superior to cotton swabs. Forensic Sci Int Genet Suppl Ser 7:468–469.
Jeffreys A, Brookfield J, Semeonoff R. 1985. Positive identification of an immigration test-case
using human DNA fingerprints. Nature 317:818–819.
Joël J, Glanzmann B, Germann U, Cossu C. 2015. DNA extraction of forensic adhesive tapes—
A comparison of two different methods. Forensic Sci Int Genet Suppl Ser 5:e579–e581.
Junkin T. 2005. Bloodsworth:The True Story of One Man’s Triumph Over Injustice. Algonquin
Books.
Kamphausen T, Schadendorf D, Von Wurmb-Schwark N, Bajanowski T, Poetsch M. 2012. Good
shedder or bad shedder- The influence of skin diseases on forensic DNA analysis from
epithelial abrasions. Int J Legal Med 126:179–183.
Karantzali E, Rosmaraki P, Kotsakis A, Le Roux-Le Pajolec MG, Fitsialos G. 2019. The effect
of FBI CODIS Core STR Loci expansion on familial DNA database searching. Forensic Sci
Int Genet 43.
Kayser M. 2015. Forensic DNA Phenotyping: Predicting human appearance from crime scene
material for investigative purposes. Forensic Sci Int Genet 18:33–48.
Kayser M. 2017. Forensic use of Y-chromosome DNA: a general overview. Hum Genet
136:621–635.
Keller PE, Kouzes R. 2017. Water Vapour Permeation in Plastics. :29.
Kirgiz IA, Calloway C. 2017. Increased recovery of touch DNA evidence using FTA paper
compared to conventional collection methods. J Forensic Leg Med 47:9–15.
Kita T, Yamaguchi H, Yokoyama M, Tanaka T, Tanaka N. 2008. Morphological study of
fragmented DNA on touched objects. Forensic Sci Int Genet 3:32–36.
Kline MC, Duewer DL, Redman JW, Butler JM. 2005. Results from the NIST 2004 DNA
Quantitation Study. J Forensic Sci 50:1–8.
Kloosterman A, Sjerps M, Quak A. 2014. Error rates in forensic DNA analysis: Definition,
numbers, impact and communication. Forensic Sci Int Genet 12:77–85.
Kumar KR, Cowley MJ, Davis RL. 2019. Next-Generation Sequencing and Emerging
Technologies. Semin Thromb Hemost 45:661–673.
Ladd C, Adamowicz MS, Bourke MT, Scherczinger CA, Lee HC. 1999. A Systematic Analysis
of Secondary DNA Transfer. J Forensic Sci 44:14599J.
Lapointe M, Rogic A, Bourgoin S, Jolicoeur C, Séguin D. 2015. Leading-edge forensic DNA
analyses and the necessity of including crime scene investigators, police officers and
91
technicians in a DNA elimination database. Forensic Sci Int Genet 19:50–55.
Lawrence MG. 2005. The relationship between relative humidity and the dewpoint temperature
in moist air: A simple conversion and applications. Bull Am Meteorol Soc 86:225–233.
Lee HC, Ladd C. 2001. Preservation and collection of biological evidence. Croat Med J 42:225–
228.
Lee S, Crouse C, Kline M. 2013. Optimizing storage and handling of DNA extracts. In: Shewale
JG, editor. Forensic DNA Analysis: Current Practices and Emerging Technologies. CRC
Press. p 19–64.
Lee SB, Clabaugh KC, Silva B, Odigie KO, Coble MD, Loreille O, Scheible M, Fourney RM,
Stevens J, Carmody GR, Parsons TJ, Pozder A, Eisenberg AJ, Budowle B, Ahmad T, Miller
RW, Crouse CA. 2012. Assessing a novel room temperature DNA storage medium for
forensic biological samples. Forensic Sci Int Genet 6:31–40.
Li B, Wang ZW, Lin QB, Hu CY. 2017. Molecular dynamics simulation of three plastic
additives’ diffusion in polyethylene terephthalate. Food Addit Contam Part A 34:1086–
1099.
Li C. 2018. Forensic genetics. Forensic Sci Res 3:103–104.
Linacre A, Pekarek V, Swaran YC, Tobe SS. 2010. Generation of DNA profiles from fabrics
without DNA extraction. Forensic Sci Int Genet 4:137–141.
Lowe A, Murray C, Whitaker J, Tully G, Gill P. 2002. The propensity of individuals to deposit
DNA and secondary transfer of low level DNA from individuals to inert surfaces. Forensic
Sci Int 129:25–34.
Lyons LA, Grahn RA, Kun TJ, Netzel LR, Wictum EE, Halverson JL. 2014. Acceptance of
domestic cat mitochondrial DNA in a criminal proceeding. Forensic Sci Int Genet 13:61–
67.
Machado H, Granja R. 2020. Forensic Genetics in the Governance of Crime.
Machado H, Silva S. 2019. What influences public views on forensic DNA testing in the
criminal field? A scoping review of quantitative evidence. Hum Genomics 13:23.
Maia J, Rodriguez-Bernaldo de Quirós A, Sendón R, Cruz JM, Seiler A, Franz R, Simoneau C,
Castle L, Driffield M, Mercea P, Oldring P, Tosa V, Paseiro P. 2016. Determination of key
diffusion and partition parameters and their use in migration modelling of benzophenone
from low-density polyethylene (LDPE) into differnt foodstuffs. Food Addit Contam Part A
33:715–724.
Mandel P, Metais P. 1948. Les acides nucleiques du plasma sanguin chez l’homme. CR Seances
Soc Biol Fil 142:241–243.
Mapes AA, Kloosterman AD, van Marion V, de Poot CJ. 2016. Knowledge on DNA Success
Rates to Optimize the DNA Analysis Process: From Crime Scene to Laboratory. J Forensic
Sci 61:1055–1061.
Marrone A, Ballantyne J. 2010. Hydrolysis of DNA and its molecular components in the dry
state. Forensic Sci Int Genet 4:168–177.
McCartney C, Shorter L. 2020. Police retention and storage of evidence in England and Wales.
92
Int J Police Sci Manag 22:123–136.
McCord BR, Gauthier Q, Cho S, Roig MN, Gibson-Daw GC, Young B, Taglia F, Zapico SC,
Mariot RF, Lee SB, Duncan G. 2019. Forensic DNA Analysis. Anal Chem 91:673–688.
Milne E, Van Bockxmeer FM, Robertson L, Brisbane JM, Ashton LJ, Scott RJ, Armstrong BK.
2006. Buccal DNA collection: Comparison of buccal swabs with FTA cards. Cancer
Epidemiol Biomarkers Prev 15:816–819.
Mistek E, Fikiet MA, Khandasammy SR, Lednev IK. 2019. Toward Locard’s Exchange
Principle: Recent Developments in Forensic Trace Evidence Analysis. Anal Chem 91:637–
654.
Moore MK, Frazier K. 2019. Humans Are Animals, Too: Critical Commonalities and
Differences Between Human and Wildlife Forensic Genetics. J Forensic Sci 64:1603–1621.
Moretti TR, Moreno LI, Smerick JB, Pignone ML, Hizon R, Buckleton JS, Bright JA, Onorato
AJ. 2016. Population data on the expanded CODIS core STR loci for eleven populations of
significance for forensic DNA analyses in the United States. Forensic Sci Int Genet 25:175–
181.
Morgan TH. 1917. The Theory of the Gene. Am Nat 51:513–544.
Mozayani A, Fisher CP eds. 2017. Forensic Evidence Management: From the Crime Scene to the
Courtroom. CRC Press.
Mueller UG, Wolfenbarger LL. 1999. AFLP genotyping and fingerprinting. Trends Ecol Evol
14:389–394.
Murphy E. 2018. Forensic DNA typing. Annu Rev Criminol 1:497–515.
Nachman MW, Crowell SL. 2000. Estimate of the mutation rate per nucleotide in humans.
Genetics 156:297–304.
Nelson M. 2010. Making sense of DNA backlogs: myths vs. reality (Report No. NCJ 232197).
National Insitute of Justice, Office of Justice Programs, U.S. Department of Justice.
Ng HH, Ang HC, Hoe SY, Lim M-L, Tai HE, Soh RCH, Syn CK-C. 2018. Simple DNA
extraction of urine samples: Effects of storage temperature and storage time. Forensic Sci
Int 287:36–39.
Noël S, Lagace K, Rogic A, Granger D, Bourgoin S, Jolicoeur C, Séguin D. 2016. DNA transfer
during laundering may yield complete genetic profiles. Forensic Sci Int Genet 23:240–247.
Novroski NMM, Wendt FR, Woerner AE, Bus MM, Coble M, Budowle B. 2019. Expanding
beyond the current core STR loci: An exploration of 73 STR markers with increased
diversity for enhanced DNA mixture deconvolution. Forensic Sci Int Genet 38:121–129.
Oliveira TP, Nogueira TLS, Valentin ESB, Santos OCL, Carvalho EF, Silva DA. 2015.
Evaluation of collection and extraction methodologies of latent fingerprints for military
application. Forensic Sci Int Genet Suppl Ser 5:e474–e475.
van Oorschot RAH. 2012. Assessing DNA Profiling Success Rates: Need for More and Better
Collection of Relevant Data. Forensic Sci Policy Manag An Int J 3:37–41.
van Oorschot RAH, Ballantyne KN, Mitchell RJ. 2010. Forensic trace DNA: A review. Investig
Genet 1:1–17.
93
van Oorschot RAH, Jones MK. 1997. DNA fingerprints from fingerprints. Nature 387:766–767.
Ostojic L, Wurmbach E. 2017. Analysis of fingerprint samples, testing various conditions, for
forensic DNA identification. Sci Justice 57:35–40.
Pang BCM, Cheung BKK. 2007. Double swab technique for collecting touched evidence. Leg
Med 9:181–184.
Panneerchelvam S, Norazmi MN. 2003. Forensic DNA profiling and database. Malaysian J Med
Sci 10:20–26.
Paunescu D, Puddu M, Soellner JO, Stoessel PR, Grass RN. 2013. Reversible DNA
encapsulation in silica to produce ROS-resistant and heat-resistant synthetic DNA’fossils’.
Nat Protoc 8:2440–2448.
Pickrahn I, Kreindl G, Müller E, Dunkelmann B, Zahrer W, Cemper-Kiesslich J, Neuhuber F.
2017. Contamination incidents in the pre-analytical phase of forensic DNA analysis in
Austria—Statistics of 17 years. Forensic Sci Int Genet 31:12–18.
Pizzamiglio M, Mameli A, My D, Garofano L. 2004. Forensic identification of a murderer by
LCN DNA collected from the inside of the victim’s car. Int Congr Ser 1261:437–439.
Plaza DT, Mealy JL, Lane JN, Parsons MN, Bathrick AS, Slack DP. 2016. Nondestructive
Biological Evidence Collection with Alternative Swabs and Adhesive Lifters. J Forensic Sci
61:485–488.
Poetsch M, Bajanowski T, Kamphausen T. 2013. Influence of an individual’s age on the amount
and interpretability of DNA left on touched items. Int J Legal Med 127:1093–1096.
Poetsch M, Pfeifer M, Konrad H, Bajanowski T, Helmus J. 2018. Impact of several wearers on
the persistence of DNA on clothes—a study with experimental scenarios. Int J Legal Med
132:117–123.
Pourazar A. 2007. Red cell antigens: Structure and function. Asian J Transfus Sci 1:24–32.
Prasad MSS, Vardhanan YS. 2018. Evaluation of efficacy of collection techniques for human
genomic DNA MAOA-uVNTR polymorphism. Int J Sci Res Biol Sci 5:6–11.
Quinlan A. 2020. Visions of Public Safety, Justice, and Healing: The Making of the Rape Kit
Backlog in the United States. Soc Leg Stud 29:225–245.
Quinones I, Daniel B. 2012. Cell free DNA as a component of forensic evidence recovered from
touched surfaces. Forensic Sci Int Genet 6:26–30.
Quinque D, Kittler R, Kayser M, Stoneking M, Nasidze I. 2006. Evaluation of saliva as a source
of human DNA for population and association studies. Anal Biochem 353:272–277.
Raina A, Pramanik P, Dogra TD. 2004. Effect of storage conditions of seminal stains on
different textures of clothes in relation to DNA yield. Indian Congr Forensic Med Toxicol 2.
Ravanat J-L, Douki T, Cadet J. 2001. Direct and indirect effects of UV radiation on DNA and its
components. J Photochem Photobiol 63:88–102.
Raymond JJ, van Oorschot RAH, Gunn PR, Walsh SJ, Roux C. 2009. Trace evidence
characteristics of DNA: A preliminary investigation of the persistence of DNA at crime
scenes. Forensic Sci Int Genet 4:26–33.
Reich DE, Schaffner SF, Daly MJ, McVean G, Mullikin JC, Higgins JM, Richter DJ, Lander ES,
94
Altshuler D. 2002. Human genome sequence variation and the influence of gene history,
mutation and recombination. Nat Genet 32:135–142.
Richter C, Park JW, Ames BN. 1988. Normal oxidative damage to mitochondrial and nuclear
DNA is extensive. Proc Natl Acad Sci U S A 85:6465–6467.
Roberston B, Vignaux GA, Berger CEH. 2016. Explaining the Strength of Evidence. In:
Interpreting Evidence: Evaluating forensic science in the courtroom. 2nd ed. John Wiley &
Sons, Ltd. p 55–67.
Roewer L. 2013. DNA fingerprinting in forensics: Past, present, future. Investig Genet 4:1–10.
Rutty GN, EAM G. 2005. DNA/risk of contamination. In: Payne-James J, Byard RW, Corey TS,
Henderson C, editors. Encyclopedia of forensic and legal medicine. volume 2. . p 189–198.
Safarikova M, Kubena AA, Frankova V, Zima T, Kalousova M. 2021. The effects of different
storage conditions and repeated freeze/thaw cycles on the concentration, purity and integrity
of genomic DNA. Folia Biol (Czech Republic) 67:10–15.
Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA. 1986. Analysis of enzymatically
amplified β-globin and HLA-DQα DNA with allele-specific oligonucleotide probes. Nature
324:163–166.
Samuel G, Prainsack B. 2019. Forensic DNA phenotyping in Europe: views “on the ground”
from those who have a professional stake in the technology. New Genet Soc 38:119–141.
Sessa F, Salerno M, Bertozzi G, Messina G, Ricci P, Ledda C, Rapisarda V, Cantatore S,
Turillazzi E, Pomara C. 2019. Touch DNA: Impact of handling time on touch deposit and
evaluation of different recovery techniques: An experimental study. Sci Rep 9:1–9.
Shewale JG, Liu RH eds. 2013. Forensic DNA analysis: current practices and emerging
technologies. CRC Press.
Shi B, Shin YK, Hassanali AA, Singer SJ. 2015. DNA Binding to the Silica Surface. J Phys
Chem B 119:11030–11040.
Shikama K. 1965. Effect of freezing and thawing on the stability of double helix of DNA. Nature
207:529–530.
Sirker M, Schneider PM, Gomes I. 2016. A 17-month time course study of human RNA and
DNA degradation in body fluids under dry and humid environmental conditions. Int J Legal
Med 130:1431–1438.
Slabbert N, Heathfield LJ. 2018. Ethical, legal and social implications of forensic molecular
phenotyping in South Africa. Dev World Bioeth 18:171–181.
Stannett V, Williams JL. 2007. The permeability of poly(ethyl methacrylate) to gases and water
vapor. J Polym Sci Part C Polym Symp 10:45–59.
Steinlechner M, Berger B, Niederstätter H, Parson W. 2002. Rare failures in the amelogenin sex
test. Int J Legal Med 116:117–120.
Storm KJ, Ropero-Miller J, Jones S, Sikes N, Pope M, Horstmann N. 2009. The 2007 Survey of
Law Enforcement Forensic Evidence Processing. Rockville.
Strom KJ, Hickman M. 2010. Processing in Police Departments. Criminol Public Policy 9:381–
404.
95
Sweet D, Hildebrand D. 1999. Saliva from cheese bite yields DNA profile of burglar: A case
report. Int J Legal Med 112:201–203.
Sweet D, Lorente M, Lorente JA, Valenzuela A, Villanueva E. 1997. An Improved Method to
Recover Saliva from Human Skin: The Double Swab Technique. J Forensic Sci 42:14120J.
Talpaert-Borlè M. 1987. Formation, detection and repair of AP sites. Mutat Res 181:45–56.
Tan X, Ge L, Zhang T, Lu Z. 2021. Preservation of DNA for data storage. Russ Chem Rev
90:280–291.
Taylor D, Bright JA, Buckleton J. 2014. Interpreting forensic DNA profiling evidence without
specifying the number of contributors. Forensic Sci Int Genet 13:269–280.
ThermoFisher Scientific. 2021. 2021 Global Insights Survey of Forensics Labs: The impact of
SARS-CoV-2 on challenges, priorities, and opportunities.
Thomas A, Holben B, Dueño K, Snow M. 2018. Mitochondrial DNA extraction from burial soil
samples at incremental distances: a preliminary study. J Forensic Sci.
Tilstone WJ, Savage KA, Clark LA. 2006. Forensic Science: An Encyclopedia of History,
Methods, and Techniques. ABC-CLIO.
Toom V, Wienroth M, M’Charek A, Prainsack B, Williams R, Duster T, Heinemann T, Kruse C,
MacHado H, Murphy E. 2016. Approaching ethical, legal and social issues of emerging
forensic DNA phenotyping (FDP) technologies comprehensively: Reply to “Forensic DNA
phenotyping: Predicting human appearance from crime scene material for investigative
purposes” by Manfred Kayser. Forensic Sci Int Genet 22:e1–e4.
Tredoux S, Mfolozi S, Shires K. 2015. Efficiency of Buccal DNA Sampling Device in the
Mortuary. J Forensic Investig 3.
Turnbough MA, Eisenberg AJ, Schade L, Shewale JG. 2013. Training of Forensic DNA
Scientists - A Commentary. In: Shewale JG, Liu RH, editors. Forensic DNA Analysis:
Current Practices and Emerging Technologies. CRC Press. p 381–389.
U.S. Government Accountability Office. 2019. DNA EVIDENCE: DOJ Should Improve
Performance Measurement and Properly Design Controls for Nationwide Grant Program.
Vandewoestyne M, Van Hoofstat D, Franssen A, Van Nieuwerburgh F, Deforce D. 2013.
Presence and potential of cell free DNA in different types of forensic samples. Forensic Sci
Int Genet 7:316–320.
de Vargas Wolfgramm E, de Carvalho FM, da Costa Aguiar VR, De Nadai Sartori MP,
Hirschfeld-Campolongo GCR, Tsutsumida WM, Louro ID. 2009. Simplified buccal DNA
extraction with FTA® Elute Cards. Forensic Sci Int Genet 3:125–127.
Verdon TJ, Mitchell RJ, Van Oorschot RAH. 2014. Evaluation of tapelifting as a collection
method for touch DNA. Forensic Sci Int Genet 8:179–186.
Vickar T, Bache K, Daniel B, Frascione N. 2018. The use of the M-Vac® wet-vacuum system as
a method for DNA recovery. Sci Justice 58:282–286.
Visser R, Hampikian G. 2012. When DNA Won’t Work. Ida Law Rev 49:40–67.
Vitoševic K, Todorovic D, Slovic Z, Zivkovic-Zaric R, Todorovic M. 2019. Forensic genetics
and genotyping. Serbian J Exp Clin Res 20:75–86.
96
Weathered L, Wright K, Chaseling J. 2020. Dealing with DNA evidence in the courtroom: a
plain English review of current issues with identification, mixture and activity level
evidence. In: The Wrongful Conviction Law Review. . p 59–73.
Westhof E. 1988. Water: an integral part of nucleic acid structure. Annu Rev Biophys Biophys
Chem 17:125–144.
Wickenheiser RA. 2002. Trace DNA: a review, discussion of theory, and application of the
transfer of trace quantities of DNA through skin contact. J Forensic Sci 47:442–50.
Widen H, Leufven A, Nielsen T. 2004. Migration of Model Contaminants from PET Bottles:
Influence of Temperature, Food Simulant and Functional Barrier. Food Addit Contam
21:993–1006.
Wienroth M. 2018. Governing anticipatory technology practices. Forensic DNA phenotyping
and the forensic genetics community in Europe. New Genet Soc 37:137–152.
Wood I, Park S, Tooke J, Smith O, Morgan RM, Meakin GE. 2017. Efficiencies of recovery and
extraction of trace DNA from non-porous surfaces. Forensic Sci Int Genet Suppl Ser
6:e153–e155.
Wyner N, Barash M, Mcnevin D, Linacre AM. 2020. Forensic Autosomal Short Tandem Repeats
and Their Potential Association With Phenotype. 11:1–7.
Yang Q, Shen Y, Shao C, Liu Y, Xu H, Zhou Y, Liu Z, Sun K, Tang Q, Xie J. 2020. Genetic
analysis of tri-allelic patterns at the CODIS STR loci. Mol Genet Genomics 295:1263–
1268.
Zahra A, Hussain B, Jamil A, Ahmed Z, Mahboob S. 2018. Forensic STR profiling based smart
barcode, a highly efficient and cost effective human identification system. Saudi J Biol Sci
25:1720–1723.
Zascage RR, Shewale SJ, Planz J V. 2013. Deep-Sequencing Technologies and Potential
Applications in Forensic DNA Testing. In: Shewale JG, Liu RH, editors. Forensic DNA
Analysis: Current Practices and Emerging Technologies. CRC Press.
Zech WD, Malik N, Thali M. 2012. Applicability of DNA Analysis on Adhesive Tape in
Forensic Casework. J Forensic Sci 57:1036–1041.
Zhang J, Zhang J, Tao R, Jiang L, Chen L, Li X, Li C, Zhang S. 2020. A newly devised
multiplex assay of novel polymorphic non-CODIS STRs as a valuable tool for forensic
application. Forensic Sci Int Genet 48.
Zoppis S, Muciaccia B, D’Alessio A, Ziparo E, Vecchiotti C, Filippini A. 2014. DNA
fingerprinting secondary transfer from different skin areas: Morphological and genetic
studies. Forensic Sci Int Genet 11:137–143.
Zuidberg M, Bettman M, Aarts LHJ, Sjerps M, Kokshoorn B. 2019. Targeting relevant sampling
areas for human biological traces: Where to sample displaced bodies for offender DNA? Sci
Justice 59:153–161.
97
Chapter 5: Methods
The methods of this research expand upon the preliminary study by focusing on the
potential reasoning behind cross-contamination (Ramey, 2019). This chapter outlines the
preliminary study, hypotheses, and methods of this project. The samples used for testing were
either exposed to male salvia or remain unopened, then placed into storage then extracted,
followed by Qubit®, qPCR, and Y-STR analysis to determine if contamination is present in the
blank swab samples.
The preliminary study for this research tested to see if contamination was possible during
the storage process. Two collection materials were tested: buccal swabs and Whatman cards.
Extracted pig DNA was introduced to the samples as a proxy for human DNA for the study to
ensure any detected contamination was not from the researchers. The variables analyzed were
sample drying time prior to storage and storage time. Contamination was detected in a significant
number of samples in both types of collection materials. There was no significance in the drying
time; however, contamination was observed, which could result from the reabsorption of water
to air-dried DNA during storage (Colotte et al., 2011; Ramey, 2019). There was significant
contamination the longer samples were in storage. Therefore, DNA is moving, but it is not
understood how it moves.
Hypotheses
Hypothesis I: The likelihood of DNA migration through a material is not influenced by an
increase in temperature.
Temperature is one of the elements typically controlled in forensic storage. The
temperature has been found to affect the packaging material, the diffusion of molecules, and
98
increase the molecular motion mobility (Maia et al., 2016; Brandsch, 2017). Nevertheless,
migration of molecules through materials is thought to be random; however, it is more plausible
for migration at a higher temperature when the flexibility of the material and molecular mobility
is higher meaning the migration will not be random (Triantafyllou et al., 2005; Wang et al.,
2010, 2012; de Fátima Poças et al., 2011). An increase in temperature would demonstrate a
linear rate of increased contamination with increased temperature. If the contamination is not
influenced by temperature, then contamination rates will be nonlinear.
Hypothesis II: At a given temperature, neither vapor pressure nor humidity influences the
movement of DNA.
Humidity is another of the elements typically controlled in forensic storage. The diffusion
of molecules can occur in either the liquid or air phase, and the difference between the phases is
the diffusion rate (de Fátima Poças et al., 2011; Maia et al., 2016; Fang and Vitrac, 2017).
Evaporation will occur when the vapor pressure is greater than water vapor. Condensation will
occur when the vapor pressure is less than the water vapor, which was observed during the
preliminary study (Anderson, 1936; Marek and Straub, 2001; Kryukov et al., 2014; Ramey,
2019). One study found the migration rate of the molecule benzophenone increases with
humidity after 30 days, which can be applied to DNA using a similar model despite
benzophenone and DNA having different physical principles to migration (Barnkob and
Petersen, 2013). With this hypothesis, an increase in contamination with an increase in humidity
would be caused by condensation.
99
Materials and Methods
Survey
A survey was sent to fifty-five government and privately funded forensic laboratories
across the United States. The survey was an anonymous questionnaire with seventeen questions,
which helped determine the research’s protocols, drying method, storage environment, and
storage time for this project. The twelve responses provided insight into the process of placing
evidence into storage beyond the information found within various forensic laboratory
handbooks. With the survey, it was determined that room temperature storage, followed by
refrigeration, are the primary temperatures forensic laboratories use for storage of evidence. How
laboratories dry genetic evidence was almost evenly distributed between the answers; however, a
swab dryer was the more often used method of drying. The majority of the forensic laboratories
use the Qiagen EZ1 extraction kit, which requires a specific automatic machine to utilize to
complete DNA extractions.
Questionnaire
1. How is biological evidence mailed to the lab? Select all that apply.
a. UPS; FedEx; USPS; dropped off; picked up by the lab; overnight; express;
first-class; priority; standard; other
2. How long does it take biological evidence from being delivered to the lab to being
placed into storage prior to extraction (from point A to point B)?
a. Less than 24 hours; less than 72 hours; a week; 2-3 weeks; a month; 2-3
months; 6 months; other
3. What temperature does your lab store genetic evidence prior to extraction?
a. Refrigerated; room temperature; controlled room temperature; frozen
100
4. What regulation protocol does your lab use? Check all that apply.
a. Lab’s own protocol; International Association for Property and Evidence
(IAPE); The Biological Evidence Preservation Handbook (NIST and NIJ);
Quality Assurance Standards for Forensic DNA Testing Laboratories; Other
5. What is the average length of time (in weeks) genetic evidence remains in storage at
your lab prior to extraction?
6. Does extracted evidence go back into storage prior to analysis? If yes, what is the
average length of time (in days) genetic evidence is left in storage between extraction
and analysis?
7. What is the average number of swab(s) taken from evidence?
a. 1; 2; 3; 4; 5+
8. How does your lab dry genetic evidence?
a. Swab rack on counter; swab rack in hood; swab dryer; other
9. How is dried biological fluid evidence stored BEFORE extraction in your lab? Select
all that apply.
a. Refrigerator; freezer; evidence locker; high density mobile shelving; open
shelving; other
10. How is dried biological fluid evidence stored AFTER extraction in your lab? Select
all that apply.
a. Refrigerator; freezer; evidence locker; high density mobile shelving; open
shelving; other
11. How is extracted evidence stored long term. Select all that apply.
101
a. Large plastic container; large cardboard container; medium plastic container;
medium cardboard container; small plastic container; small cardboard
container; large evidence envelope; large glassine envelope; medium evidence
envelope; medium glassine envelope; small evidence envelope; small glassine
envelope; evidence tape; plastic tube; evidence-pro blank security bags; other
12. Are genetic evidence samples stored in proximity to other materials, or other
samples? If yes, is the genetic evidence stored in proximity to other evidence from the
same case or non-related case?
a. No; Yes, same case; Yes, non-related case
13. What is the approximate distance between genetic evidence samples? Select all that
apply.
a. Multiple samples stored together in a box; multiple samples stored together in
an envelope; storage containers physically touching one another; other
14. What DNA extraction protocol does your lab use? Write in Answer.
15. Do you ever have to re-extract samples?
a. Frequently; occasionally; rarely; never; other
16. Does the lab randomly test for contamination of samples left in long term storage?
a. Yes; no; other
17. How is your lab funded?
a. Government; private; both; other
Storage Setup
The questionnaire allowed for the research methods to be refined to provide a more
accurate setup based on forensic laboratories across the United States. For the project, each
102
tested variable consisted of two types of boxes. The first type consisted of the ten control swabs,
which remained unopened until the swabs were removed from storage for extractions. The
second type consisted of five blank swabs and five known swabs (Table 5.1). The blank swabs
were left unopened until the swabs were removed from storage for extractions and stored in the
same container as the known swabs, which were introduced to DNA prior to storage. BD BBL™
CultureSwab™ Sterile swabs were used for this study, and each swab was labeled with the
sample type, reference number, and entry date, utilizing a sharpie on the paper label on the
exterior.
Table 5.1. Sample types and numbers used for analysis.
Box Type
Sample Type
Number of Swabs
1: Controls
Control
10
2: Known/Blank
Blank
5
2: Known/Blank
Known
5
For this study, the saliva was collected from one male volunteer. The Institutional
Review Board approved the study (Reference No.: 118-20), and informed written consent was
obtained from the participant. Saliva was collected using a sterile 15 mL tube, which 50 𝜇𝐿
would then be applied to each of the known swabs. The known swabs were then dried for twenty
minutes using the Dry-Fast Swab Dryer, which had been cleaned with DNA Away on the
internal and external surfaces then left to dry (Figure 5.1). The swabs were then placed into the
microcentrifuge tube rack, which was Velcroed to the bottom of the box, based on the layout
displayed in figure 5.2 (appendix A,B). The blank swabs were removed from its packaging,
labeled, and immediately placed in the tube rack, without having broken the paper seal, after the
known swabs had been placed. This seal visually indicates if a swab has been opened and
103
potentially exposed to air. Before placing the samples into storage, the box, tube rack,
TraceableGo™ Datalogger, and any Barska dehumidifier bags were cleaned with DNA Away and
allowed to dry completely. The samples then remained in storage for 45 days before being
extracted. The length of time was derived from an analysis of the survey and my prior
preliminary research. Based on the survey, the most frequent average time in storage was 6
weeks. This was similar to the original 45 days used in the preliminary research (Ramey, 2019).
The boxes were stored in different locations (Modern Laboratory, Ancient DNA Laboratory,
antechamber, or my apartment) to ensure no cross-contamination occurred between the two
boxes.
Figure 5.1. Five swabs are drying for 20 minutes in the Dry-Fast Swab Dryer after applying
saliva.
104
Figure 5.2. The placement of the swabs during the storage process. Red indicates swabs with
known DNA, and black are blank swabs.
TraceableGo Datalogger
Relative humidity and temperature levels were tracked with a TraceableGo™ Datalogger
Hygrometer (Cole-Parmer Instrument Company: Traceable® Products., 2018). With the use of
the hygrometer, the temperature was monitored with an accuracy of +/- 0.4 °C between -10 °C to
70 °C and with an accuracy of +/- 0.5 °C between -10 °C and -20 °C. Humidity was monitored
with an accuracy of +/- 3% between 5 and 75 percent relative humidity and an accuracy of +/-
5% outside that range (Cole-Parmer Instrument Company: Traceable® Products., 2018).
The datalogger was secured within the container with magnets on one of the side walls.
Four dataloggers were utilized: CC6537-6374, CC6537-6380, CC6537-6333, CC6537-6302. The
dataloggers had the same configured settings for the start/stop mode, memory mode, unit
preference, and logging interval. The levels were logged every hour and one minute throughout
the storage process (Appendix C).
Temperature
Three temperatures were tested: room temperature, freezer, and refrigeration (Figure 5.3-
5.5). Each box contained one TraceableGo™ Datalogger Hygrometer, one Barska dehumidifier
bag, and the samples. Type one boxes were stored in either the anti-chamber or the ancient DNA
laboratory at the University of Montana. Type two boxes were stored in the modern laboratory at
the University of Montana. The modern laboratory has a workbench table and a counter opposite
XX X X
XX
XX X X
105
each other, which allowed separation between setup and the storage area. As indicated in storage
setup, type one boxes contain the controls, while type two boxes contain the blank and known
swabs (Table 5.1). The room temperature controls were stored in the antechamber that leads into
the Ancient DNA Laboratory at the University of Montana. The antechamber is a small room
that stores supply from the laboratories and separates the Ancient DNA Laboratory from the rest
of the building. A UV light is utilized in the antechamber to eliminate DNA from highly
contaminated objects. The container was wrapped in aluminum foil to ensure the UV light in the
room did not interfere with the samples. Prior to storage the counter or shelving were cleaned
with DNA Away.
Figure 5.3. Type two box of the room temperature samples on the modern lab counter.
106
Figure 5.4. A representation of the samples in the modern lab freezer.
Figure 5.5. Type two box of refrigerated samples in the modern lab refrigerator.
Humidity
Three relative humidity levels were tested: approximately zero, 35%, and 50-60%. To
ensure no contamination during the storage process the box types were stored in different
locations. Type one boxes were stored in the anti-chamber, and type two box were stored in the
107
modern laboratory at the University of Montana, apart from the 50-60% relative humidity
samples, which were stored in an enclosed closet in my apartment to provide access to secure
Wi-Fi for a Kasa Smart Wi-Fi Plug Mini (for the humidifier) while maintaining a consistent
environment. As indicated in storage setup, type one boxes contain the controls while type two
boxes contain the blank and known swabs (Table 5.1). A UV light is utilized in the antechamber
to eliminate DNA from highly contaminated objects. The container was wrapped in aluminum
foil to ensure the UV light in the room did not interfere with the samples.
The relative humidity was monitored to achieve approximately zero humidity throughout
the storage process. Each box for these samples contained two Barska dehumidifier bags (Figure
5.6). Periodically throughout the storage process, the box was opened to reset the humidity levels
by microwaving the dehumidifier bags because the relative humidity levels would slowly
increase. Prior to opening the box, the area around the box was cleaned with DNA Away. The
box was briefly opened to obtain the dehumidifier bags to reset the humidity levels. Then the box
was closed while the dehumidifier bags were microwaved for 4 minutes each. The dehumidifier
bags were then wiped with DNA Away and placed on paper towels to cool down for another 4
minutes before being placed back into the boxes. Type one box was opened once on November
9th. While type two box was opened on three occasions, October 6th, October 8th, and November
2nd.
A 25% relative humidity set of samples was going to be conducted. However, after
conducting the storage of the room temperature samples the humidity levels were at
approximately 25%. The setup of the room temperature samples was the same as the humidity
samples, except for the number of Barska dehumidifier bags within the box. The humidity
108
samples were also stored at room temperature. Therefore, these samples double as a test for the
temperature and humidity variables.
Figure 5.6. Type one box of approximately zero relative humidity in the process of being placed
into storage in the anti-chamber with aluminum foil placed around the container.
Figure 5.7. Type two box of relative humidity 35% on the modern lab counter
The 35% relative humidity samples did not contain a Barska dehumidifier bag within the
box. The type one box was wrapped in aluminum foil to protect from UV rays and stored in the
109
anti-chamber. The type two box was stored in the modern lab at the University of Montana
(Figure 5.7).
The two 50-60% relative humidity boxes were stored in the same location, but at separate
times, due to the setup required to achieve the humidity levels. Type one box was stored first to
ensure that the environment was not the potential cause of contamination. Type one box and two
of the 50-60% percent relative humidity samples were stored in an enclosed closet with an outlet,
cleaned with Clorox Bleach, and lined with aluminum foil. A small slit was cut into the seal tape,
coving an opening in the box, to allow the Zoo Reptile Fogger Terrarium humidifier tubing to be
inserted into the box (Figure 5.8). A Kasa Smart Wi-Fi Plug Mini was used to control the
humidifier throughout the storage process and allowed for more accurate control over the relative
humidity (Table 5.2).
Table 5.2. The automatic on/off schedule using the Kasa Smart plug to control the humidifier
ON OFF
12:00 AM 12:03 AM
1:36 AM 1:39 AM
3:15 AM 3:18 AM
4:51 AM 4:54 AM
6:27 AM 6:30 AM
8:06 AM 8:09 AM
9:42 AM 9:45 AM
11:18 AM 11:21 AM
12:54 PM 12:57 PM
2:34 PM 2:37 PM
4:10 PM 4:13 PM
5:46 PM 5:49 PM
7:22 PM 7:25 PM
8:57 PM 9:00 PM
10:36 PM 10:39 PM
110
Figure 5.8. Type two box of relative humidity 50/60% in enclosed closet with aluminum foil
underneath.
Extractions
Extractions were conducted in the Ancient DNA Laboratory at the University of
Montana. The lab is an enclosed room with an antechamber separating the laboratory from the
rest of the building. Before entering the lab, the individual must be wearing proper attire
consisting of a coverall, hairnet, mask, gloves, and protective arm sleeves. The lab was wiped
down with bleach and DNA Away, eliminating DNA from highly contaminated objects. In
addition, between every utilization of the laboratory the overhead UV lights are activated to
eliminate DNA from highly contaminated objects.
The two boxes were extracted on separate days (Table 5.3). The swabs from type one
box, the controls, were extracted with two extraction control blank swabs. The swabs from type
two box were split into two groups for extraction. The first group, from type two box, contained
the blank swabs and an extraction control blank swab followed by the known swabs and an
extraction control blank swab — the extraction control blank swab was present to try and
111
determine if potential contamination originated from the extraction process. Between the blank
swabs and known swabs excitation, the workbench was wiped down again with DNA Away.
Table 5.3. The three extraction groups listing what box the samples originated, the sample type,
and number of extraction control swabs were extracted along with the samples.
Box Type
Sample Type
Extraction Controls
1
Control
2
2
Blank
1
2
Known
1
DNA extraction from the swabs was done following a ChargeSwitch™ gDNA Buccal
Cell Kit protocol (Invitrogen, Carlsbad, CA, USA). The extractions resulted in twelve separate
sets. Twenty-four low binding tubes, twelve collection tubes, two 15 mL tubes, and dog nail
clippers were placed into the UV crosslinker to allow UV radiation to degrade any potential
contaminating DNA on their surfaces. The 15 mL tubes were removed from the UV crosslinker
after 5 minutes. The low binding tubes and collection tubes were removed from the UV
crosslinker right before use within the extraction process, with the first set of tubes being
removed after approximately 10 minutes. The dog nail clippers were removed from the UV
crosslinker after 10 minutes. A bead bath (aluminum beads to substitute for a water bath) was
heated to 37°C. While the bath heated, the lysis mix and purification mix were prepared. The
lysis mix contained 12 mL of ChargeSwitch® lysis buffer and 120 𝜇𝐿 of proteinase K, that was
then inverted three times. The purification mix, for twelve and a half samples, contained
500 𝜇𝐿 of fully resuspended ChargeSwitch® magnetic beads and 1250 𝜇𝐿 of ChargeSwitch®
purification buffer.
112
Preparing the lysate was the first step in the extraction process. The first set of twelve low
binding tubes and clippers were removed from the UV crosslinker. The tubes were then labeled
on the exterior with a sharpie based on the sample number. A swab would be opened and placed
with the tip side down into the corresponding tube, cutting the stem with the clippers as close to
the tip as possible. The clippers were cleaned with DNA Away and then placed in the UV
crosslinker for 20 seconds between each sample. Next, 1 mL of the lysis mix prepared prior was
added to each tube, ensuring that the swab tip was completely immersed. The samples were then
incubated in the bead bath for 20 minutes.
Binding DNA was the next step in the extraction process. Once the samples were
removed from the bead bath, the supernatant was removed from the low bind tubes and placed
into the corresponding collection tubes. The purification mix that was prepared was then
resuspended so that the magnetic beads were evenly distributed throughout it. Then 140 𝜇𝐿 of
the mix was added to the collection tube and pipetted gently fifteen times using a 300 𝜇𝐿
pipette. Next, the samples were incubated for a minute at room temperature. This was
followed by the collection tubes being placed in a magnetic rack for a minute. Once the
beads formed a pellet, the supernatant was collected and discarded without disturbing the
pellet.
Washing DNA was the next step in the extraction process. With the collection tubes
still in the magnetic rack, 1 mL of ChargeSwitch® washer buffer was added to the tube. The
magnetic pellet should have been resuspended when the buffer was added. The tubes remained in
the magnetic rack for a minute to allow the magnetic pellet to reform. Then, the supernatant was
be collected and discarded without disturbing the pellet. The washing DNA steps were repeated
once for a total of two washes.
113
The final step was eluting the DNA. The collection tubes were removed from the
magnetic rack, and 150 𝜇𝐿 of ChargeSwitch® elution buffer was added to the tube and pipetted
gently 10 times. Next, the samples were incubated at room temperature for a minute. The tubes
were placed back onto the magnetic rack for a minute. The supernatant was collected and placed
into a new low bind, labeled tube. After extractions were finished, the lab was wiped down with
bleach and DNA Away, and the UV light would be activated for an hour to eliminate DNA from
highly contaminated objects.
Qubit
The Qubit® dsDNA HS Assay was used to determine if there was quantifiable DNA. The
analysis provides an expected range and quality of the double stranded DNA present in the
sample (Nakayama et al., 2016). The Qubit® tubes were placed into the UV crosslinker for an
hour prior to use. Two standards were quantified for each group of samples. The standards
consisted of 190 𝜇𝐿 of Qubit® and 10 𝜇𝐿 of Qubit® dsDNA HS of either standard #1 or standard
#2 in a 0.5 mL thin-walled tube. Then 195 𝜇𝐿 of Qubit® and 5 𝜇𝐿 of the corresponding sample
were added to a 0.5 mL tube for the samples. Next, each tube was vortex for 10 seconds before
incubating for 2 minutes at room temperature. Following this, a Qubit4 was used to measure the
concentration of DNA in each sample.
qPCR
Quantitative PCR was conducted using the Plexor® HY System kit with the Stratagene
Mx3000P® instrument to quantify the DNA extract’s Y-chromosome quantity. The qPCR tubes
and lids were placed into the UV crosslinker for thirty minutes prior to use to allow UV radiation
to degrade any potential contaminating DNA on their surfaces. While the tubes were in
crosslinker, the reaction mix for quantification assays was prepared which consisted of 18 𝜇𝐿 per
114
reaction plus two additional reactions to account for pipetting error. Prior to creating the mix, the
Plexor® HY 2X Master Mix, Water Amplification Grade, and Plexor® HY 20X Primer/IPC Mix
were thawed at room temperature and then vortexed for 10 seconds. For each sample, the mix
consisted of 10 𝜇𝐿 of Plexor® HY 2X Master Mix, 7 𝜇𝐿 of water, and 1 𝜇𝐿 of Plexor® HY 20X
Primer/IPC Mix, that was then vortexed for 10 seconds. Next, the bench was wiped down with
DNA Away. Then, 18 𝜇𝐿 of the reaction mix was added to the qPCR tubes followed by 2 𝜇𝐿 of
the extracted samples.
The preparation of the qPCR samples were dependent on the swab type. The extracted
samples were prepared before the standards and the no-template controls to reduce the chance
contamination during the setup of the qPCR mix. The blank samples were done in duplicate to
account for variability in pipetting, sampling, or amplification, as well as to increase the chances
of potentially picking up any DNA. The control (no DNA ever in the samples or boxes) and
extraction control (swabs used for to aid in detecting extraction contamination) samples were not
duplicated because these samples should not have any DNA present in the samples. The known
samples were also not duplicated because a high concentration of DNA was expected to be
present in the samples. The extracted samples were then sealed and placed to the side.
Following the preparation of the extracted samples, the standards and no-template
controls were prepared. A TE-4 buffer was created by combining 1 mL of Tris-HCI, 20 𝜇𝐿 of
0.5M EDTA, and 99 mL of DI water. A serial dilution was created by thawing Plexor® HY Male
Genomic DNA Standard 50 ng/ 𝜇𝐿 and then vortexed for 10 seconds. The dilution was started by
adding 10 𝜇𝐿 of the Male Genomic Standard to a 0.2 mL tube followed by 40 𝜇𝐿 of TE-4 buffer
to make a concentration of 10 ng/ 𝜇𝐿. This dilution was then vortex for 10 seconds. Next, 10 𝜇𝐿
of the 10 ng/𝜇𝐿 concentration was added to the next 0.2 mL tube followed by 40 𝜇𝐿 of TE-4
115
buffer to make the 2 ng/ 𝜇𝐿, which was then vortexed for 10 seconds. This continued another
four times, with each concentration reducing by twenty percent (Table 5.4). Then, 18 𝜇𝐿 of the
reaction mix was added to the qPCR tubes followed by either 2 𝜇𝐿 of one of the standard dilution
or TE-4 buffer. The standards and no-template controls were done in duplicate. The unknown and
standard samples were then placed into an opaque, plastic, yellow bag to protect the samples
from the light as it was transported from the Ancient DNA laboratory to the Genomics Core
across campus of the University of Montana.
Table 5.4. Dilution of Plexor® HY Genomic DNA Standard
Concentration
Volume of DNA
Volume of TE-4 Buffer
50 ng/ 𝜇𝐿
Undiluted DNA
0 𝜇𝐿
10 ng/ 𝜇𝐿
10 𝜇𝐿 of undiluted DNA
40 𝜇𝐿
2 ng/ 𝜇𝐿
10 𝜇𝐿 of 10 ng/𝜇𝐿 dilution
40 𝜇𝐿
0.4 ng/ 𝜇𝐿
10 𝜇𝐿 of 2 ng/𝜇𝐿 dilution
40 𝜇𝐿
0.08 ng/ 𝜇𝐿
10 𝜇𝐿 of 0.4 ng/𝜇𝐿 dilution
40 𝜇𝐿
0.016 ng/ 𝜇𝐿
10 𝜇𝐿 of 0.08 ng/𝜇𝐿 dilution
40 𝜇𝐿
0.0032 ng/ 𝜇𝐿
10 𝜇𝐿 of 0.016 ng/𝜇𝐿 dilution
40 𝜇𝐿
The qPCR samples were run with the Mx3000P® software using the SYBR Green (with
Dissociation Curve) experiment type. In the optics configuration window, the dyes definitions
were listed as name “CO560” and filter set “HEX-JOE filter set.” They dye were then assigned
for each filter set type. Filter set CY5 was assigned dye IC5, ROX filter was assigned dye
CR610, filter HEX-JOE was assigned dye CO560, and FAM filter was assigned dye FAM. The
filter gains were then set to CY5 x1, ROX x1, HEX-JOE x1, and FAM x2. Next, on the plate
setup tab the wells that were being used were highlighted and designated as unknown well type.
Then, the FAM, CO560, and CR610 fluorescence data were selected and the IC5 was designated
as the reference dye for each well.
116
Continuing in the Mx3000P® software, the thermal cycling profile was set to the Plexor®
kit’s specifications for three segments. The first segment was the initial denaturation step that
reached 95˚C for two minutes. The second segment was the denaturation step that ran for 38
cycles. The annealing phase reached 95˚C for 5 seconds followed by the extension phase that
decreased to 60˚C for 40 seconds. During the extension phase endpoint data was collected at two
points. The third segment was the melt temperature curve step that ran for 48 cycles. The initial
temperature for this segment was 65˚C with a 0.6˚C increase each cycle for 40 seconds. Endpoint
data was collected at two points during the third segment.
The qPCR results were then analyzed using the Plexor® Analysis Software. The data was
imported as a new run with the Stratagene Mx3000P® selected and the autosomal target assigned
the FAM dye for amplification and melt, Y target assigned C0560 for amplification and melt,
IPC target assigned CR610 dye for amplification and melt, and passive reference target assigned
IC5 dye. The wells containing the extracted samples were selected and defined as unknown. The
wells containing TE-4 buffer were selected and defined as no-template control. The wells
containing standards were selected and assigned the specific concentration of DNA. After this
was completed, the melt threshold temperature of the correct amplicon was adjusted to the
expected target melt temperature based on the specific dye type: FAM target range 79-81˚C,
CO560 target range 81-83˚C, and CR610 target range 79-81˚C. The d(RFU)/dT, which measures
the change in fluorescence in relation to temperature, was set to the default threshold of 25
percent. This threshold is calculated and based on the standard samples used in a single qPCR
analysis. Then, a standard curve was generated by selecting all the samples to determine the
DNA concentration of the unknown samples. The standard curve calculated the concentration of
DNA present in the sample, which was used to determine the samples that required an STR
117
analysis. Due to low concentration of DNA, an additional melt curve threshold was analyzed to
determine if Y-chromosomal DNA was present in the sample at a lower threshold, which was set
at 5 percent.
STRs
STR analysis was conducted using PowerPlex® Fusion System with the Applied
Biosystems® instrument to corroborate the results from the qPCR analyses and establish the
genetic identity of the DNA located in the non-DNA exposed samples. The PowerPlex® Fusion
5X Master Mix, PowerPlex® Fusion 5X Primer Pair Mix, and Water Amplification Grade were
thawed and vortexed for 15 seconds. After the samples thawed, the PCR amplification mix was
prepared which consisted of 10 𝜇𝐿 per reaction. The mix consisted of 5 𝜇𝐿 of PowerPlex® Fusion
5X Master Mix and 5 𝜇𝐿 PowerPlex Fusion 5X Primer Mix. The amplification mix was then
vortexed for 10 seconds. Next, 10 𝜇𝐿 of the amplification mix was added to each well. Then 15
𝜇𝐿 of extracted DNA was added to the corresponding well. For the positive amplification control
it was vortexed for 10 seconds and diluted to 0.5ng.
Using a 96-well thermocycler (ThermoFisher) the cycle was set based on the Plexor®
Fusion kit’s specifications for four segments. The first segment reached 96˚C for one minute that
ran for one cycle. The second segment contained three plateaus: the first reached 94˚C for 10
seconds, the second decreased to 59˚C for one minute, and the third reached 72˚C for 30
seconds. The second segment ran for 32 cycles. The third segment decreased to 60˚C for 10
minutes, which ran for one cycle. The fourth segment decreased to 4˚C on a hold.
Once the thermal cycle completed the fragment analysis was completed. A loading mix
was prepared by combining 1 𝜇𝐿 of internal lane standard and 9.5 𝜇𝐿 formamide per sample. The
loading mix was then vortexed for 15 seconds. Then, 10 𝜇𝐿 of the loading mix and 1 𝜇𝐿 of
118
amplified sample was added to each well. The wells were then briefly centrifuged. Next, the
samples were denatured at 95˚C for 3 minutes followed by a freezer plate block bath for 3
minutes before being placed into the ABI 3031 instrument in the UM Genomics Core. Then,
analysis was conducted using GeneMapper version 3.6.
119
References:
Anderson DB. 1936. Relative Humidity or Vapor Pressure Deficit. Ecol Soc Am 17:277–282.
Barnkob L, Petersen J. 2013. Effect of relative humidity on the migration of benzophenone from
paperboard into the food simulant Tenax® and modeling hereof. Food Addit Contam Part A
30:395–402.
Brandsch R. 2017. Probabilistic migration modelling focused on functional barrier efficiency and
low migration concepts in support of risk assessment. Food Addit Contam Part A 34:1743–
1766.
Cole-Parmer Instrument Company: Traceable® Products. 2018. TraceableGO, Version: 2.0.3.
Colotte M, Coudy D, Tuffet S, Bonnet J. 2011. Adverse Effect of Air Exposure on the Stability
of DNA Stored at Room Temperature. Biopreserv Biobank:47–50.
Fang X, Vitrac O. 2017. Predicting diffusion coefficients of chemicals in and through packaging
materials. Crit Rev Food Sci Nutr 57:275–312.
de Fátima Poças M, Oliveria JC, Peteira JR, Brandsch R, Hogg T. 2011. Modelling migration
from paper into a food simulant. Food Control 22:303–312.
Kryukov AP, Levashov VY, Pavlyukevich N V. 2014. Kinetic Theory of Transfer Processes. J
Eng Phys Thermophys 87:237–245.
Maia J, Rodriguez-Bernaldo de Quirós A, Sendón R, Cruz JM, Seiler A, Franz R, Simoneau C,
Castle L, Driffield M, Mercea P, Oldring P, Tosa V, Paseiro P. 2016. Determination of key
diffusion and partition parameters and their use in migration modelling of benzophenone
from low-density polyethylene (LDPE) into differnt foodstuffs. Food Addit Contam Part A
33:715–724.
Marek R, Straub J. 2001. Analysis of the evaporation coefficient and the condensation
coefficient of water. Int J Heat Mass Transf 44:39–53.
Plexor® HY System for the Stratagene Mx3000P® and Mx3005P® Quantitative PCR Systems
Technical Manual #TM294, Promega Corporation, 2007.
Promega Corporation, PowerPlex® Fusion System Technical Manual, TMD039, Revision 10/12.
Nakayama Y, Yamaguchi H, Einaga N, Esumi M. 2016. Pitfalls of DNA quantification using
dnabinding fluorescent dyes and suggested solutions. PLoS One 11:1–12.
Ramey SL. 2019. DNA integrity in forensic samples.
Triantafyllou V, Akrida-Demertzi K, Demertzi P. 2005. Determination of partition behavior of
organic surrogates between paperboard packaging materials and air. J Chromatogr A
1077:74–79.
Wang Z, Wang P, Hu C. 2010. Molecular dynamics simulation on diffusion of 13 kinds of small
molecules in polyethylene terephthalate. Packag Technol Sci 23:457–469.
Wang Z, Wang P, Hu C. 2012. Investigation in influence of types of polypropylene material on
diffusion by using molecular dynamics simulation. Packag Technol Sci 25:329–339.
120
Chapter 6: Results
Questionnaire Survey
The questionnaire was sent to different forensic facilities across the United States, twelve
of the laboratories that were contacted responded to the survey, of which 75% were government
funded. The responses to the questionnaire help develop the research and determine what areas
to focus on within the storage process. There were a series of six questions on the questionnaire
that pertained to operations of the DNA analysis facilities. The Quality Assurance Standards for
Forensic DNA Testing Laboratories was utilized by 54.5% of the facilities (Figure 6.1). When
asked what DNA extraction protocols the facility uses, 75% responded with Qiagen EZ1
protocol (Figure 6.2). The drying method varied amongst the facilities; however, the swab dryer
was the most frequent method at 33.3% (Figure 6.3). Facilities rarely re-extract samples or test
for contamination after the initial evidence analysis (Figure 6.4-6.5).
Figure 6.9. What regulation protocol does your lab use? Check all that apply. Multiple Choice.
121
Figure 6.10. What DNA extraction protocol does your lab use? Open response.
Figure 6.11. How does your lab dry genetic evidence? Multiple Choice.
Figure 6.12. Do you ever have to re-extract samples? Multiple Choice.
0 2 4 6 8 10
EZ1
Organic
Chelex
Bone Protocol
PrepFiler Express
Qiacube
Extraction Protocol
122
Figure 6.13. Does the lab randomly test for contamination of samples left in long term storage?
Multiple Choice.
There were a series of three questions on the questionnaire that pertained to the delivery
of evidence to the facilities. Evidence arrives to these facilities in a wide array of methods; the
top three methods were UPS, dropped off, and overnighted at 14.1% (Figure 6.6). Once
delivered, evidence is placed into storage within 24 hours of being received by the facilities
69.2% of the time (Figure 6.7). Two swabs are the average number of swabs used to collect
evidence, which was reported by 66.7% of the facilities.
There were a series of eight questions on the questionnaire that pertained to storage of
evidence at the facilities. Prior to extractions, genetic evidence was stored at room temperature
by 38.9% of facilities, refrigeration by 27.8% of facilities, and either freezer or controlled room
temperature 16.7% of facilities. Evidence lockers were used to store dried evidence before and
after extractions by 58.3% of facilities. While 41.6% of facilities used open shelving. The
average time evidence is stored prior to extractions was 17.92 weeks, with 2 weeks being the
lowest time in storage and 51 weeks being the highest. However, 6 weeks was the most frequent
length of time reported. Genetic evidence was reported to be stored with multiple samples in one
box by 75% of the facilities and in proximity to non-related cases by 58.3% of the facilities
(Figure 6.8-6.9). Extracted evidence was reported to be stored in plastic tubes by 41.6% of
123
facilities and in small evidence envelopes by 33.3% of facilities. The average time extracted
evidence is placed back into storage prior to analysis was 3.56 days, with 14 days being the
longest time between extraction and analysis.
Figure 6.14. How is biological evidence mailed to the lab? Select all that apply. Multiple
Choice.
Figure 6.15. How long does it take biological evidence from being delivered to the lab to being
placed into storage prior to extraction (from point A to point B)? Multiple Choice.
124
Figure 6.16. What is the approximate distance between genetic evidence samples? Select all that
apply. Multiple Choice.
Figure 6.17. Are genetic evidence samples stored in proximity to other materials or other
samples? If yes, is the genetic evidence stored in proximity to other evidence from the same case
or non related case? Multiple Choice.
The results of this study consists of two types of analyses: qubit and qPCR. The
following sections discuss the results found from performing those tests.
125
Qubit Results
Temperature
The Qubit was able to detect double stranded DNA in all of the known samples ranging
from 0.780 ng/𝜇𝐿 to 2.19 ng/𝜇𝐿 (Table 6.1-6.3). There was no doubled stranded DNA detected in
the refrigerated and frozen blank samples. The Qubit did detect DNA in two freezer controls and
one room temperature extraction control. In the blank samples, DNA was only detected in one
room temperature sample. The analysis provides an expected range (0.1 to 120 ng) and quality of
the double stranded DNA present in the sample (Nakayama et al., 2016).
Humidity
The Qubit was able to detect double stranded DNA in all of the known samples ranging
from 0.122 ng/𝜇𝐿 to 3.31 ng/𝜇𝐿 (Table 6.4-6.6). There was no doubled stranded DNA detected in
any of the blank samples. The Qubit did detect DNA in one zero percent humidity extraction
control that was extracted alongside the known samples of this variable.
Table 6.5. Room Temperature. Samples beginning with ‘B’ are the extraction controls, which are
listed below its extraction group.
Sample Number
Quantifiable DNA
Sample Number
Quantifiable DNA
1 Known
1.66 ng/𝜇𝐿
6
Out of Range
2 Known
0.972 ng/𝜇𝐿
7
Out of Range
3 Known
1.91 ng/𝜇𝐿
8
Out of Range
4 Known
1.91 ng/𝜇𝐿
9
Out of Range
5 Known
1.62 ng/𝜇𝐿
10
Out of Range
B4
Out of Range
B1
0.0228 ng/𝜇𝐿
11 Blank
0.166 ng/𝜇𝐿
16
Out of Range
12 Blank
Out of Range
17
Out of Range
13 Blank
Out of Range
18
Out of Range
14 Blank
Out of Range
19
Out of Range
15 Blank
Out of Range
20
Out of Range
B3
Out of Range
B2
Out of Range
126
Table 6.6. Refrigeration. Samples beginning with ‘B’ are the extraction controls, which are
listed below its extraction group.
Sample Number
Quantifiable DNA
Sample Number
Quantifiable DNA
306 Known
1.14 ng/𝜇𝐿
301
Out of Range
307 Known
2.19 ng/𝜇𝐿
302
Out of Range
308 Known
1.47 ng/𝜇𝐿
303
Out of Range
309 Known
1.35 ng/𝜇𝐿
304
Out of Range
310 Known
0.780 ng/𝜇𝐿
305
Out of Range
B16
Out of Range
B7
Out of Range
316 Blank
Out of Range
311
Out of Range
317 Blank
Out of Range
312
Out of Range
318 Blank
Out of Range
313
Out of Range
319 Blank
Out of Range
314
Out of Range
320 Blank
Out of Range
315
Out of Range
B15
Out of Range
B8
Out of Range
Table 6.7. Freezer. Samples beginning with ‘B’ are the extraction controls, which are listed
below its extraction group.
Sample Number
Quantifiable DNA
Sample Number
Quantifiable DNA
506 Known
1.66 ng/𝜇𝐿
501
Out of Range
507 Known
0.972 ng/𝜇𝐿
502
0.0356 ng/𝜇𝐿
508 Known
1.91 ng/𝜇𝐿
503
Out of Range
509 Known
1.91 ng/𝜇𝐿
504
Out of Range
510 Known
1.62 ng/𝜇𝐿
505
0.0620 ng/𝜇𝐿
B18
Out of Range
B13
Out of Range
516 Blank
Out of Range
511
Out of Range
517 Blank
Out of Range
512
Out of Range
518 Blank
Out of Range
513
Out of Range
519 Blank
Out of Range
514
Out of Range
520 Blank
Out of Range
515
Out of Range
B17
Out of Range
B14
Out of Range
127
Table 6.8. Zero percent relative humidity. Samples beginning with ‘B’ are the extraction
controls, which are listed below its extraction group.
Sample Number
Quantifiable DNA
Sample Number
Quantifiable DNA
101 Known
1.82 ng/𝜇𝐿
106
Out of Range
102 Known
2.18 ng/𝜇𝐿
107
Out of Range
103 Known
1.52 ng/𝜇𝐿
108
Out of Range
104 Known
1.78 ng/𝜇𝐿
109
Out of Range
105 Known
0.215 ng/𝜇𝐿
110
Out of Range
B6
0.122 ng/𝜇𝐿
B9
Out of Range
111 Blank
Out of Range
116
Out of Range
112 Blank
Out of Range
117
Out of Range
113 Blank
Out of Range
118
Out of Range
114 Blank
Out of Range
119
Out of Range
115 Blank
Out of Range
120
Out of Range
B5
Out of Range
B10
Out of Range
Table 6.9. 35% relative humidity. Samples beginning with ‘B’ are the extraction controls, which
are listed below its extraction group.
Sample Number
Quantifiable DNA
Sample Number
Quantifiable DNA
201 Known
0.150 ng/𝜇𝐿
206
Out of Range
202 Known
3.31 ng/𝜇𝐿
207
Out of Range
203 Known
2.01 ng/𝜇𝐿
208
Out of Range
204 Known
0.676 ng/𝜇𝐿
209
Out of Range
205 Known
1.28 ng/𝜇𝐿
210
Out of Range
B12
Out of Range
B21
Out of Range
211 Blank
Out of Range
216
Out of Range
212 Blank
Out of Range
217
Out of Range
213 Blank
Out of Range
218
Out of Range
214 Blank
Out of Range
219
Out of Range
215 Blank
Out of Range
220
Out of Range
B11
Out of Range
B22
Out of Range
128
Table 6.10. 55% relative humidity. Samples beginning with ‘B’ are the extraction controls,
which are listed below its extraction group.
Sample Number
Quantifiable DNA
Sample Number
Quantifiable DNA
401 Known
0.186 ng/𝜇𝐿
406
Out of Range
402 Known
0.123 ng/𝜇𝐿
407
Out of Range
403 Known
0.208 ng/𝜇𝐿
408
Out of Range
404 Known
2.64 ng/𝜇𝐿
409
Out of Range
405 Known
4.56 ng/𝜇𝐿
410
Out of Range
B24
Out of Range
B19
Out of Range
411 Blank
Out of Range
416
Out of Range
412 Blank
Out of Range
417
Out of Range
413 Blank
Out of Range
418
Out of Range
414 Blank
Out of Range
419
Out of Range
415 Blank
Out of Range
420
Out of Range
B23
Out of Range
B20
Out of Range
qPCR
The Plexor® analysis, which was used to interpret the qPCR results from the Mx3000P®,
provided insight into the samples and allowed for quantifying different types of DNA potentially
present. After establishing the standard curve for the assigned dyes FAM (autosomal DNA) and
CO560 (Y-chromosomal DNA), the concentration is calculated based on the DNA type. The
quantitation cycle, during which the DNA is detected, is reported by the Cq. If the melt threshold
is in the expected target melt temperature range (81-83˚C for CO565; 79-81˚C for FAM or
CR610), then the Tm? will indicate with a ‘Yes’, ‘No’, or ‘No Call’. A ‘Yes’ result indicates the
amplification is within the expected melt temperature range and has crossed the melt threshold.
While a ‘No’ result indicates the amplification is not within the expected melt temperature range.
A ‘No Call’ result indicates the amplification is within the expected melt temperature range, but
the amplification does not cross the melt threshold (Plexor® HY System Manual). The melt
threshold is determined by a percentage in which the denaturing double stranded DNA amplicon
129
signal must surpass the expected change in fluorescence over change in temperature. The melt
curve is influenced by the number of amplicons in the sample (Steffen, 2016).
Temperature
The Plexor® analysis was conducted on the known samples for the temperature variable.
Autosomal DNA was detected in all the known samples, which the concentrations ranged from
5.696 E-01 to 9.354 ng/𝜇𝐿. Y-chromosomal DNA was detected in all the known samples, which
the concentrations ranged from 2.162 E0 to 6.527 E-01ng/𝜇𝐿 (Appendix D).
The Plexor® analysis was conducted on the blank samples for the temperature variable.
Autosomal DNA was detected in some of the blank samples (Appendix D). In the room
temperature samples, swabs 11 and 13 detected Autosomal DNA. In the refrigerated samples,
swabs 316, 318, and 320 detected autosomal DNA. In the freezer samples, swabs 517, 518, 519,
and 520 detected autosomal DNA. However, these samples did not detect Y-chromosomal DNA.
Y-chromosomal DNA was only detected in swab 15 at a concentration of 6.19E-04 ng/𝜇𝐿, which
was a room temperature sample (Table 6.7). However, the amplification is not within the
expected target melt temperature when the threshold is at 25%. When the threshold was reduced
to 5%, sample 15 changed to a ‘No Call,’ demonstrating that the sample carried too little DNA to
amplify past the melt threshold.
130
Table 6.11. CO560-Y qPCR results of the blank temperature samples. Samples that cross the
amplification threshold have a Cq listed, which indicates the quantitation cycle. The
concentration is of the Y-chromosomal DNA. Tm? indicates if the melt threshold is in the
expected target melt temperature range. Two thresholds were analyzed 25% and 5%.
Sample
Number
Sample
Type
Temperature
Cq
Concentration
Tm?
25%
Tm?
5%
11
Blank
Room
N/A
N/A
NO
NO
12
Blank
Room
N/A
N/A
NO
NO
13
Blank
Room
N/A
N/A
NO
NO
14
Blank
Room
N/A
N/A
NO
NO
15
Blank
Room
36.36
6.19E-04
NO
NO CALL
316
Blank
Refrigeration
N/A
N/A
NO
NO
317
Blank
Refrigeration
N/A
N/A
NO
NO
318
Blank
Refrigeration
N/A
N/A
NO
NO
319
Blank
Refrigeration
N/A
N/A
NO
NO
320
Blank
Refrigeration
N/A
N/A
NO
NO
516
Blank
Freezer
N/A
N/A
NO
NO
517
Blank
Freezer
N/A
N/A
NO
NO
518
Blank
Freezer
N/A
N/A
NO
NO
519
Blank
Freezer
N/A
N/A
NO
NO
520
Blank
Freezer
N/A
N/A
NO
NO
The Plexor® analysis was conducted on the control samples for the temperature variable.
An initial test was conducted of the control samples. Based on the qPCR results of controls, the
entire plate of samples had been contaminated at some point during the qPCR preparations.
Therefore, another plate of the control samples needed to be conducted to determine if
contamination occurred during the storage process. However, because of the limited number
solution left in the Plexor® HY System kit only 4 of the 10 control swabs of each temperature
variable could be tested. Autosomal DNA was detected in some of the control samples
(Appendix D). During the first qPCR run of the controls, all the samples detected autosomal
DNA. Except for swabs 511 and 502, which are freezer samples, Y-chromosomal DNA was also
detected during this run. In the room temperature control samples, swabs 6 and 18 detected
autosomal DNA. In the freezer control samples, swab 511 detected autosomal DNA. The
extraction swab for the blank refrigerator samples, B15, detected autosomal DNA. Y-
131
chromosomal was detected in some of the samples. In the room temperature controls, swabs 6
and 20 detected Y-chromosomal DNA ranging from 3.706 E-03 to 2.957 E-02 ng/𝜇𝐿. In the
freezer samples, swab 504 detected Y-chromosomal DNA at a concentration of 4.597 E-03
ng/𝜇𝐿. The extraction swab B15 also detected Y-chromosomal DNA at a concentration of 1.311
E-02 ng/𝜇𝐿. However, the amplification is not within the expected target melt temperature when
the threshold is at 25%. When the threshold was reduced to 5%, samples 20 and 315 changed to
a ‘No Call’. Of those samples, 20 was the only sample with a Y-chromosomal DNA
concentration (Table 6.8).
Table 6.12. CO560-Y qPCR results of the control temperature samples from the second run.
Samples that cross the amplification threshold have a Cq listed, which indicates the quantitation
cycle. The concentration is of the Y-chromosomal DNA. Tm? indicates if the melt threshold is in
the expected target melt temperature range. Two thresholds were analyzed 25% and 5%.
Sample
Number
Sample
Type
Temperature
Cq
Concentration
Tm?
25%
Tm?
5%
6
Control
Room
33.04
2.957 E-02
NO
NO
17
Control
Room
N/A
N/A
NO
NO
18
Control
Room
N/A
N/A
NO
NO
20
Control
Room
33.04
3.706 E-03
NO
NO CALL
303
Control
Refrigeration
N/A
N/A
NO
NO
304
Control
Refrigeration
N/A
N/A
NO
NO
311
Control
Refrigeration
N/A
N/A
NO
NO
315
Control
Refrigeration
N/A
N/A
NO
NO CALL
501
Control
Freezer
N/A
N/A
NO
NO
504
Control
Freezer
36.52
4.597 E-03
NO
NO
511
Control
Freezer
N/A
N/A
NO
NO
513
Control
Freezer
N/A
N/A
NO
NO
B15
Extraction
Control:
Blank
Refrigeration
34.56
1.311 E-02
NO
NO
Humidity
The Plexor® analysis was conducted on the known samples for the humidity variable.
Autosomal DNA was detected in all the known samples, which the concentrations ranged from
132
5.352 E-03 to 6.670 ng/𝜇𝐿. Y-chromosomal DNA was detected in all the known samples, which
the concentrations ranged from 8.245 E-03 to 2.164 ng/𝜇𝐿 (Appendix D).
The Plexor® analysis was conducted on the blank samples for the humidity variable.
Autosomal DNA was detected in some of the blank samples, which had not been exposed to
DNA (Appendix D). In the approximate zero humidity samples, swabs 114 and 115 detected
Autosomal DNA. In the 35% humidity samples, swabs 212, 213, and 214 detected autosomal
DNA. In the 55% samples, swabs 412, 413, 414, and 415 detected autosomal DNA. However,
the swabs 114, 115, 212, and 413 did not detect Y-chromosomal DNA. Y-chromosomal DNA
concentration was detected in at least one swab of each tested humidity level. In the approximate
zero humidity samples, swab 111 detected 2.472E-04 ng/𝜇𝐿 of Y-chromosomal DNA. In the
35% humidity samples, swab 213 detected 2.896E-04 ng/𝜇𝐿 of Y-chromosomal DNA. In the
55% humidity samples, swabs 411, 412, 414, and 415 detected Y-chromosomal DNA that
ranged from 3.289E-03 to 3.656E-04 ng/𝜇𝐿. However, the amplification is not within the
expected target melt temperature when the threshold is at 25%. When the threshold was reduced
to 5%, samples 113, 115, 411, and 413 changed to a ‘No Call’. Of those samples, 411 was the
only sample with a Y-chromosomal DNA concentration (Table 6.9).
133
Table 6.13. CO560-Y qPCR results of the blank humidity samples. Samples that cross the
amplification threshold have a Cq listed, which indicates the quantitation cycle. The
concentration is of the Y-chromosomal DNA. Tm? indicates if the melt threshold is in the
expected target melt temperature range. Two thresholds were analyzed 25% and 5%.
Sample
Number
Sample
Type
Humidity
Cq
Concentration
Tm?
25%
Tm?
5%
111
Blank
~0
37.82
2.472E-04
NO
NO
112
Blank
~0
N/A
N/A
NO
NO
113
Blank
~0
N/A
N/A
NO
NO CALL
114
Blank
~0
N/A
N/A
NO
NO
115
Blank
~0
N/A
N/A
NO
NO CALL
211
Blank
35
N/A
N/A
NO
NO
212
Blank
35
N/A
N/A
NO
NO
213
Blank
35
37.57
2.896E-04
NO
NO
214
Blank
35
N/A
N/A
NO
NO
215
Blank
35
N/A
N/A
NO
NO
411
Blank
55
37
3.998E-04
NO
NO CALL
412
Blank
55
33.59
3.289E-03
NO
NO
413
Blank
55
N/A
N/A
NO
NO CALL
414
Blank
55
33.8
2.893E-03
NO
NO
415
Blank
55
37.13
3.656E-04
NO
NO
The Plexor® analysis was conducted on the control samples for the humidity variable. An
initial test was conducted of the control samples. Based on the qPCR results of controls, the
entire plate of samples had been contaminated at some point during the qPCR preparations.
Therefore, another plate of the control samples needed to be conducted to determine if
contamination occurred during the storage process. However, because of the limited number
solution left in the Plexor® HY System kit only 4 of the 10 control swabs of each humidity
variable could be tested. Autosomal DNA was detected in some of the control samples
(Appendix D). In the 35% humidity samples, swabs 206, 207, 217, and 218 detected autosomal
DNA at a concentration ranging from 7.128 E-04 to 1.123 E-02 ng/𝜇𝐿. Swabs 206, 207, and 218
also detected Y-chromosomal DNA ranging from 4.506 E-03 to 1.927 E-03 ng/𝜇𝐿. There was no
detection of autosomal of Y-chromosomal DNA for the 35% humidity extraction control
134
samples. In the 55% humidity extraction controls, autosomal DNA was detected in swab B24 at
a concentration of 5.816 E-04 ng/𝜇𝐿. During the first qPCR run of the controls, all the samples
detected autosomal DNA. Except for swabs 118, 408, B10, and B19 detected Y-chromosomal
DNA during this run. However, the amplification is not within the expected target melt
temperature when the threshold is at 25%. When the threshold was reduced to 5%, samples 410
and 418 changed to a ‘No Call’ and 409 changed to a ‘Yes’, which none of the samples indicated
a Y-chromosomal DNA concentration (Table 6.10).
Table 6.14. CO560-Y qPCR results of the control humidity samples from the second run.
Samples that cross the amplification threshold have a Cq listed, which indicates the quantitation
cycle. The concentration is of the Y-chromosomal DNA. Tm? indicates if the melt threshold is in
the expected target melt temperature range. Two thresholds were analyzed 25% and 5%.
Sample
Number
Sample
Type
Humidity
Cq
Concentration
Tm?
25%
Tm?
5%
106
Control
~0
37.07
3.419 E-03
NO
NO
108
Control
~0
37.65
2.506 E-03
NO
NO
119
Control
~0
37.93
2.163 E-03
NO
NO
120
Control
~0
N/A
N/A
NO
NO
409
Control
55
N/A
N/A
NO
YES
410
Control
55
N/A
N/A
NO
NO CALL
416
Control
55
N/A
N/A
NO
NO
418
Control
55
N/A
N/A
NO
NO CALL
B5
Extraction
Control:
Blank
~0
N/A
N/A
NO
YES
B10
Extraction
Control:
Control
~0
N/A
N/A
NO
NO
206
Control
35
35.32
7.866 E-03
NO
NO
207
Control
35
34.20
2.603 E-03
NO
NO
218
Control
35
35.57
1.768 E-02
NO
CALL
NO
220
Control
35
N/A
N/A
NO
NO
135
STR
STR analysis was attempted using the PowerPlex® Fusion System kit, which were then
ran through GeneMapper version 3.6. However, results came back inconclusive and could not
generate an STR profile for neither standards nor the known samples.
136
References:
Nakayama Y, Yamaguchi H, Einaga N, Esumi M. 2016. Pitfalls of DNA quantification using
dnabinding fluorescent dyes and suggested solutions. PLoS One 11:1–12.
Plexor® HY System for the Stratagene Mx3000P® and Mx3005P® Quantitative PCR Systems
Technical Manual #TM294, Promega Corporation, 2007.
Steffen L. 2016. Introduction to Real-Time PCR: Basic Principles and Chemistries. :1–46.
Available from: https://www.promega.com/-/media/files/promega-worldwide/north-
america/promega-us/webinars-and-events/2016/introduction-to-qpcr-basics.pdf?la=en
137
Chapter 7: Discussion & Conclusion
This chapter discusses the observations, findings, and readdresses the hypotheses of this
research on the relationship temperature and humidity has with contamination in storage. If
contamination during storage is possible, the findings suggest the DNA concentration is too low
to interfere with generating a STR profile. The following chapter addresses the observations that
led to this conclusion on storage contamination.
Qubit
The Qubit analysis provides an expected range (0.1 to 120 ng) to quantify the double-
stranded DNA in the sample (Nakayama et al., 2016). Single sample analysis can produce
varying results, as seen in some of the control samples of this study, which resulted in both
quantifiable DNA and ‘out of range’ for a single sample. Therefore, the results from a Qubit
analysis should not influence the interpretation of additional analyses. Nakayama et al. (2016)
found Qubit analysis accuracy to be dependent on the initial condition of the DNA sample
source. Compared to two other quantification methods, NanoDrop and qPCR, the Qubit analysis
was not consistently the most accurate. For instance, the Qubit quantification did not correspond
to the dilution ratio in the samples where the DNA had been extracted from frozen tissue cells.
Thus, the Qubit indicated lower values of double-stranded DNA (Nakayama et al., 2016).
Because the present study delt with an initial low DNA concentration, any contamination or
stochasticity in pipetting in the blank samples could potentially be missed with the Qubit
analysis.
The quantifiable DNA detected by the qubit in the few controls and extraction controls
could indicate two scenarios. Two controls detected quantifiable DNA and two extraction
controls (one from a known extraction group and one from a control extraction group). First, the
138
swabs used in the study potentially were contaminated during the manufacturing process,
therefore, were not DNA-free (despite being advertised as such). Second, the samples were
contaminated during the extraction process potentially caused by the lab bench or equipment not
being wiped down efficiently enough. Therefore, if a high concentration of DNA is present in the
blank or control swab results, then the contamination is more plausibly from an external source
other than storage.
Nonspecific Amplification
The presence of nonspecific amplification within the samples was indicated by the qPCR
and STR results. First, many of the qPCR blank and control swabs either detected a DNA
concentration without making a call, or did not determine a DNA concentration with made call.
Second, the attempted STR analysis was unable to provide results for the known swab samples.
But why does the presence of nonspecific amplification matter? Nonspecific amplification can be
primer-dimers, mis-priming, or inhibitors, which interfere with the results as the inhibitors will
bind to the DNA and change the shape of the melt curve. If the melt curve is outside the expected
melt temperature range, this indicates nonspecific amplification to be present in the samples
(Krenke et al., 2008; Thompson, 2010; Thompson et al., 2014). Therefore, it is crucial to
maintain the integrity of the DNA, as the analysis kit and DNA concentration influence the
downstream detection of a STR profile. The Plexor® HY kit has trouble detecting profiles from
DNA concentrations beginning between 0.62 pg/𝜇𝐿 to 0.21 pg/𝜇𝐿 (Krenke et al., 2008; Ginart et
al., 2019). If nonspecific amplification does not interfere with the samples, then any potential
contamination from storage is too low to detect within a STR profile. Therefore, any
contamination that is detected is a result from another part of the forensic investigation process,
139
such as evidence collection or DNA extraction. This data therefore reaffirms the confidence level
in the STR profiles discovered from genetic evidence.
Temperature, Storage, and Contamination
Based on the analyses, temperature does not influence contamination during storage.
Neither refrigeration nor the freezer detected Y-chromosomal DNA on the swabs in close
proximity to those that had been exposed to DNA. However, DNA concentration was detected in
a freezer control and a refrigeration extraction control. Yet, the Plexor® analysis did not call
amplification within the expected melt temperature range at both 25% and 5% melt threshold,
which indicates the detected DNA concentration is a result of primer dimer or nonspecific
amplification.
The effect room temperature has on contamination during storage needs further
investigation. During the Qubit analysis, a room temperature blank swab (Blank 11) detected
0.166 ng/𝜇𝐿 quantifiable double-stranded DNA; however, in the qPCR analysis this sample did
not detect a Y-chromosomal DNA concentration, but autosomal DNA concentration was
detected. Therefore, it is possible the DNA present in swab 11 is a result of a different source of
contamination. During qPCR analysis, another room temperature blank swab (Blank 15) detected
a Y-chromosomal DNA concentration. At a 25% melt threshold, there was no amplification
detected within the melt temperature range for swab 15. When the melt threshold was reduced to
5%, amplification was detected within the melt temperature range but did not cross the melt
threshold for swab 15. There is likely nonspecific amplification interfering with this sample as
STR analysis could not provide results or identification of the DNA source. If Y-chromosomal
DNA was present in swab 15, which would suggest storage contamination at room temperature,
then it cannot be parsed further because of the amplification interference.
140
However, two control swabs detected a Y-chromosomal DNA concentration. Yet, the
Plexor® analysis did not call amplification within the expected melt temperature range, which
indicates the detected DNA concentration is a result of primer dimer or nonspecific
amplification. Control swab 20, resulted in a ‘No Call’ (amplification detected within the
temperature range, but does not pass the melt threshold) when the melt threshold was at 5%.
Therefore, it is possible that contamination occurred during qPCR setup or through the kit itself,
as the high concentration of the standard DNA included with the Plexor® is located within the
same, small box as the other solution materials.
Humidity, Storage, and Contamination
Based on the analyses, humidity potentially influences contamination during storage.
Neither zero percent humidity nor 35% humidity detected Y-chromosomal DNA at a 25% melt
threshold. Zero percent humidity blank swabs 113 and 115 did result in a ‘No Call’ when the
melt threshold was at 5%, however, these swabs did not detect a DNA concentration, so primer
dimer is likely present in these samples. A DNA concentration was detected in swabs 111 and
213, but there was no amplification detected within the melt temperature range, suggesting the
concentration is a result of nonspecific amplification.
The effect 55% humidity has on contamination during storage needs further investigation.
Four of the five blank swabs detected a concentration of Y-chromosomal DNA. However, there
was no amplification detected within the melt temperature range at a 25% melt threshold. At a
5% melt threshold, there was amplification detected within the melt temperature range but did
not cross the melt threshold for swab 411 and 413s. There is likely nonspecific amplification
interfering with this sample as STR analysis could not provide results. If Y-chromosomal DNA
was present in swab 411 and 413, which would suggest storage contamination when humidity is
141
above 55%, then it cannot be parsed further because of the amplification interference. Despite
this, the increased detection of a Y-chromosomal concentration in the 55% humidity swabs
reflects the effects humidity has on DNA beginning at 50% relative humidity. If high levels of
humidity causes contamination, then the effects of rehydration and denaturation of DNA should
be analyzed further (Bonnet et al., 2009; Colotte et al., 2011; Tan et al., 2021).
The qPCR results for the humidity controls indicates interference. Three of the 0% and
35% humidity controls swabs detected a DNA concentration, which were not amplified within
the melt temperature range. A 55% humidity control swab and a 0% extraction control detected
amplification above a 5% melt threshold despite there being no DNA concentration detected.
Another two 55% humidity control swabs detected amplification within the melt temperature
range that did not cross the 5% melt threshold, despite there being no DNA concentration
detected. These detections of amplification are likely an indication of primer dimer, which is a
PCR by-product (Dash et al., 2020).
Hypotheses
After addressing my findings, we need to readdress the original hypotheses set out by this
research, in chapter 5, to consider how the results should be assessed. Based on this research the
hypotheses must be accepted until more in-depth research can be conducted.
Hypothesis I: The likelihood of DNA migration through a material is not influenced by an
increase in temperature.
The hypothesis is potentially disproven based on the results. If hypothesis I were true,
then contamination would be present at all temperatures or absent altogether. The results indicate
contamination during the storage method used does not occur when the temperature is below
8˚C. However, the qPCR result for one room temperature blank swab detected DNA
142
amplification within the melt temperature range, but the amplification did not cross the melt
threshold, thus resulting in a ‘No Call’ on the DNA concentration in the sample. Therefore,
temperatures above 8 ˚C potentially influences the movement of DNA.
Hypothesis II: At a given temperature, neither vapor pressure nor humidity influences the
movement of DNA.
The hypothesis is potentially partially disproven based on the results. If hypothesis II
were true, then contamination would be consistently present at different humidity levels or
absent altogether. The results indicate humidity does not influence the movement of DNA below
35% humidity. However, the qPCR results for one 55% humidity blank swab detected DNA
amplification within the melt temperature range, but the amplification did not cross the melt
threshold, thus resulting in a ‘No Call’ on the DNA concentration in the sample. Therefore, high
humidity potentially influences the movement of DNA.
Limitations
With forensic evidence, there is variation in how the evidence is stored, collected, and
packaged. Because of this, the results of the research would need to be tested with different
collection material and packaging to determine if similar results occur under different conditions.
Sample size is another limitation. Each variable tested for contamination of five blank
swabs amongst five known swabs. If contamination were to occur in the variables, whose
samples did not detect contamination, then the possibility of contamination potentially happens
at a lower rate.
Implications
Despite being unable to determine if temperature or humidity influence storage
contamination, there are three implications of the research. First, contamination does not occur
143
during these storage conditions when the temperature is below 8˚C. This could be a result of a
restriction in movement of DNA based on the temperature, which has been observed in leaching
studies (Hebsgaard et al., 2009; Arnold et al., 2011; Andersen et al., 2012). In addition, less
water vapor is needed to reach 100% relative humidity as temperature decreases, therefore, there
is more water vapor present in storage at room temperature than in storage below 8˚C. If
forensic facilities can access either refrigerators or freezers, then those should be preferred
storage method for buccal swabs. Using refrigeration or freezers over room temperature will add
another layer to maintaining evidence integrity.
Second, room temperature potentially has an influence on contamination during storage.
This is inferred by the humidity results detecting a DNA concentration in at least one sample for
each humidity level tested along with one of the room temperature swabs. Therefore, further
research is needed to analyze the effects room temperature has on DNA in relation to
contamination. Since room temperature is not a specifically designated range within forensic lab
manuals, all temperatures above 8˚C should be tested.
Lastly, high levels of humidity potentially have an influence on contamination during
storage. As discussed above, humidity begins to have an effect on DNA beginning at 50%
relative humidity (Bonnet et al., 2009; Colotte et al., 2011; Tan et al., 2021). However, room
temperature storage conditions do not control for humidity, nor temperature-controlled storage
conditions which allow humidity up to 60% (Ballou et al., 2013). If room temperature storage
must be used, then the maximum humidity levels need to be reduced to below 50% humidity.
Further research is needed to analyze the effects high levels of humidity has on DNA in relation
to contamination. The humidity levels tested should focus two points where DNA begins to
rehydrate and the DNA structure changes (Westhof, 1988; Bonnet et al., 2009): from 45% to
144
50% relative humidity, and 70% to 75% relative humidity at 1% increments. Then between 50%
to 70% test at 5% increments. This would help determine if potential contamination is a result of
the rehydration of DNA or its structural change and the need to determine alternative storage
methods to prevent those changes. If rehydration was the cause of contamination, this would
further support the need to ensure genetic evidence is dried prior to packaging.
