OPTIMIZING THE ANALYSIS OF TRACE DNA FROM FINGERNAIL EVIDENCE
By
Ashley Elizabeth Doran

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Forensic Science—Master of Science
2014

ABSTRACT
OPTIMIZING THE ANALYSIS OF TRACE DNA FROM FINGERNAIL EVIDENCE
By
Ashley Elizabeth Doran
During a physical assault, biological material can be transferred between an assailant and
victim. When victims defend themselves with their hands, biological material from the assailant
may accumulate underneath their fingernails. Currently, research on fingernail evidence is
limited; there is no optimized method regarding fingernail evidence processing. It is unknown
what type of packaging best prevents loss and degradation of DNA during transport, and what
procedures are best for recovering the maximum amount of exogenous DNA while limiting the
amount of nail DNA. In this research, exogenous DNA recovery was optimized. Factors
investigated included: comparison of soaking solutions; length of soaking time; length of
digestion; vortexing; the effect of pelleting exogenous material for extraction; and the
comparison of improved organic and silica-based extractions. Female volunteers then scratched
male volunteers, and the improved procedure was applied. Next, different kinds of packaging
were compared, noting if DNA degradation and loss occurred. DNAs were quantified and
autosomal and Y-STRs were analyzed. Soaking the fingernail for 15 min in digestion buffer
prior to extraction removed most exogenous DNA. Pelleting exogenous material led to DNA
loss, so exogenous material was not pelleted. Instead, DNAs were extracted from the soaking
solutions, followed by a 2-h digestion and organic extraction. Mixtures were common when
analyzing autosomal STRs, so Y-STR analysis is suggested. Microfuge tubes were better for
transporting fingernail evidence, and there was little degradation after storage for 6 months.
These optimizations helped increase exogenous DNA recovery from fingernail evidence.

To my family for their love and support; particularly my grandparents, who always believed I
could accomplish anything. I love you.

iii

ACKNOWLEDGEMENTS

First and foremost, I would like to thank my advisor, Dr. David Foran, for all of his
guidance throughout this process; I am forever grateful that I was given this opportunity to fulfill
the dream I had since high school. I would also like to thank my committee members, Dr. David
Carter and Ms. Bethanie De La Ossa, for taking time to offer me helpful comments and
suggestions. This project was in part supported by Award No. 2010-DN-BX-K224, awarded by
the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. The
opinions, findings, and conclusions or recommendations expressed in this publication are those
of the author and do not necessarily reflect those of the Department of Justice. Further, this
research could not have been possible without the help of my many volunteers, including my
former students from BS171 and BS172, as well as former and current members of the Michigan
State University Forensic Biology Laboratory, including Amanda Fazi, Sarah Rambadt, Lisa
Hebda, Michelle Metchikian, Mac Hopkins, Tim Antinick, Becca Ray, Drew Fischer, and Molly
Lynch. Special thanks goes to Ellen Jesmok, Barb Fallon, and Ashley Mottar, who both
supported me and kept me sane throughout my last year of graduate school. I am also deeply
thankful for those who helped edit my thesis, including NSC 840, Dr. Renate Snider, and Lisa
Hebda. Last but not least, I would like to thank my family and friends for their love and support
throughout these past couple years.

iv

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................................ vii
LIST OF FIGURES .........................................................................................................................x
INTRODUCTION ...........................................................................................................................1
Sources of DNA Mixtures from Fingernail Evidence .........................................................2
Techniques for Collecting DNA Evidence from Fingernails ..............................................3
Methods for Fingernail Evidence Processing ......................................................................4
DNA Isolation and Purification Techniques .......................................................................5
Quantitative PCR Analysis of Exogenous and Endogenous DNA ......................................6
Current Forensic DNA Analysis Methods: Autosomal and Y Chromosome STRs ..........11
Recovery of Exogenous DNA from Fingernails ...............................................................14
Packaging and Transportation of Fingernail Evidence ......................................................16
Study Objectives ................................................................................................................16
MATERIALS AND METHODS ...................................................................................................18
DNA Isolation and Purification .........................................................................................18
Quantification of DNAs .....................................................................................................19
Autosomal and Y-STR Analyses ......................................................................................20
Sample Collection .............................................................................................................21
Digestion Modifications for DNA Isolation ......................................................................22
Soaking Solutions for Exogenous DNA recovery .............................................................23
Soaking Times for Exogenous DNA recovery ..................................................................23
Silica-Based Kit Extraction vs. Organic Extraction with RNA-Pre-Treated Filters .........24
Exogenous Material Recovery from Pellets, Fingernails, and Soak Solutions .................24
Scratchings Utilizing Optimized Procedures for STR Analysis ........................................25
DNA Degradation in Microfuge Tubes .............................................................................25
Assessment of DNA Loss during Transportation ..............................................................26
Statistical Analyses of DNA Quantifications and STR Profile Percentages .....................27
RESULTS ......................................................................................................................................28
Soaking Modifications for Exogenous Cell Recovery from Fingernail Evidence ............28
Silica-Based Kit Extraction vs. Organic Extraction utilizing RNA-Treated Filters ..........32
Comparison of Digestion Modifications for Exogenous Cell Recovery ...........................32
STR Profiles from Scratchings using Optimized Procedures ............................................34
Assessment of DNA Degradation in Microfuge Tubes .....................................................38
Assessment of DNA Recovery from Scratchings after Transportation .............................39
DISCUSSION ................................................................................................................................41
APPENDICES ...............................................................................................................................53
APPENDIX A: DNA QUANTIFICATION DATA ..........................................................54
v

APPENDIX B: AUTOSOMAL STR PROFILES FROM BLOOD DEPOSITION ON
FINGERNAILS .................................................................................................................57
APPENDIX C: STR PROFILES FROM SCRATCHINGS ..............................................63
APPENDIX D: STR PROFILES OF TRANSPORTED SCRATCHINGS IN COIN
ENVELOPES, BINDLE PAPERS, AND PLASTIC MICROFUGE TUBES ..................67
APPENDIX E: Y-STR PROFILES FOR DNA STORAGE AND DEGRADATION .....75
REFERENCES ..............................................................................................................................76

vi

LIST OF TABLES

Table 1. Components of the TaqMan® qPCR assay....................................................................... 8
Table 2. Median exogenous and total (endogenous + exogenous) DNA concentrations (pg/µL)
for various soaking times (min) from fingernails harboring blood prior to digestion and
extraction (n=48 total). Significant differences existed among exogenous (p=0.013) and
endogenous (p=0.0003) DNA concentrations. ............................................................................. 31
Table 3. Pairwise comparisons (Mann-Whitney U; α=0.05) showing significance values among
the average exogenous DNA concentrations (n=48 total). Significant p-values are in red. ......... 31
Table 4. Pairwise comparisons (Mann-Whitney U; α=0.05) showing significance values among
the average total DNA concentrations (n=48 total). Significant p-values are in red. ................... 31
Table 5. Average exogenous DNA yields (pg) for commercial kit extractions and organic
extractions coupled with RNA-pre-treated filters (n=28 total). T-test showed no significant
differences between types of extraction methods or soaking times: ap=0.738734; bp=0.215766;
c
p=0.222789; and dp=0.7480818................................................................................................... 32
Table 6. Comparison of autosomal STR profiles from blood on fingernails for different digestion
periods (h) utilizing a 15 min soak (n=7 total). The average % of alleles consistent with the
exogenous profile was calculated, as was the % of loci that contained alleles not consistent with
the exogenous DNA profile. ......................................................................................................... 33
Table 7. Summary of quantifications and STR analyses for scratchings utilizing optimized DNA
collection and extraction procedures (n=18 total). Red italicized number indicates an outlier
(Grubbs; α=0.05), and the numbers in parentheses indicate averages calculated without inclusion
of the outlier. ................................................................................................................................. 35
Table 8. Median exogenous DNA concentrations (pg/µL) from fingernails stored for 1 week in
microfuge tubes (n=15 total). After deposition, blood was allowed to dry for 0 min, 6 h, or 24 h
prior to placement in microfuge tubes (Kruskal-Wallis; p=0.075). .............................................. 38
Table 9. Median exogenous DNA concentrations (pg/µL) from fingernails with 1 µL of male
blood, stored at room temperature in microfuge tubes for various time periods (Kruskal-Wallis;
p=0.617) (n=21 total). ................................................................................................................... 38
Table 10. Exogenous DNA recovered (pg/µL) and percent Y-alleles called consistent with the
known exogenous profile from fingernail scratchings transported in coin envelopes, bindle
papers, and microfuge tubes (Kruskal-Wallis; p=0.639) (n=45 total). Fingernails transported
vii

within the microfuge tubes were not transferred to fresh tubes for analysis. Red italicized number
indicates an outlier (Grubbs; α=0.05), and the numbers in parentheses indicate averages
calculated without inclusion of the outlier. ................................................................................... 40
Table A1. DNA recovered (pg/µL) from a 5 min soak in Tris/EDTA/SDS (n=12). …………..54
Table A2. DNA recovered (pg/µL) from a 15 min soak in Tris/EDTA/SDS (n=12). ……….…54
Table A3. DNA recovered (pg/µL) from a 30 min soak in Tris/EDTA/SDS (n=12). ………….55
Table A4. DNA recovered (pg/µL) from a 60 min soak in Tris/EDTA/SDS (n=12). ………….55
Table A5. Exogenous DNA concentrations (pg/µL) from fingernails containing 1 µL of male
blood, stored at room temperature for various time periods (n=21). ……………………………56
Table A6. Exogenous DNA concentrations (pg/µL) from fingernails containing 1 µL of male
blood, stored at room temperature for 1 week after allowing to dry for certain time periods
(n=15). …………………………………………………………………………………………...56
Table A7. Exogenous DNA yields (pg) after a 15 min soak and extracted utilizing optimized kit
protocol (Hebda et al. 2014) or an organic extraction with RNA-pre-treated filters (n=14). …...56
Table A8. Exogenous DNA yields (pg) after a 60 min soak and extracted utilizing optimized kit
protocol (Hebda et al. 2014) or an organic extraction with RNA-pre-treated filters (n=14)…….56
Table B1. Autosomal STR profiles of fingernails containing blood that underwent a 15 min soak
and had varying digestion times (n=7). Also includes the % of exogenous alleles called, as well
as the % of loci that contain mixture (alleles that do not match the exogenous DNA profile). ...57
Table B2. Autosomal STR profiles for various exogenous: endogenous DNA concentration
ratios (listed below each sample), utilizing a 5 min soak period and a 2 h digestion (n=4). Also
includes the % of exogenous alleles called, as well as the % of loci that contain mixture (alleles
that do not match the known exogenous DNA profile)………………………………………….59
Table B3. Autosomal STR profiles for various exogenous: endogenous DNA concentration
ratios (listed below each sample), utilizing a 15 min soak period and a 2 h digestion (n=3). Also
includes the % of exogenous alleles called, as well as the % of loci that contain mixture (alleles
that do not match the known exogenous DNA profile). ………………………………………...60
Table B4. Autosomal STR profiles for various exogenous: endogenous DNA concentration
ratios (listed below each sample), utilizing a 30 min soak period and a 2 h digestion (n=4). Also
includes the % of exogenous alleles called, as well as the % of loci that contain mixture (alleles
that do not match the known exogenous DNA profile)………………………………………….61
Table B5. Autosomal STR profiles for various exogenous: endogenous DNA concentration
ratios (listed below each sample), utilizing a 60 min soak period and a 2 h digestion (n=4). Also

viii

includes the % of exogenous alleles called, as well as the % of loci that contain mixture (alleles
that do not match the known exogenous DNA profile)………………………………………….62
Table C1. Autosomal STR profiles from the 18 scratchings that contained exogenous DNA
(n=11). Samples not containing exogenous alleles were not included within the table. The % of
exogenous alleles called, as well as the % of loci that contain mixture (alleles that do not match
the known exogenous DNA profile) are included. Each profile comes from exogenous DNA
collected from a separate fingernail……………………………………………………………...63
Table C2. Y-STR profiles from the 18 scratchings (n=18). Sample number correlates with the
sample number from Table 1C (above). Includes profiles from scratchings that quantified at 0
pg/µL and samples that did not contain any exogenous alleles in the autosomal profile. Includes
the % of exogenous alleles called………………………………………………………………..65
Table D1. Autosomal STR profiles from fingernail clippings transported in coin envelopes
(n=15). Includes the % of exogenous alleles called, as well as the % of loci that contain mixture
(alleles that are not consistent with the exogenous DNA profile). Dashes indicate there were not
sufficient exogenous DNA quantifications, so samples were not analyzed……………………..67
Table D2. Y- STR profiles from fingernail clippings transported in coin envelopes (n=15).
Includes the % of exogenous alleles called………………………………………………………69
Table D3. Autosomal STR profiles from fingernail clippings transported in bindle papers
(n=15). The % of exogenous alleles called, as well as the % of loci that contain mixture (alleles
that do not match the known exogenous DNA profile) are included. Dashes indicate there were
not sufficient exogenous DNA quantifications, so samples were not analyzed…………………70
Table D4. Y- STR profiles from fingernail clippings transported in bindle papers (n=15).
Includes the % of exogenous alleles called………………………………………………………71
Table D5. Autosomal STR profiles from fingernail clippings transported in microfuge tubes,
with fingernail removed before processing (n=15). Includes the % of exogenous alleles called, as
well as the % of loci that contain mixture (alleles not consistent with the exogenous DNA
profile). Dashes indicate there were not sufficient exogenous DNA quantifications, so samples
were not analyzed………………………………………………………………………………..72
Table D6. Y- STR profiles from fingernail clippings transported in microfuge tubes (n=15).
Includes the % of exogenous alleles called………………………………………………………74
Table E1. Y-STR profiles of exogenous DNA recovered from fingernail clippings containing
blood after storage at room temperature for various time periods (n=7). Exogenous alleles were
present for all samples. ………………………………………………………………………….75

ix

LIST OF FIGURES

Figure 1. The hyponychium, or the distal end of the fingernail. When scratching an assailant,
exogenous material has the potential to accumulate on or under this part of the nail. Taken from
Seidel 2013...................................................................................................................................... 3
Figure 2. Methods of fingernail evidence collection, including (a) clipping the fingernail, (b)
swabbing the fingernail, or (c) scraping under the fingernail for exogenous material. .................. 4
Figure 3. The QIAamp® DNA Investigator Kit is an example of a commercial method that
utilizes a silica-based extraction. The cells are lysed, DNA is bound to the silica filter, unwanted
components are washed away, and DNA is eluted. Taken from QIAGEN 2012. .......................... 6
Figure 4. Diagram of the TaqMan® assay. The TaqMan® probe (purple) anneals to a specific
DNA sequence between the forward (orange) and reverse (red) primers. As a new strand of DNA
is produced, DNA polymerase cleaves the probe, separating the reporter dye (green ‘R’) from the
quencher (blue ‘Q’), increasing fluorescence. The more DNA present, the more fluorescence
detected. Taken from Kumar 2013. ................................................................................................ 9
Figure 5. Exemplary qPCR data. The y-axis shows the RFUs, and the x-axis shows the PCR
cycle number. The horizontal green line represents the critical threshold. The more starting DNA
present, the more fluorescence produced and the faster the Ct value is reached. ......................... 10
Figure 6. Exemplary IPC data. The y-axis shows the RFUs, and the x-axis shows the PCR cycle
number. The horizontal green line represents the critical threshold. The close proximity of the
curves to one another indicates that the assay is working correctly and the DNA samples do not
contain PCR inhibitors. ................................................................................................................. 10
Figure 7. Exemplary IPC data displaying PCR inhibition. The y-axis shows the RFUs, and the xaxis shows the PCR cycle number. The horizontal green line represents the critical threshold.
Two samples (represented by red and yellow lines) did not cross the threshold, and are not in
close proximity to the other samples that crossed the threshold, indicating amplification was
inhibited. ....................................................................................................................................... 11
Figure 8. Example of STR repeat units. This STR has a 3 bp repeat unit (GAC). The top
example has 3 repeat units, while the bottom has 4 repeat units. This individual would type as a
3, 4 at this locus. ........................................................................................................................... 12
Figure 9. Exemplary STR profiles; (a) represents a mixed autosomal STR profile. The tall ‘X’
peak indicates a mixture predominantly originating from a female; (b) represents the same DNA
sample after Y-STR analysis, and there is no indication of DNA mixture. In both instances, the
x

gray boxes above the peaks are the loci, and the numbers below the peaks are the number of
repeats present. .............................................................................................................................. 13
Figure 10. Paternal lineage of Y-STRs. Squares indicate male, circles indicate female. The solid
black fill represents the inheritance pattern. (A) shows that the Y-chromosome is passed down to
the males in the lineage. (B) shows that the Y chromosome is not present. ................................. 13
Figure 11. Female volunteer scratching a male volunteer with her three center fingers, applying
three pounds of force. ................................................................................................................... 22
Figure 12. Packaging used in transportation study; (from the left) coin envelope, bindle paper,
and a plastic 1.5-mL microfuge tube. ........................................................................................... 26
Figure 13. Comparison of average exogenous DNA concentrations (pg/µL) after soaking in
various solutions (n=11 total). TE, Tris/EDTA/SDS, and ATL buffer/Pro K recovered the most
exogenous DNA. ATL buffer and PBS did not recover as much exogenous DNA as the other
soaking solutions. .......................................................................................................................... 28
Figure 14. Comparison of average total (endogenous + exogenous) DNA concentrations (pg/µL)
after soaking in various solutions (n=11 total). The addition of proteinase K greatly increased the
total DNA concentration. .............................................................................................................. 29
Figure 15. Comparison of soak times (h) for exogenous DNA recovery using TE (n=4 total).
DNA was extracted from the pelleted material; the figure shows that the longer the soaking time,
the less DNA recovered from the pellet. A positive control (+) was also analyzed, containing 1
µL of blood. .................................................................................................................................. 30
Figure 16. Comparison of relative exogenous DNA recovery (%) after soaking in TE or
Tris/EDTA/SDS from the pellet, fingernail, or soak solution, after a 5 min soaking period (n=12
total). ............................................................................................................................................. 30
Figure 17. The influence of digestion time on exogenous and total (endogenous + exogenous)
DNA concentrations (pg/µL) after 0.5 h, 1 h, 2 h, and 24 h (n=4 total). A positive control (+)
containing 1 µL of blood was also analyzed. The average exogenous DNA concentration after
0.5 h was the lowest; after a 1 hr digestion period, the average exogenous DNA concentration
plateaued around 400 pg/µL. Total DNA concentration increased substantially at 24 h. ............ 33
Figure 18. Comparison of average exogenous and total (endogenous + exogenous) DNA
concentrations (pg/µL) after implementing different amounts and frequencies of vortexing for a
2-h digestion period (n=6 total). Includes standard error bars. ..................................................... 34
Figure 19. Exemplary autosomal STR profile from scratchings. The gray boxes above the peaks
represent the name of the locus. The boxes below the peaks represent the number of repeats for
xi

that allele. The endogenous (female) profile is dominant, as indicated by the tall ‘X’ peak at the
amelogenin (AMEL) locus. .......................................................................................................... 36
Figure 20. Exemplary Y-STR profile of exogenous DNA following scratching. The gray boxes
above the peaks represent the name of the locus. The boxes below the peaks represent the
number of repeats for that allele. The peaks are consistent with the exogenous DNA profile. .... 37

xii

INTRODUCTION

In 2012, more than 1 million violent crimes occurred in the United States, over 62% of
which were aggravated assaults, and more than 25% of which were committed using personal
weapons such as hands, fists, and feet. Other reported violent crimes included forcible rape
(6.9%) and murder (1.2%), both of which potentially included use of personal defense weapons
by the victims (FBI Uniform Crime Report 2012). During such violent attacks, it is possible that
the victim and the assailant exchange biological materials, including hairs, saliva, blood, and
epithelial cells. In some instances the victim may scratch the attacker, resulting in the
accumulation of the assailant’s skin beneath the fingernails. Depending on the amount of force
exerted, blood from the attacker may be drawn as well. When exogenous (foreign) material is
obtained from fingernails, the genetic information may be useful for identifying an assailant if
enough of their DNA is present.
Two problems exist that have the potential to complicate DNA analysis when collecting
fingernail evidence. The first is lack of standardization for the collection of exogenous material.
The most current edition of A National Protocol for Sexual Assault Medical Forensic Examiners
(U.S. Department of Justice 2013) recommends that practitioners first ask if any scratching
occurred, followed by a visual examination of the fingernails for loose fibers or exogenous
biological material. Then, the nails may be clipped and collected, or either scrapings or
swabbings of the nails may be obtained. These collection methods have not been systematically
compared, so it is not known which is most advantageous for obtaining the maximum amount of
exogenous cells/DNA while minimizing the amount of endogenous (from the fingernail itself)
cells/DNA retrieved.

1

Transportation of fingernail evidence to the DNA testing facility also has the potential to
impair analysis. There are many ways to package fingernail evidence: some practitioners use
coin envelopes for transport (e.g., Sparrow Forensic Pathology), while others use bindle papers
(Alaska Scientific Crime Detection Laboratory 2012). Since the evidence is not processed on
site, it must reach the testing facility without being lost or compromised. During transportation,
exogenous cells have the potential to become dislodged from the fingernail, and recovery from
the packaging may be overlooked or impossible.

Sources of DNA Mixtures from Fingernail Evidence
From a forensic biology standpoint, a mixture exists when more than one source of DNA
is present in a sample. A major source of DNA mixture from fingernail evidence is endogenous
cells that are extracted along with the desired exogenous cells (Allouche et al. 2008, Bengtsson
et al. 2011). The majority of endogenous DNA is likely derived from the keratinous part of the
fingernail (the nail itself), however DNA consistent with the nail may come from epithelial cells
that accumulate on or under the fingernail hyponychium (Figure 1) as it grows (Bengtsson et al.
2011), or from rubbing or scratching one’s self; therefore, the vast majority of DNA from
fingernail evidence may come from the victim (Wickenheiser 2002). Foreign DNA beneath
fingernails has been found to be rare except between intimate partners (Cook and Dixon 2006,
Matte el al. 2011). However, during a violent attack, exogenous DNA may accumulate
underneath the fingernails, the amount of which is dependent on the force of the scratches and
whether or not the skin is broken (Matte et al. 2011). The biological material collected following
an attack may provide genetic information useful for identifying an assailant.

2

Figure 1. The hyponychium, or the distal end of the fingernail. When scratching an assailant,
exogenous material has the potential to accumulate on or under this part of the nail. Taken from
Seidel 2013.

Techniques for Collecting DNA Evidence from Fingernails
Following a physical assault, qualified personnel such as sexual assault nurse examiners
(SANEs) may collect fingernail evidence. If the victim is deceased, fingernail evidence is
collected by the pathologist during autopsy. This evidence can be obtained through clipping
(Figure 2a), swabbing (Figure 2b), or scraping (Figure 2c); the methodology used depends upon
the agency (FBI 1999, Alaska Scientific Crime Detection Laboratory 2012, U.S. Department of
Justice 2013). The evidence is then packaged and transported to the crime laboratory. There,
forensic biologists may soak the nails in order to remove exogenous material for DNA isolation
and analysis (Harbison et al. 2003, Alaska Scientific Crime Detection Laboratory 2012, Hebda et
al. 2014). It is also common to swab the clippings, either individually or cumulatively (Alaska

3

Scientific Crime Detection Laboratory 2012). During cumulative swabbing, one swab is used for
multiple nails, such as those from one hand.

Figure 2. Methods of fingernail evidence collection, including (a) clipping the fingernail,
(b) swabbing the fingernail, or (c) scraping under the fingernail for exogenous material.

Methods for Fingernail Evidence Processing
Swabbing, scraping beneath the nail, or clipping the fingernails each have advantages and
disadvantages. Swabbing is most frequently used in crime laboratories, since it is easy to perform
and the practitioner can visualize the exogenous material recovered from the nail. However, this
can result in many swabs to process, and not all exogenous material may be recovered.
Cumulatively swabbing fingernails/fingernail clippings requires less effort than processing each
fingernail individually, and potentially increases DNA yields; however, it could also result in
cross-contamination, mixtures, and DNA loss from one fingernail to the next (Cook and Dixon
2006, Hebda et al. 2014). Nail scrapings are processed directly, and the applicator may also be
analyzed for any adhering genetic material. Hebda et al. (2014) found that less endogenous DNA
is recovered through scraping than soaking or swabbing, reducing its contribution to the mixture.
On the other hand, scrapings may also result in exogenous DNA loss when not all biological
material is removed from the fingernail. Hebda at al. (2014) also discovered that soaking

4

fingernail clippings recovered the most exogenous DNA, however a large amount of endogenous
DNA was recovered as well. The lack of standardization in collecting fingernail evidence is
problematic, since the relative amount of DNA recovered using these methods has not been
thoroughly examined.

DNA Isolation and Purification Techniques
The next step in nail evidence processing, DNA isolation, has four main goals: (1)
minimize handling to prevent contamination, (2) maximize DNA yield, (3) remove any
unwanted components, and (4) prevent degradation of DNA. A common DNA isolation
technique is phenol/chloroform (organic) extraction (Comey et al. 1994). First, an aqueous
solution containing a detergent and proteinase is used to lyse cells. Following an incubation
period, phenol and chloroform are added to separate the water-soluble DNA from other organic
compounds, including proteins, lipids, and other cellular components. Still, deleterious materials
that interfere with DNA analysis can remain within the aqueous layer, although these can
potentially be removed through further purification. One common technique for accomplishing
this is centrifugal filtration (e.g., Edson et al. 2004, Bille et al. 2009); these filter devices purify
DNA by allowing contaminants to pass through, while the DNA remains within the retentate.
Another common DNA isolation and purification technique is silica-based extraction,
which is included as part of some commercial kits (e.g., Greenspoon et al. 1998). As shown in
Figure 3, cells are lysed using a tissue lysis buffer and proteinase K to release DNA. The solution
is transferred to the silica column; under high salt conditions DNA is bound to the silica (Melzak
et al. 1996), while the other cellular components pass through when washed with ethanol-based
buffers. Once the unwanted materials are removed, the DNA is eluted from the silica using a low

5

salt solution. However, DNA may remain on the silica filter, in which case multiple elutions can
be implemented (e.g., Hebda et al. 2014). Unfortunately, the relative utility of any of these
techniques for processing fingernail evidence has not been thoroughly investigated.

Figure 3. The QIAamp® DNA Investigator Kit is an example of a commercial method that
utilizes a silica-based extraction. The cells are lysed, DNA is bound to the silica filter, unwanted
components are washed away, and DNA is eluted. Taken from QIAGEN 2012.

Quantitative PCR Analysis of Exogenous and Endogenous DNA
Before analyzing DNA from fingernail evidence, the forensic biologist needs to
determine how much of it is present in the sample. The polymerase chain reaction (PCR) is a
6

technique that creates billions of copies of a target sequence of DNA, making its subsequent
analysis possible. Quantitative PCR (qPCR) is an offshoot of PCR, and is used to estimate the
amount of DNA present in a sample. One common qPCR method is a TaqMan® assay.
Components of the assay (Table 1) include primers for target DNA amplification, and a
TaqMan® probe for detection of the amplified sequence. As shown in Figure 4, the probe
consists of a reporter dye, such as FAM® or VIC®, at the 5ʹ end, and a non-fluorescent quencher
attached to the 3ʹ end. A laser is used to excite the dye, and its fluorescence can be detected.
When the probe is intact, the close proximity of the reporter dye to the quencher results in the
absorbance of the dye’s fluorescence. As target DNA is synthesized during PCR, the probe
anneals to a complementary sequence located between the forward and reverse primers. The
probe is cleaved in the presence of a DNA polymerase, separating the dye from the quencher,
and fluorescence can be detected. As more PCR product is synthesized, the amount of
fluorescent signal increases. Figure 5 shows an example of qPCR data. The fluorescent signal is
measured in relative fluorescent units (RFUs) on the y-axis. The critical threshold (Ct) is placed
above the background noise, and is used as a relative measure of the concentration of the target
DNA within the PCR reaction.
Another component of many TaqMan® assays is the inclusion of an internal PCR control
(IPC), which is used to verify that the assay and detection instrumentation are working properly.
The IPC includes a synthetic DNA sequence, to which a separate probe can anneal (Table 1)
(Quantifiler® kit manual 2012). When the assay is working properly, the IPC will have a similar
Ct value in all samples (Figure 6). If any samples are inhibited (something within the extract is
slowing or prohibiting amplification), the IPC will not produce the expected result (Figure 7).

7

Examples of kits that utilize TaqMan® technology are the Quantifiler® Human DNA
Quantification kit (for total DNA) and the Quantifiler® Y Human Male DNA Quantification kit
(for male DNA). Even though these kits cannot be compared directly because they utilize
separate reaction mixtures (Life Technologies 2012), relative amounts of male DNA to the total
DNA can be compared to roughly determine how much female DNA is present.
Table 1. Components of the TaqMan® qPCR assay.
Template DNA
Target Specific
Assay

Human DNA

IPC Assay

Synthetic DNA

Primers
Specific for
amplification of
human DNA
Specific for
amplification of
synthetic DNA

8

TaqMan® Probe
Dye
FAM®
VIC®

Figure 4. Diagram of the TaqMan® assay. The TaqMan® probe (purple) anneals to a specific
DNA sequence between the forward (orange) and reverse (red) primers. As a new strand of DNA
is produced, DNA polymerase cleaves the probe, separating the reporter dye (green ‘R’) from the
quencher (blue ‘Q’), increasing fluorescence. The more DNA present, the more fluorescence
detected. Taken from Kumar 2013.

9

Figure 5. Exemplary qPCR data. The y-axis shows the RFUs, and the x-axis shows the PCR
cycle number. The horizontal green line represents the critical threshold. The more starting DNA
present, the more fluorescence produced and the faster the Ct value is reached.

Figure 6. Exemplary IPC data. The y-axis shows the RFUs, and the x-axis shows the PCR cycle
number. The horizontal green line represents the critical threshold. The close proximity of the
curves to one another indicates that the assay is working correctly and the DNA samples do not
contain PCR inhibitors.

10

Figure 7. Exemplary IPC data displaying PCR inhibition. The y-axis shows the RFUs, and the xaxis shows the PCR cycle number. The horizontal green line represents the critical threshold.
Two samples (represented by red and yellow lines) did not cross the threshold, and are not in
close proximity to the other samples that crossed the threshold, indicating amplification was
inhibited.

Current Forensic DNA Analysis Methods: Autosomal and Y Chromosome STRs
Short tandem repeat (STR) analysis is undertaken after quantification, producing an
individual’s DNA profile. STRs are repeated DNA segments 2 – 6 bp in length, exemplified in
Figure 8. Amplification of STRs can be successful even when DNAs are degraded because of
their small size. Each person has two STR alleles at a given locus (location on a chromosome),
one from each parent. The number of repeats for each allele is variable among individuals. When
multiple loci are analyzed, the level of differentiation between individuals increases. Thirteen
core STR loci have been established by the FBI and are included within the national DNA
database. When all 13 loci are tested, the probability of two unrelated people having a matching
STR profile is roughly 1 in 100 trillion (Budowle et al. 1998). The probability becomes even less
likely when additional loci are included in the analysis, resulting in STR profiles that are unique
to each individual, excluding identical twins.

11

Figure 8. Example of STR repeat units. This STR has a 3 bp repeat unit (GAC). The top
example has 3 repeat units, while the bottom has 4 repeat units. This individual would type as a
3, 4 at this locus.

When a physical assault is committed, there is a possibility of DNA mixture within the
autosomal STR profile, as exemplified in Figure 9a. Utilizing STRs found only on the Y
chromosome increases the chance of obtaining a single profile consistent with the attacker
(Figure 9b), if the attacker was male and the victim female. Y-STRs are particularly useful in
instances where small amount of male DNA may be present in a background containing larger
amounts of female DNA. However, unlike autosomal STRs, Y-STRs are not individualizing
since they are passed down identically from father to son (Figure 10).

12

Figure 9. Exemplary STR profiles; (a) represents a mixed autosomal STR profile. The tall ‘X’
peak indicates a mixture predominantly originating from a female; (b) represents the same DNA
sample after Y-STR analysis, and there is no indication of DNA mixture. In both instances, the
gray boxes above the peaks are the loci, and the numbers below the peaks are the number of
repeats present.

Figure 10. Paternal lineage of Y-STRs. Squares indicate male, circles indicate female. The solid
black fill represents the inheritance pattern. (A) shows that the Y-chromosome is passed down to
the males in the lineage. (B) shows that the Y chromosome is not present.

13

Recovery of Exogenous DNA from Fingernails
Researchers have studied various collection methods for fingernail evidence, but the
majority did not directly compare multiple methodologies. Harbison et al. (2003) recovered
fingernail evidence from two female homicide victims that had been submerged in water (one in
bath water and the other in saltwater) for 2 to 3 hours. The fingernails were clipped and soaked
in 1 mL of distilled water for 30 minutes, then centrifuged to pellet the exogenous material. The
pellet was digested overnight to lyse any cells, and DNAs were isolated using a Chelex®
extraction. Mixtures were present in both cases; the dominant profile belonged to a male, with
the minor profile belonging to the victim. Soaking the clipped fingernails successfully removed
exogenous DNA, although it was not determined if DNA remained in the soaking solution
following centrifugation.
Cook and Dixon (2007) focused on the frequency of mixed DNA profiles in the “general
population,” based on 50 male and 50 female donors of varying occupations and ages. The
underside of the fingernails were swabbed, and DNA was isolated from them using QIAGEN™
QIAamp Mini Kits. Background levels of exogenous DNA were assessed utilizing autosomal
STRs to establish the likelihood of obtaining a mixed DNA profile. Out of 200 DNA profiles, 30
contained exogenous alleles. This included 12 swabs containing four or more foreign alleles and
18 swabs with less than four. The exogenous DNA was consistent with an intimate partner in the
majority of these cases. The authors concluded that exogenous DNA underneath fingernails is
rare unless the subjects have an intimate relationship.
Matte et al. (2011) examined the frequency of exogenous DNA beneath fingernails, as
well as its persistence. Nails from university students were either scraped or clipped and
swabbed, and DNAs were isolated using an organic extraction. One objective was to compare the

14

amount of exogenous DNA from students who lived alone or with other individuals. Another
was to determine the persistence of exogenous cells derived from scratching another person, in
which DNA was either collected immediately or 5 – 6 h after scratching. Overall, foreign DNA
under fingernails was uncommon; more than casual contact was required for fingernails to
accumulate exogenous material. In addition, exogenous DNA rarely persisted beneath the
fingernail 5 – 6 h after scratching.
Cline et al. (2003) focused on retrieving exogenous material from fingernails and
reducing or eliminating recovery of endogenous DNA. Fingernail clippings were soaked in
sterile 25 mM ethylenediaminetetraacetic acid (EDTA), deionized water, 1% sodium dodecyl
sulfate (SDS), or 5% Terg-A-Zyme® for one hour, vortexing the sample occasionally. The
soaking solution was transferred to a new tube for digestion, followed by organic extraction.
EDTA was most effective in reducing the amount of endogenous DNA as it did not break down
the actual nail.
Hebda et al. (2014) compared various methods for removal of exogenous material from
nails in an effort to standardize techniques for collection and analysis. Soaking clipped nails
directly in digestion buffer recovered more exogenous DNA than did swabbing or scraping.
However, soaking was problematic for autosomal STR analysis because the endogenous alleles
were more pronounced than exogenous alleles. Swabbing or scraping reduced the amount of
endogenous DNA, but less exogenous DNA was recovered. Swabbing, and even more so
scraping, resulted in loss of exogenous STR alleles, relative to soaking.

15

Packaging and Transportation of Fingernail Evidence
Fingernail evidence must be packaged and transported to the crime laboratory following
its collection, which has the potential to result in exogenous DNA loss and/or crosscontamination. The effects of packaging and transportation of fingernail evidence were
investigated by Hebda et al. (2014). Applying two pounds of force, females scratched male
volunteers three times with their three middle fingers. Immediately after scratching, fingernails
were clipped, placed individually into coin envelopes, and carried in a backpack for 5 days. After
transportation, the nails were removed and placed into separate microfuge tubes. Further, the
coin envelopes containing the fingernails were swabbed. Nails and coin envelope swabs were
processed using a QIAGEN QIAamp ® DNA Investigator Kit, followed by quantification and YSTR analysis. There was significant exogenous DNA loss from the fingernails (p<0.001), yet
DNA was not recovered from the packaging. On average, only 25% of the possible exogenous
alleles were present in Y-STR profiles.

Study Objectives
From the research presented above, it is clear that: (1) exogenous DNA is uncommon
underneath fingernails; (2) there is not a standard method of collecting and analyzing fingernail
evidence; (3) soaking fingernail clippings recovers the maximum amount of exogenous DNA,
although a substantial amount of endogenous DNA can be recovered along with it; (4)
exogenous DNA can be lost during transportation of fingernail evidence; and (5) methods of
packaging other than coin envelopes need to be evaluated.
The goals of this research were to continue to contribute to the study of Hebda et al.
(2014) by:

16



Further optimizing soaking for exogenous cell recovery, including:
o Comparison of different soaking solutions and soaking times for maximum
exogenous DNA recovery;
o Applying the optimized methodology to simulated forensic evidence, in the form
of controlled scratchings.



Determine which type of packaging preserves fingernail evidence most effectively by:
o Reducing DNA loss during transportation;
o Preventing DNA degradation during transportation.

By determining the best way to maximize exogenous DNA yields while minimizing endogenous
DNA, the probative value of fingernail evidence increases. Further, by improving packaging and
transportation methods, exogenous DNA loss will be reduced en route to the crime laboratory,
potentially becoming more useful in forensic investigations.

17

MATERIALS AND METHODS

The Michigan State University Institutional Review Board approved the procedures for
the collection and use of biological samples in this study (IRB# 08-526). All samples were
deidentified. Fingernails and buccal swabs were collected from volunteers, and anonymity was
maintained through randomly assigned numbers and letters. Blood from a single male donor was
used throughout all optimization-related studies. Scratchings were implemented to simulate
forensic evidence, in which female volunteers scratched male volunteers.

DNA Isolation and Purification
A QIAamp® DNA Investigator Kit (QIAGEN, Hilden, Germany) was used for DNA
isolation, following the modified protocols used in Hebda et al. (2014). Supplies and kit buffers
were UV irradiated for 5 min (approximately 2.5 J/cm2) before use. Four hundred microliters of
the kit tissue lysis buffer (ATL) and 10 µL of proteinase K (provided in the kit) were added to
each tube and incubated at 55ºC. Once digestion was complete, 400 µL of buffer AL plus 1 µL
of carrier RNA (provided in the kit) were added to the tubes, which were vortexed for 15 sec and
incubated at 70ºC in a water bath for 10 min. Two hundred microliters of 200 proof ethanol was
added and incubated for 3 min at room temperature. The lysate was transferred to a silica filter
containing a 2 mL collection tube, provided by the kit manufacturer. DNAs were washed with
500 µL of buffer AW1, 700 µL of buffer AW2, and 700 µL of 200 proof ethanol (not included in
the kit), respectively, and centrifuged for 1 min at 6000 x g. The column membrane was dried by
centrifugation at full speed (21,130 x g), followed by incubation at room temperature for 10 min.
Twenty microliters of buffer ATE was added to the center of the membrane, incubated for 5 min

18

at room temperature, and centrifuged at full speed (repeated 3 times), to elute DNAs from the
column.
Supplies and solutions for organic extractions were also UV irradiated prior to use (5
min, approximately 2.5 J/cm2). In 1.5-mL microfuge tubes containing the samples, 400 µL of
digestion buffer (20 mM Tris, pH 7.5; 50 mM EDTA; 0.1% SDS) was added, followed by the
addition of 5 µL of proteinase K (20 mg/mL). A reagent blank was produced by adding 400 µL
of digestion buffer and 5 µL of proteinase K. Samples were incubated at 55ºC for 2 h, after
which 400 µL of phenol was added, vortexed for 20 sec, and centrifuged at full speed for 5 min.
The aqueous layer was transferred to 1.5-mL microfuge tubes containing 400 µL of chloroform.
Samples were vortexed for 20 sec and centrifuged for 5 min at full speed. The aqueous layer was
transferred to 30 kDa Amicon® Ultra-0.5 centrifugal filter devices (Millipore, Billerica, MA) and
centrifuged at 14,000 x g for 10 min. Filter devices were pre-treated with 1 µL of 10 µg/mL
RNA in 499 µL of low TE, and centrifuged at 14,000 x g for 10 min (previously validated in our
laboratory). The filters were washed with 300 µL of low TE (10 mM Tris, pH 7.5; 0.1 mM
EDTA) and centrifuged at 14,000 x g for 10 min, for a total of three washes. The flow-through
was discarded each time. The filters were inverted and placed into clean Amicon® tubes, and
centrifuged at 1000 x g for 3 min to collect the retentate. The final volume of the DNA extracts
was approximately 28 µL.

Quantification of DNAs
Total (endogenous and exogenous) DNA and male (exogenous) DNA were quantified
using Quantifiler™ and Quantifiler™ Y Human Male DNA Quantification kits (Life
Technologies, Foster City, CA), respectively. Standards were created through serial dilution of

19

the Quantifiler™ Human DNA Standard, according to the manufacturer’s protocols. Reactions
were comprised of 7.5 µL of the Quantifiler™ PCR reaction mix, 6.3 µL of either the
Quantifiler™ Human Primer mix (for total DNA) or the Quantifiler™ Y Human Male Primer
mix, and 1.2 µL of either DNA extract or standard, for a total reaction volume of 15 µL.
An iCycler™ Thermal Cycler (Bio-Rad, Hercules, CA) was used for qPCR, and
fluorescence was detected with an iQ™5 Multicolor Real-Time PCR Detection System (BioRad). Cycling parameters were set per the manufacturer’s protocol. Data were analyzed using the
iQ™5 Optical System Software (Bio-Rad).

Autosomal and Y-STR Analyses
Autosomal STR analysis was performed using an AmpFISTR® Identifiler® PCR
Amplification kit (Life Technologies), while Y-STR analysis was completed using an
AmpFISTR® Yfiler® PCR Amplification kit. Reactions for both PCR methods were modified to
10 µL, which was previously validated at the Michigan State University Forensic Biology
Laboratory (data not shown). The Identifiler® PCR buffer volume was scaled down to 2 µL per
sample, and the primers were scaled down to 1 µL per sample. The Yfiler® PCR buffer was
reduced to 1.85 µL per sample, with the primers at 1 µL per sample. Both STR kit manufacturers
recommend the addition of 1 ng of template DNA; the volume of DNA extract added to the PCR
reaction for both types of STR analyses was based on the male DNA yield. The DNA input
volume had to be maximized in some instances (particularly the samples from the scratching
experiments) because of low DNA quantities.
PCR products were electrophoresed on a 3500 Genetic Analyzer with a 50 cm 3500
Capillary Array (Applied Biosystems). Nine microliters of Hi-Di formamide (Applied

20

Biosystems) and 0.3 µL of GeneScan™-500-LIZ™ Size Standard (Applied Biosystems) were
added to 1 µL of PCR product. Allelic ladders were included for each injection (AmpFISTR®
Identifiler® Allelic Ladder or AmpFISTR® Yfiler® Allelic Ladder). POP-7™ (384) Performance
Optimized Polymer (Applied Biosystems) was utilized for electrophoresis. GeneMapper v4.1
was used for data analysis. The threshold for both STR analyses was set at 150 RFUs; alleles
lower than this value were not included in analysis.

Sample Collection
Optimizations of the fingernail evidence collection and DNA isolation were performed
on clipped female fingernails, with 1 µL of male blood (from a single donor) deposited on the
clipping. Prior to blood deposition, all sides of the fingernail clipping were cleaned with a
Kimwipe (Kimberly-Clark, Irving, TX) wetted with 40 µL of distilled water. Once dirt and other
exogenous materials had been removed, male blood was deposited and left to dry overnight. A
positive control was run along with each experiment, and consisted of 1 µL of male blood which
was thawed and directly added to the microfuge tube for digestion.
Scratchings were implemented after DNA isolation optimization. Male volunteers placed
their forearms across an Eat Smart Precision PRO Kitchen Scale (Health Tools LLC, Wyckoff,
NJ), with their palms facing up, and the scale was zeroed. Females scratched male volunteers
with their three center fingers, three times, applying three pounds of force. The amount of force
applied to the volunteer was enough to redden, but not break the skin. Force was measured using
the kitchen scale, which was zeroed after each scratch (Figure 11). The fingernails from the
female volunteer were cut with scissors and placed in separate packaging.

21

Figure 11. Female volunteer scratching a male volunteer with her three center fingers, applying
three pounds of force.
Digestion Modifications for DNA Isolation
Digestion times for exogenous DNA isolation were examined utilizing a DNA
Investigator Kit. Fingernail clippings with blood were placed in 400 µL of ATL buffer and 10
µL of proteinase K, and incubated at 55ºC for 30 min, 1 h, 2 h, or 24 h. A positive control,
consisting of 1 µL of male blood directly added to a 1.5-mL microfuge tube with 400 µL of ATL
buffer and 10 µL of proteinase K, was incubated for 24 hours at 55ºC. A reagent blank (400 µL
of ATL buffer and 10 µL of proteinase K) was also incubated at 55ºC for 24 h. DNA isolation
was performed as described above. Subsequently, total and male DNAs were quantified.
Agitation during a 2-h digestion period was also investigated. The basic digestion
parameters were as described above, only differing in the amount of agitation during the
digestion period, which were as follows: (1) no agitation during the digestion period, (2) brief
agitation every 30 min of the digestion period, and (3) agitation only at the end of the digestion
period. DNA isolation was performed, followed by quantification of total and male DNAs.

22

Soaking Solutions for Exogenous DNA recovery
Fingernail clippings containing 1 µL of blood were soaked in various solutions; distilled
water, 1x phosphate buffered saline (PBS) (0.9%, 10mM), Tris/EDTA (TE) (10 mM Tris, pH
7.5; 1 mM EDTA), Tris/EDTA/SDS (digestion buffer) (20 mM Tris, pH 7.5; 50 mM EDTA;
0.1% SDS), ATL buffer without the addition of proteinase K, and ATL buffer plus proteinase K.
Two hundred microliters of each solution was added to 1.5-mL microfuge tubes containing a
fingernail clipping, and were soaked at room temperature (~25ºC) for 1 h, during which they
were briefly agitated every 15 min. The clippings were transferred to clean microfuge tubes. The
soaking solution was centrifuged for 5 min at full speed to form a pellet, after which the liquid
was removed. DNAs were extracted from the pellet as described above, implementing a 2-h
digestion period. DNAs were also extracted from the microfuge tubes containing the fingernails
to determine if any residual exogenous DNA remained after soaking. Total and male DNAs were
quantified, followed by autosomal STR analysis.

Soaking Times for Exogenous DNA recovery
Soaking times were initially tested using female fingernail clippings with 1 µL of male
blood deposited on them, and were soaked in 400 µL of TE, sufficient to submerge the entire
nail. Fingernails were soaked for 5 min, 10 min, 15 min, 20 min, 30 min, 1 h, 1.5 h, or 2 h, and
briefly agitated four times during the soaking period. The clippings were transferred to clean
microfuge tubes, and the soaking solution was centrifuged at full speed to pellet the exogenous
material. The liquid was removed, and DNAs were extracted from the pellet. Total and male
DNAs were quantified.

23

Female fingernail clippings containing 1 µL of male blood were soaked in 400 µL of
digestion buffer for either 5 min, 15 min, 30 min, or 1 h. Samples were briefly agitated four
times during soaking. Fingernails were removed and 5 µl of proteinase K was added to the
solution. Samples were incubated at 55ºC for 2 h, followed by organic extraction (as outlined
above). Total and male DNAs were quantified.

Silica-Based Kit Extraction vs. Organic Extraction with RNA-Pre-Treated Filters
DNAs were extracted from fingernails containing blood utilizing the silica-based kit
extraction (described above) or with an organic extraction incorporating RNA-pre-treated
centrifugal filtration devices (also described above). Positive controls of 1 µL of male blood
were run along with the fingernail samples. Male DNA was quantified, and yields were
converted to picograms (pg) per volume.

Exogenous Material Recovery from Pellets, Fingernails, and Soak Solutions
Fingernails with 1 µL of male blood were soaked in 400 µL of TE or digestion buffer for
5 min, with brief agitation every min. The fingernails were transferred to clean 1.5-mL
microfuge tubes. Exogenous material left in the soaking solution was pelleted by centrifugation
at full speed for 5 min. The soaking solution was removed from the pellet and placed in a clean
1.5-mL microfuge tube. Four hundred microliters of digestion buffer and 10 µL of proteinase K
were added to the pellets. Proteinase K (10 µL) was added to the digestion-buffer soaking
solution. Digestion buffer and proteinase K (400 µL and 10 µL, respectively) were also added to
the fingernails that had been removed after pelleting. Ten percent SDS (4.2 µL) was added to the
samples containing TE, followed by 10 µL of proteinase K. A positive control containing 1 µL

24

of male blood, 400 µL of digestion buffer, and 10 µL of proteinase K was also created. All
samples were incubated at 55ºC for 2 h, followed by an organic extraction. Total and male DNAs
were quantified.

Scratchings Utilizing Optimized Procedures for STR Analysis
Female volunteers scratched male volunteers as described previously. Fingernails were
cut with scissors and individually placed into 1.5-mL microfuge tubes. Four hundred microliters
of digestion buffer was added to each tube and the fingernails were soaked for 15 min, with brief
agitation every 4 min. The fingernail clipping was discarded. Proteinase K (5 µL) was added to
the soaking solution, which was incubated at 55ºC for 2 h. DNAs were isolated via organic
extraction. Total and male DNAs were quantified, and autosomal and Y-STRs were analyzed.

DNA Degradation in Microfuge Tubes
Fingernails containing 1 µL of blood were prepared as described above, placed into
individual 1.5-mL microfuge tubes, and stored at room temperature for 0 d, 2 d, 1 wk, 2 wk, 1
mo, 3 mo, and 6 mo. DNAs were extracted after each time period using a DNA Investigator Kit,
followed by male DNA quantification and Y-STR analysis.
Fingernails containing male blood were allowed to dry for 0 min, 6 h, or 24 h prior to
storage. Samples were stored at room temperature for one week. A positive control was created,
consisting of 1 µL of male blood directly added to the digestion buffer. DNAs were extracted by
organic extraction, followed by male DNA quantification.

25

Assessment of DNA Loss during Transportation
Female volunteers scratched male volunteers, using the scratching protocol described
above. Fingernails were clipped and placed into coin envelopes, bindle papers (folded at the time
of packaging), or 1.5-mL microfuge tubes (Figure 12), which were placed in a brown paper bag
and carried in a backpack for 5 days. Fingernails were removed from packaging and placed into
new 1.5-mL microfuge tubes. The coin envelopes and bindle papers were swabbed, and DNAs
were extracted by organic extraction. DNAs were extracted from both the empty microfuge tube
and the fingernail. Fingernail clippings were also transported in microfuge tubes and DNAs were
extracted directly from the nail in the tube. Exogenous material was recovered from the
fingernail, following the optimized procedure described above, and isolated via organic
extraction. Male DNA was quantified, followed by autosomal and Y-STR analysis.

Figure 12. Packaging used in transportation study; (from the left) coin envelope, bindle paper,
and a plastic 1.5-mL microfuge tube.

26

Statistical Analyses of DNA Quantifications and STR Profile Percentages
Kruskal-Wallis was used to calculate significance; in the event of a significant difference
(p<0.05), the Mann-Whitney U test was utilized for pairwise comparisons. Outliers were
detected using Grubb’s Test (α=0.05) from graphpad.com (GraphPad Software, Inc.). A t-test
was used to determine significance (p<0.05) between the mean DNA yields of kit and organic
extractions.
The percentage of exogenous alleles called (autosomal and Y-STRs) was calculated by
dividing the number of alleles called by the total number of alleles from the known exogenous
profile, multiplied by 100. The percent of mixture for autosomal STR analysis was calculated by
dividing the number of loci containing endogenous alleles by the total number of loci and
multiplied by 100.

27

RESULTS

Soaking Modifications for Exogenous Cell Recovery from Fingernail Evidence
Among soaking solutions (Figure 13), ATL buffer and PBS resulted in the lowest
exogenous DNA recovery from blood on nails, while water, TE, Tris/EDTA/SDS, and ATL
buffer/proteinase K, respectively, recovered more. Addition of proteinase K to the ATL buffer
greatly increased the concentration of endogenous DNA (Figure 14), while water recovered the
least. Tris/EDTA/SDS and TE were utilized to recover exogenous DNA from fingernails in

Average Exogenous DNA
Concentration (pg/μL)

further experiments.
500
450
400
350
300
250
200
150
100
50
0

Soaking Solution
Figure 13. Comparison of average exogenous DNA concentrations (pg/µL) after soaking in
various solutions (n=11 total). TE, Tris/EDTA/SDS, and ATL buffer/Pro K recovered the most
exogenous DNA. ATL buffer and PBS did not recover as much exogenous DNA as the other
soaking solutions.

28

Average Total DNA Concentration
(pg/μL)

6000
5000
4000
3000
2000
1000
0
-1000

Soaking Solution
Figure 14. Comparison of average total (endogenous + exogenous) DNA concentrations (pg/µL)
after soaking in various solutions (n=11 total). The addition of proteinase K greatly increased the
total DNA concentration.

Pelleting exogenous material following soaking led to DNA loss. Figure 15 shows that
less exogenous DNA was recovered after increased soaking time in TE. Further, the
concentration of the positive control was higher than any of those recovered from the fingernails.
An ancillary study showed that soaking fingernail clippings with blood in TE for 5 min resulted
in 75% of exogenous DNA recovered from the pellet, 16% came from the fingernail, and 9%
was from the soaking solution (Figure 16). When soaked in Tris/EDTA/SDS, 33% of the
exogenous DNA existed within the pellet, 7% on the fingernail, and 60% in the soaking solution.
Additional experiments utilized Tris/EDTA/SDS as the soaking solution.

29

Exogenous DNA Concentration (pg/µL)

500
450
400
350
300
250
200
150
100
50
0

0.5

1

1.5
Soak Time (h)

2

(+)

Figure 15. Comparison of soak times (h) for exogenous DNA recovery using TE (n=4 total).
DNA was extracted from the pelleted material; the figure shows that the longer the soaking time,
the less DNA recovered from the pellet. A positive control (+) was also analyzed, containing 1
µL of blood.
80

Exogenous DNA
Recovered (%)

70
60
50
40

TE

30

Tris/EDTA/SDS

20
10
0
Pellet

Fingernail

Soak Solution

Figure 16. Comparison of relative exogenous DNA recovery (%) after soaking in TE or
Tris/EDTA/SDS from the pellet, fingernail, or soak solution, after a 5 min soaking period (n=12
total).

30

Soaking time had a significant influence on both exogenous and total DNA recovery
from nails harboring blood (Table 2; p=0.013 and 0.0003 respectively). Pairwise comparisons
(Mann-Whitney U; α=0.05) showed that more DNA was recovered after 30 min than 15 or 60
min, and almost significantly more than 5 min (Table 3). Total DNA concentrations were also
significantly higher after 5 and 30 min compared to 15 and 60 min (Table 4).

Table 2. Median exogenous and total (endogenous + exogenous) DNA concentrations (pg/µL)
for various soaking times (min) from fingernails harboring blood prior to digestion and
extraction (n=48 total). Significant differences existed among exogenous (p=0.013) and
endogenous (p=0.0003) DNA concentrations.
Median Exogenous DNA
Median Total DNA
Time (min)
Concentration (pg/µL)
Concentration (pg/µL)
(Kruskal-Wallis; p=0.013)
(Kruskal-Wallis; p=0.0003)
555
1885
5
263
878
15
824
2000
30
302
675
60
Table 3. Pairwise comparisons (Mann-Whitney U; α=0.05) showing significance values among
the average exogenous DNA concentrations (n=48 total). Significant p-values are in red.
Time (min)
15
30
60
p=0.237
p=0.061
p=0.371
5
p=0.004
p=0.885
15
p=0.012
30
Table 4. Pairwise comparisons (Mann-Whitney U; α=0.05) showing significance values among
the average total DNA concentrations (n=48 total). Significant p-values are in red.
Time (min)
15
30
60
p=0.002
p=0.583
p=0.002
5
p=0.004
p=0.954
15
p=0.004
30

31

Silica-Based Kit Extraction vs. Organic Extraction utilizing RNA-Treated Filters
There was no statistical difference between commercial kit and organic extraction DNA
yields from blood on fingernails, nor between the soaking times (Table 5). However, the organic
extraction using treated filters had higher average exogenous yields after both soaking times. The
15 min soak prior to organic extraction resulted in less endogenous DNA recovery than the 60
min soak (see Appendix A for individual DNA quantifications). Given this, 15 min soaking
times were utilized for further experiments.

Table 5. Average exogenous DNA yields (pg) for commercial kit extractions and organic
extractions coupled with RNA-pre-treated filters (n=28 total). T-test showed no significant
differences between types of extraction methods or soaking times: ap=0.738734; bp=0.215766;
c
p=0.222789; and dp=0.7480818.
Soak Time (min)
Average Exogenous DNA Yield (pg)
Kit Extraction
Organic Extraction
a,d
4640
5100a,c
15
b,d
5080
6900b,c
60

Comparison of Digestion Modifications for Exogenous Cell Recovery
Digestion time impacted both exogenous and endogenous DNA recovery from fingernails
containing blood (Figure 17). The 0.5-h time frame had the lowest exogenous and total DNA
concentrations. The total DNA concentration increased with time, which was accentuated after a
24-h digestion period. The amount of exogenous DNA recovered never reached that of the
positive control.

32

Exogenous and Total DNA
Concentrations (pg/µL)

4000
3500

3000
2500

2000

Exogenous DNA

1500

Total DNA

1000
500

0
0.5

1

2
24
Digestion Time (h)

(+)

Figure 17. The influence of digestion time on exogenous and total (endogenous + exogenous)
DNA concentrations (pg/µL) after 0.5 h, 1 h, 2 h, and 24 h (n=4 total). A positive control (+)
containing 1 µL of blood was also analyzed. The average exogenous DNA concentration after
0.5 h was the lowest; after a 1 hr digestion period, the average exogenous DNA concentration
plateaued around 400 pg/µL. Total DNA concentration increased substantially at 24 h.
Comparison of autosomal STR profiles following different digestion periods is shown in
Table 6. All digestion times resulted in almost 100% of autosomal alleles present. The 1-h and
24-h periods had slightly higher percent mixtures (66 and 57%, respectively) than the 2-h
digestion, which had 54% mixture. All samples contained some loci with mixtures (primarily
alleles from the fingernail donor); however, there was no indication that an increased
endogenous DNA concentration resulted in a higher percentage of loci containing mixture (See
Appendix B). Subsequent experiments utilized a 2-h digestion period.

Table 6. Comparison of autosomal STR profiles from blood on fingernails for different digestion
periods (h) utilizing a 15 min soak (n=7 total). The average % of alleles consistent with the
exogenous profile was calculated, as was the % of loci that contained alleles not consistent with
the exogenous DNA profile.
Digestion Period (h)
1
2
24
Exogenous Profile
100
100
99
(Average % alleles)
Mixture
66
54
57
(Average % loci)
33

Exogenous DNA concentrations did not change substantially with brief vortexing, all
falling within 400 – 550 pg/µL (Figure 18). Briefly vortexing after digestion resulted in a slightly
higher average total DNA yield than did samples that were not vortexed, whereas vortexing
every 30 min greatly increased the concentration of endogenous DNA recovered. Vortexing after

Average DNA Concentrations (pg/µL)

digestion was utilized in further experiments.

3000
2500
2000
1500
Exogenous DNA
1000

Total DNA

500
0
None

Briefly every 30
Briefly after
min of digestion
digestion
Amount and Frequency of Vortexing

Figure 18. Comparison of average exogenous and total (endogenous + exogenous) DNA
concentrations (pg/µL) after implementing different amounts and frequencies of vortexing for a
2-h digestion period (n=6 total). Includes standard error bars.

STR Profiles from Scratchings Using Optimized Procedures
Following scratchings, on average 209 pg/µL of exogenous DNA was recovered from
individual fingernails utilizing the optimized methodology, ranging from 0 to over 600 pg/µL
(Table 7). Forty-eight percent of profiles contained autosomal alleles consistent with the
exogenous DNA (range = 0 – 100%). There were seven instances when no exogenous alleles

34

were detected despite relatively high exogenous DNA concentrations. Sixty-one percent of Yalleles consistent with the exogenous donor were detected (range = 18 – 100%). All samples that
underwent Y-STR analysis had alleles present, including those that had exogenous DNA
concentrations of 0 pg/µL. In most autosomal STR profiles, mixtures were dominated by the
endogenous DNA profile (e.g., Figure 19). When Y-STRs were analyzed for the same samples,
(e.g., Figure 20) the resulting profile contained only a single source of DNA consistent with the
exogenous DNA profile (see Appendix C for more information).

Table 7. Summary of quantifications and STR analyses for scratchings utilizing optimized DNA
collection and extraction procedures (n=18 total). Red italicized number indicates an outlier
(Grubbs; α=0.05), and the numbers in parentheses indicate averages calculated without inclusion
of the outlier.
Autosomal Alleles
Y-Alleles
Endogenous DNA Exogenous DNA
Consistent with
Consistent with
Sample
Concentration
Concentration
Exogenous
Exogenous
(pg/µL)
(pg/µL)
Profile (%)
Profile (%)
529
23
52
94
1
197
97
0
18
2
244
345
0
47
3
403
260
0
47
4
270
609
68
88
5
279
356
97
100
6
857
443
0
100
7
569
223
79
76
8
4360
367
72
65
9
1900
409
79
29
10
307
273
69
65
11
426
158
0
41
12
380
3
90
71
13
133
0
0
24
14
411
128
100
100
15
412
4
80
29
16
595
30
80
76
17
431
26
0
29
18
Median
412
191
69
65
Average
706 (491)
209
48
61

35

Figure 19. Exemplary autosomal STR profile from scratchings. The gray boxes above the peaks represent the name of the locus.
The boxes below the peaks represent the number of repeats for that allele. The endogenous (female) profile is dominant, as
indicated by the tall ‘X’ peak at the amelogenin (AMEL) locus.

36

Figure 20. Exemplary Y-STR profile of exogenous DNA following scratching. The gray boxes above the peaks represent the name
of the locus. The boxes below the peaks represent the number of repeats for that allele. The peaks are consistent with the exogenous
DNA profile.

37

Assessment of DNA Degradation in Microfuge Tubes
There was no significant difference in exogenous DNA concentrations for blood
deposited on fingernails (Kruskal-Wallis; p=0.075) among the blood-drying time periods (Table
8). However, when allowed to dry for 24 h, there was a higher median DNA concentration than
when they were placed immediately into microfuge tubes after deposition. Zero minutes had the
lowest median exogenous DNA concentration.

Table 8. Median exogenous DNA concentrations (pg/µL) from fingernails stored for 1 week in
microfuge tubes (n=15 total). After deposition, blood was allowed to dry for 0 min, 6 h, or 24 h
prior to placement in microfuge tubes (Kruskal-Wallis; p=0.075).
Drying Time Period
0 min
6 hr
24 hr
Median Exogenous
2120
3410
3520
DNA Concentration
(pg/µL)

Storing fingernails with blood up to 6 months (Table 9) did not produce significantly
different exogenous DNA concentrations (Kruskal-Wallis; p=0.617). Median exogenous DNA
concentrations ranged from 97 – 320 pg/µL. However, the median concentration of exogenous
DNA after 6 months was the lowest. The median concentration for 1 week was also lower than
the other time periods. One-hundred percent of Y-STR alleles from the exogenous profile were
present for all samples (Appendix E).

Table 9. Median exogenous DNA concentrations (pg/µL) from fingernails with 1 µL of male
blood, stored at room temperature in microfuge tubes for various time periods (Kruskal-Wallis;
p=0.617) (n=21 total).
0
2
1
2
1
3
6
Time Period
days days week weeks month months months
Median Exogenous DNA
218
320
200
226
234
234
97
Concentration (pg/µL)

38

Assessment of DNA Recovery from Scratchings after Transportation
Table 10 shows DNA concentrations recovered from fingernails transported in coin
envelopes, bindle papers, or microfuge tubes following scratchings; exogenous DNA
concentrations did not differ significantly (Kruskal-Wallis; p=0.639). The average and median
exogenous DNA concentrations from fingernails transported in microfuge tubes were higher than
those transported in coin envelopes or bindle papers. Additionally, the average percent of Yalleles present was higher for those transported within the coin envelopes than those transported
in bindle papers or microfuge tubes. However, fingernails transported in microfuge tubes had the
highest median percent of Y-alleles detected. DNA recovered from fingernails transported in the
bindle papers had the lowest average DNA concentrations (with inclusion of the outlier) and Yalleles called (see Appendix D for details).

39

Table 10. Exogenous DNA recovered (pg/µL) and percent Y-alleles called consistent with the
known exogenous profile from fingernail scratchings transported in coin envelopes, bindle
papers, and microfuge tubes (Kruskal-Wallis; p=0.639) (n=45 total). Fingernails transported
within the microfuge tubes were not transferred to fresh tubes for analysis. Red italicized number
indicates an outlier (Grubbs; α=0.05), and the numbers in parentheses indicate averages
calculated without inclusion of the outlier.
Sample
Coin Envelope
Bindle Paper
Microfuge Tube
DNA
YDNA
YDNA
YConcentration Alleles Concentration Alleles Concentration Alleles
(pg/µL)
(%)
(pg/µL)
(%)
(pg/µL)
(%)
10
82
10
24
30
100
1
240
100
0
0
10
88
2
10
100
30
35
10
0
3
0
12
0
12
0
6
4
0
100
0
0
0
6
5
0
71
0
100
530
100
6
166
100
182
100
420
100
7
0
53
18
24
42
100
8
0
6
7
18
100
100
9
7
94
0
18
0
0
10
55
82
3
47
0
18
11
5
100
12
59
3
82
12
22
59
0
88
53
94
13
0
18
44
76
313
100
14
62
100
188
100
0
6
15
Median
7
82
7
35
10
88
Average
38 (24)
72 (70)
33
47
101
60

40

DISCUSSION

The goal of the research presented here was to further optimize DNA extraction and
analysis techniques from fingernail evidence. During earlier research at the Michigan State
Forensic Biology Laboratory, Hebda et al. (2014) noted several things:


Soaking fingernail clippings recovered more exogenous material than did swabbing or
scraping.



A silica-based extraction utilizing three elutions recovered more exogenous DNA than
did an organic extraction coupled with centrifugal filtration.



Transporting fingernails together within a package did not result in cross-contamination
but did result in DNA loss, particularly when the exogenous material was loosely
adhering to the fingernail clippings, such as epithelial cells from scratching.



Y-STR analysis was more effective than autosomal STR analysis at distinguishing the
exogenous DNA profile when male DNA was underneath a female fingernail.

It was important to have a systematic way to analyze exogenous DNA in order to address
the aforementioned points and quantitatively compare exogenous cell recovery. This could not
be done via scratching as the quantity of exogenous material that accumulates is highly variable,
but instead was accomplished by depositing a consistent amount of cells on the fingernails.
Placement of 1 µL male blood onto female fingernails allowed for differentiation of exogenous
(male) and endogenous (female) DNA. Note that this procedure does not mimic all assaults;
instead, it was utilized because it provided a simple and direct way to measure exogenous DNA
recovery through Y chromosome quantification.

41

Once exogenous cell recovery methods were optimized, scratchings were investigated.
The amount of exogenous material recovered potentially depends on multiple factors, such as the
time of year (dryer skin in the winter compared to the summer), the force behind the scratches,
and the size of the nails. Loss of exogenous material could also occur during nail clipping or
transfer to the microfuge tubes for extraction. Several strategies were or potentially could be
implemented to mitigate these variables. First, scratchings for each experiment should ideally be
collected around the same time of year to reduce variation based on climate. However, since
volunteers were limited, samples were collected when available, and could have been a source of
variation. Second, the force of scratchings was measured with a kitchen scale in an attempt to
standardize it. However, an individual with small nails will necessarily be distributing that force
onto a smaller area of skin, thus scratching harder. To overcome this, nails could have been
measured prior to scratching, and fingernails similar in size could have been utilized if/when
there were multiple volunteers available. Another option would be to figure out a correction
factor for the force applied and utilize that for all scratches to standardize them. Third, if the
exogenous material adhered loosely to the fingernail, it might fall off when clipped or
transferred. Fingernails should have been clipped over clean paper, which would collect any
exogenous material that had fallen off of the nails; the paper could then be emptied into the
transportation container (ideally a tube or similar) containing the fingernail. Additionally,
swabbing the clippers and extracting DNA from the swabs along with DNA from the fingernails
could overcome some of this loss, however this technique was not implemented in this research.
The primary goal of the current study was to optimize the soaking technique for recovery
of exogenous biological material. Hebda et al. (2014) soaked fingernail clippings in
Tris/EDTA/SDS buffer and proteinase K overnight. The current research showed that after 2 h,

42

the amount of exogenous DNA recovered plateaued, and that vortexing slightly helped with
exogenous material recovery. However, exogenous DNA concentrations from the fingernails
were at least 100 pg/µL less than the positive control, indicating that either the exogenous DNA
was not being fully recovered, or that it was lost. As seen in the pelleting experiment, after
soaking fingernails in TE and Tris/EDTA/SDS, over 7% of the exogenous material remained on
the fingernails, even though none was present visually. It is possible that the exogenous material
was not being dislodged, or it somehow became integrated into the fingernail, preventing
recovery. On the other hand, DNA may have degraded while the blood was drying either because
of bacterial contamination or exposure to nucleases, resulting in consistently lower exogenous
DNA yields from nails than positive controls. The blood used for the positive control was stored
in a freezer until the day of extraction, then immediately deposited into digestion buffer once
thawed, providing little or no opportunity for DNA to degrade.
Endogenous DNA recovery, which greatly increased after longer digestion times, was
problematic during autosomal STR analysis. Overnight digestion released so much nail DNA
that exogenous alleles were often undetectable. Endogenous DNA concentrations almost
quadrupled following a 24-h digestion, most likely because the proteinase K continued to break
down the keratin of the fingernail. Vortexing during digestion also increased endogenous DNA,
presumably by further breaking down the nail and releasing DNA.
Since one goal of this research was to minimize endogenous DNA retrieval, it was
important to assess methods that prevent the further breakdown of keratin. One way to
accomplish this was to control the sizes of fingernail clippings used for each experiment.
Fingernails differ in width and often length among fingers. Some donors had fingernails that
were longer and thicker, while others had nails that were brittle and thin. It is possible more

43

endogenous DNA was released from one set/type of fingernails than others. Fingernails similar
in size were utilized within experiments in an attempt to minimize such differences, however
nails from multiple individuals had to be used to increase sample size. Using nails that were
similar in size likely decreased endogenous DNA variation among nails from a donor, but there
was still substantial variation among individuals. Endogenous DNA was also minimized in the
optimized protocol by removing the fingernail after exogenous material was loosened from the
nail by soaking, then adding proteinase K to break down cells that were in solution. Another
option for reducing endogenous DNA release from the nail would be to decrease the digestion
incubation temperature. In the current study, all samples were incubated at 55°C, which helps
unfold proteins, ultimately making it easier for proteinase K to break down the keratin and
contribute to the inactivation of nucleases that degrade DNA. Lowering the temperature may
reduce endogenous DNA recovery, but it also could reduce exogenous DNA recovery. There
might also be degradation from nucleases if they are not inactivated.
Addition of proteinase K to the soaking solution also contributed to the release of
endogenous DNA from the fingernail, which was noted by Cline et al. (2003). Proteinase K is
able to digest keratin, which is why it is used by forensic biologists to break down hair for DNA
analysis. Proteinase K predominately cleaves peptide bonds of hydrophobic amino acids. When
in solution with the fingernail, nail DNA is released. Further, when proteinase K is in solution
with a denaturing agent such as SDS, its activity increases (Wolfgang et al. 1974). An option that
could be explored in future research would be to utilize proteases other than proteinase K. The
proteases investigated should not be inactivated when in solution with detergents, such as the
alkaline proteases found in Bacillus cereus (Mohan Banik and Prakash 2004). In contrast,
proteolytic enzymes from fruit juices such as papain, bromelain, etc. should be avoided since it

44

appears that they increase the breakdown of keratin (Yoshida-Yamamato et al. 2010). Other
proteases can be investigated as well, such as serine and cysteine proteases, which come from
plants, fungi, or various animals and are commonly used by biologists (Mótyán et al. 2013).
Pelleting cells from fingernail evidence has been used in casework (e.g., Harbison et al.
2003), and was reportedly successful. However, the current study showed that not all DNA
remained within the pellet. This was particularly true for soaking solutions that contained
detergents, such as the Tris/EDTA/SDS digestion buffer or potentially the ATL buffer. In a DNA
extraction, SDS helps lyse cells, so when the fingernail was soaked in buffer to remove
exogenous material, cells were likely being lysed at the same time. The kit’s ATL buffer
chemical composition is unknown, but based on its foaming it was surmised that it contained
some type of detergent. Here again, the DNA could have been in the soaking solution and not in
the pellet. When TE was further examined as a soaking solution, results showed that the longer a
fingernail soaked in it, the less exogenous DNA was recovered from the pellet, meaning it was
likely that the cells were lysed in solution. PBS was used in an attempt to prevent cell lysis;
however, less exogenous DNA was recovered, possibly due to degradation of DNA in solution,
as there was no EDTA or proteinase K to help prevent DNase activity. This could also explain
the low levels of exogenous DNA recovery from the ATL buffer. Given these results, it is clear
that it is better to extract DNA from the entire soaking solution rather than the pellet, especially
since utilizing a soaking solution containing a detergent recovers more exogenous DNA from the
nail.
Soaking solutions containing EDTA recovered more exogenous DNA than those without.
Further, the Tris/EDTA/SDS soaking solution recovered more exogenous DNA than the TE
solution, likely because the concentration of EDTA within the former differs greatly from the

45

latter (50 mM in Tris/EDTA/SDS, 1 mM EDTA in TE). EDTA chelates free magnesium, which
prevents nucleases from degrading DNA; therefore, an increased concentration of EDTA should
provide better protection against nucleases (Yoshida-Yamamato et al. 2010). Addition of SDS in
the soaking solution could also explain the higher amount of exogenous cells recovered. As
mentioned, SDS is used to help lyse cells; however detergents are also used in many cleaning
products. Along with lysing cells, SDS was probably loosening exogenous material from the
fingernail.
It was surprising that significantly more exogenous DNA was recovered after 30 min of
soaking in Tris/EDTA/SDS digestion buffer than was recovered after 15 and 60 min, because
one would assume that exogenous DNA recovery would plateau after a certain time period,
similarly to what was seen following various digestion times (exogenous DNA recovery
plateaued after an hour). It is possible that prior to 30 min, not all of the exogenous material was
recovered from the fingernail. After an hour, the DNA could have started to degrade while in
solution, resulting in significantly lower recovery. Another possibility is that that the samples
with higher yields were not cleaned thoroughly prior to blood deposition, and male cells were
already on them. Even though no exogenous material was visually nor microscopically present
after cleaning, traces could have still been adhering to the fingernail. Exogenous DNA recovered
could also have been variable because all samples for each time point were not analyzed
together. Initially, just a few samples were analyzed for each time period to see if a trend
emerged. Later, sample size was increased so significance could be evaluated. When sample
sizes were increased, there were multiple samples soaking for each of the four time periods.
Because there were so many samples soaking at one time, fingernails may not have been
removed after exactly 5 min, 30 min, et cetera, potentially causing an increase in DNA

46

quantifications. Generally, the DNA quantifications of the initial time points were lower than
those that were analyzed later. After both sets of extractions, all samples were re-quantified
together using the same qPCR master mix. Variation within time points was reduced, however
extracting and quantifying all time points at the same time would have been a better option.
Soaking for 5 min in the Tris/EDTA/SDS buffer resulted in significantly higher total
DNA recovery than when soaked for 15 and 60 min. Further, after 60 min there was significantly
less total DNA recovered than after 30 min. Analysis of the IPC curves indicated that there was
not any inhibition at any of the time points, and the assay was working properly. Increasing the
sample size, as discussed above, could have produced the higher quantifications if those samples
had soaked longer than the necessary time frame. There also could have been some DNA
degradation after 60 min (also discussed previously), since several of the quantifications were
under 1000 pg/μL. However, it seems more probable that both of these could have resulted from
variation among fingernails, particularly because they were not all from the same individual.
Previous experiments showed that the longer a fingernail soaked in Tris/EDTA/SDS buffer, the
more endogenous DNA was recovered (see Appendix A). Even though precautions were taken to
ensure the experiments were as controlled as possible (such as size of the fingernail clipping), it
is likely that disparities among nails led to higher endogenous DNA recoveries at 5 and 30 min.
Revisiting DNA extraction methods was also important to assure that the most exogenous
DNA was being recovered from the fingernail evidence. Hebda et al. (2014) compared the
optimized silica-based kit extractions to organic extractions, and found that the former recovered
more DNA. However, other research in the Michigan State Forensic Biology Laboratory (Doran
and Foran 2014) has shown that there is DNA loss when utilizing centrifugal filtration devices
because the DNA becomes trapped within the filter. To mitigate this, the filtration devices were

47

pre-treated with yeast RNA prior to the addition of the DNA extract, which coated the filter
device and lessened the adhesion of DNA. The RNA did not interfere with PCR amplification,
since it cannot act as a template. DNA recovery greatly improved, and the pre-treatment step was
implemented into laboratory protocols when extracting trace or touch DNA. The current research
compared the organic extraction with RNA treated centrifugal filtration devices to the optimized
kit extraction. Although there were no significant differences between the two extraction
methods, the organic extraction with treated filters improved DNA yields for both 15 and 60 min
soakings. Since there was not a statistical difference between 15 and 60 min exogenous DNA
yields, 15 min soaks were used in the improved protocol.
After exogenous DNA recovery, autosomal and Y-STRs were analyzed for a subset of
fingernails that contained blood and were soaked for 15 minutes, but had variable digestion
times. Results showed that every sample examined resulted in complete exogenous profiles,
indicating that digestion time did not impact STR analyses. However, this was not the case when
analyzing autosomal STRs from fingernails after scratching. In some instances, exogenous DNA
was not detected during qPCR, but a few exogenous autosomal alleles were present. Even more
exogenous alleles were detected when Y-STR analysis was utilized. A validation study of the
Yfiler™ assay by Gross et al. (2008) showed that it was more sensitive than Quantifiler® and
Quantifiler® Y qPCR assays for male DNA detection. Quantifiler® and Quantifiler® Y only
reliably detects DNA down to 23 pg/µL (QIAGEN 2012), which means exogenous DNA that did
not amplify during qPCR might still produce detectable Y-alleles.
As noted earlier, exogenous DNA yields from scratchings varied, ultimately affecting the
detection of exogenous alleles during autosomal STR analysis. Manufacturers of Identifiler® and
Yfiler® STR assays suggest input of around 1 ng of template DNA. For this research, the

48

maximum volume of DNA extract needed to be added to PCR reactions. In instances where the
endogenous DNA concentration was much higher than the exogenous, maximizing the DNA
template led to detection of the endogenous profile, while alleles from the exogenous donor were
sometimes not detected. Similarly, it has been found that in many instances of sexual assaults,
the female contribution to the mixture is much greater than the male contribution, and sometimes
the male component is undetectable (Prinz et al. 1998, Wallin et al. 1998).
There was one nonsensical result where the exogenous DNA quantified higher (345
pg/µL) than total DNA (244 pg/µL). Upon autosomal STR analysis, only the endogenous DNA
profile was detected. The IPC curves for both assays were close together, indicating that the
sample was not inhibited, and that the assays were working properly. This sample was only
quantified once; if it had been run in duplicate, or if it had been re-quantified, the exogenous
DNA concentration may have been lower (or the total DNA concentration may have been
higher). As mentioned previously, the quantification assays for total and male DNA cannot be
directly compared because they utilize different reaction mixtures, so it is likely that the
exogenous DNA quantification was artificially high. Re-quantifying the sample, or running it in
duplicate would have provided a more reliable quantification.
One problem with standard Y-STR analysis is that male relatives cannot be distinguished
from one another. Because of this, Y-STRs are not typically utilized initially; instead crime
laboratories depend on autosomal STR analysis (Roewer 2009). However, new Y-STR kits (such
as PowerPlex® Y23 [Promega] and Y-Filer® Plus [Life Technologies]) which incorporate
additional Y-STR loci are being implemented into standard operating procedures, since they are
more discriminatory than the original Y-STR kits. According to Kayser and de Knijff (2011), the
higher mutation rates of these Y-STRs provide greater differentiation between related males. In a

49

study by Ballantyne et al. (2010), 13 rapidly mutating Y-STRs were used to differentiate over
70% of closely and distantly paternally related males; the same samples analyzed with the
standard 17 Y-STRs were only able to differentiate 13%. Throughout the current study, 61% of
Y-STRs consistent with known exogenous alleles were detected, as opposed to only 48% of
autosomal STRs. By using these new Y-STR kits, more loci will be incorporated into analysis,
potentially increasing the percent of Y-STRs detected. Further, males that are related have the
potential to be differentiated from each other.
Previous research on transportation of fingernail evidence (Hebda et al. 2014) only
investigated use of coin envelopes. However, not every agency uses those; some use bindle paper
(Alaska Scientific Crime Detection Laboratory 2012), which can be tailored to any size. It was
surmised that having a smaller package for fingernails might decrease the amount of DNA lost,
because there would be less movement within the package. Another option, suggested by Hebda
et al. (2014), was use of microfuge tubes for nail transportation, which could both contain the
fingernail during transportation and be used for the DNA extraction. There was exogenous DNA
loss across all methods of transportation in the current research; however, there was less when
nails were transported within microfuge tubes. A potential problem with transporting fingernail
evidence in microfuge tubes is degradation of the biological material, since the plastic tubes
could create an environment for bacteria to grow and decay to occur. It is standard protocol to
transport biological evidence in breathable packaging so it can dry, preventing decay and DNA
degradation. The FBI’s Handbook of Forensic Services (2011) suggests not utilizing plastic
containers when collecting biological evidence. However, DNA concentrations from fingernail
evidence stored long-term in microfuge tubes did not degrade significantly. Further, the
exogenous DNA concentrations did not change regardless of blood-drying time prior to

50

placement in microfuge tubes. It is possible that the space within the 1.5-mL microfuge tube was
enough to allow the fingernails to dry without causing significant degradation. For this research
individual fingernails were placed in tubes, and it is unknown if storing a set of five fingernails
in one tube would result in degradation. An advantage of the tube storage method was that DNA
loss was minimized because extractions were performed within the microfuge tube, where the
majority of exogenous material was contained throughout transportation and extraction. Further,
fingernail evidence can be examined microscopically within the tube, again minimizing handling
and potentially preventing DNA loss.
Throughout this research, several techniques for improving the success of exogenous
DNA recovery were implemented into fingernail evidence analysis:


Soaking the fingernail in Tris/EDTA/SDS buffer for 15 min prior to DNA extraction,
with brief vortexing throughout, removed the majority of exogenous DNA from the
fingernail, while minimizing the amount of endogenous DNA.



Removing the fingernail after soaking and adding proteinase K to the Tris/EDTA/SDS
buffer recovered more exogenous DNA than pelleting, since exogenous DNA is lost to
the soak solution.



Short digestion periods of 1 or 2 h are efficient for lysing exogenous cells instead of
utilizing an overnight digestion.



A standard organic extraction coupled with centrifugal filtration recovers more
exogenous DNA than a silica-based kit extraction when the filtration devices are pretreated with RNA.



Y-STR analysis is beneficial when there is a male/female mixture.

51



Fingernail evidence can be packaged and transported in microfuge tubes, where they can
remain for microscopic analysis and DNA extraction.

Fingernail evidence can be an important part of an investigation in the event of a physical
assault when collected and analyzed properly. After an assault, fingernail evidence should be
collected so that exogenous DNA recovery is maximized. Maximizing exogenous DNA while
reducing the amount of endogenous DNA is extremely important for successful DNA profiling.
Implementing these improved methods for exogenous DNA recovery will increase the value of
fingernail evidence within the criminal justice system by making exogenous DNA recovery and
analysis more successful.

52

APPENDICES

53

APPENDIX A
DNA QUANTIFICATION DATA

Table A1. DNA recovered (pg/µL) from a 5 min soak in Tris/EDTA/SDS (n=12).
Sample Number
Total DNA (pg/µL)
Exogenous DNA (pg/µL)
3920
274
1
2560
162
2
977
194
3
1370
183
4
2150
805
5
958
791
6
4200
843
7
1830
949
8
1510
537
9
1570
574
10
1940
682
11
2010
329
12

Table A2. DNA recovered (pg/µL) from a 15 min soak in Tris/EDTA/SDS (n=12).
Sample Number
Total DNA (pg/µL)
Exogenous DNA (pg/µL)
2140
360
1
1130
323
2
814
45.4
3
341
129
4
350
189
5
395
184
6
376
203
7
1380
826
8
1510
721
9
942
851
10
420
148
11
1070
573
12

54

Table A3. DNA recovered (pg/µL) from a 30 min soak in Tris/EDTA/SDS (n=12).
Sample Number
Total DNA (pg/µL)
Exogenous DNA (pg/µL)
725
508
1
740
266
2
2190
897
3
2910
658
4
2830
666
5
1700
777
6
1810
902
7
1670
963
8
1090
871
9
9510
1160
10
4790
935
11
7240
641
12

Table A4. DNA recovered (pg/µL) from a 60 min soak in Tris/EDTA/SDS (n=12).
Sample Number
Total DNA (pg/µL)
Exogenous DNA (pg/µL)
543
122
1
937
151
2
570
302
3
608
302
4
726
317
5
623
226
6
581
195
7
814
398
8
140
44.1
9
4050
896
10
1310
973
11
1700
816
12

55

Table A5. Exogenous DNA concentrations (pg/µL) from fingernails containing 1 µL of male
blood, stored at room temperature for various time periods (n=21).
Exogenous DNA Concentration (pg/µL)
0 Days
2 Days
1 Week
2 Weeks
1 Month
3 Months
6 Months
474
------(+)
171
320
200
234
234
234
90
A
218
274
36
171
190
344
97
B
313
441
344
226
301
72
320
C

Table A6. Exogenous DNA concentrations (pg/µL) from fingernails containing 1 µL of male
blood, stored at room temperature for 1 week after allowing to dry for certain time periods
(n=15).
Exogenous DNA Concentration (pg/µL)
0 min
6 hr
24 hr
4370
--(+)
1980
3610
2160
A
2630
3410
3910
B
2120
1790
3520
C
3010
3130
3120
D
2000
3560
3760
E
Table A7. Exogenous DNA yields (pg) after a 15 min soak and extracted utilizing optimized kit
protocol (Hebda et al. 2014) or an organic extraction with RNA-pre-treated filters (n=14).
Exogenous DNA Yield (pg)
Kit Extraction
Organic Extraction
6660
10 080
1
1180
9040
2
6960
1270
3
6480
3610
4
3700
5290
5
2360
5150
6
4430
5680
7
Table A8. Exogenous DNA yields (pg) after a 60 min soak and extracted utilizing optimized kit
protocol (Hebda et al. 2014) or an organic extraction with RNA-pre-treated filters (n=14).
Exogenous DNA Yield (pg)
Kit Extraction
Organic Extraction
1480
3420
1
1370
4230
2
5160
8460
3
2080
8460
4
5830
8880
5
6120
6330
6
9120
5460
7
56

APPENDIX B
AUTOSOMAL STR PROFILES FROM BLOOD DEPOSITION ON FINGERNAILS
Autosomal STR profiles from fingernails containing blood. Italicized red numbers indicate alleles not consistent with the known
exogenous profile. An asterisk (*) indicates allelic dropout.
Table B1. Autosomal STR profiles of fingernails containing blood that underwent a 15 min soak and had varying digestion times
(n=7). Also includes the % of exogenous alleles called, as well as the % of loci that contain mixture (alleles that do not match the
exogenous DNA profile).
Locus
Digestion Time (h)
Exogenous
24A
24B
2A
2B
2C
1A
1B
Amel
X, Y
X, Y
X, Y
X, Y
X, Y
X, Y
X, Y
X, Y
13, 14, 15
13, 14, 15
13, 14, 15
13, 14, 15
13, 14, 15
D8
13, 14
13, 14
13, 14
28, 29, 30,
29, 30, 31,
28, 29, 30,
29, 30, 31,
28, 30, 31,
30, 31, 31.2,
31, 31.2,
31.2, 32.2,
31, 31.2,
D21
31, 32.2
32.2
31.2, 32.2
31.2, 32.2
32.2
33.2
32.2
32.2
8, 10, 11
8, 10, 11
8, 10, 11
8, 10, 11
8, 10, 11
D7
10, 11
10, 11
10, 11
10, 11, 12
10, 11, 12
10, 11, 12
10, 11, 12
10, 11, 12
CSF
11, 12
11, 12
11, 12
15, 16, 17,
15, 16, 17,
14, 15, 16
14, 15, 16
14, 15, 16
14, 15, 16
14, 15, 16
D3
15, 16
18
18
6, 7, 9.3
6, 7, 9.3
Tho
6, 7
6, 7
6, 7
6, 7
6, 7
6, 7
D13
12
12
12
12
12
12
12
12
9, 11, 12, 13
9, 11, 13
9, 11, 12
9, 11, 12, 13
9, 11, 13
D16
9, 11
9, 11
9, 11
22, 23, 24,
23, 24, 25
23, 24, 25
23, 24, 25
D2
23, 25
23, 25
23, 25
23, 25
25
13, 14, 15
13, 14, 15
13, 14, 15
13, 14, 15
13, 14, 15
13, 14, 15
D19
14, 15
14, 15
15, 16, 17,
15, 16, 17,
15, 16, 17,
15, 16, 17,
15, 16, 17,
15, 16, 17
15, 16, 17
vWA
15, 16
18
18
18
18
18
8, 10, 12
TPOX
8, 10
8, 10
8, 10
8, 10
8, 10
8, 10
8, 10
14, 15, 16,
14, 15, 16,
14, 15, 16,
14, 15, 16,
14, 15, 16,
D18
16, 18
16, 18
16, 18
18
18
18
18
18
11, 12, 13
11, 12, 13
D5
11, 12
11,12
11, 12
11, 12
11, 12
11, 12
57

Table B1 (cont’d)
FGA
23, 25
Exogenous Alleles
(% Called)
Mixture
(% Loci)

23, 25

22, 23, 25

22, 23, 25

18, 23, 25

23, 25

22, 23, 25

22, 23, 25

97

100

100

100

100

100

100

44

69

69

56

38

63

69

58

Table B2. Autosomal STR profiles for various exogenous: endogenous DNA concentration ratios (listed below each sample), utilizing
a 5 min soak period and a 2 h digestion (n=4). Also includes the % of exogenous alleles called, as well as the % of loci that contain
mixture (alleles that do not match the known exogenous DNA profile).
Locus
Sample
Exogenous
A
B
C
D
DNA
Amel
X, Y
X, Y
X, Y
X, Y
X, Y
12, 13, 14, 15
10, 12, 13, 14
10, 12, 13, 14
10, 12, 13, 14
D8
13, 14
28, 30, 31, 32.2
31, 31.2, 32.2
30, 30.2, 31, 32.2
30, 30.2, 31, 32.2
D21
31, 32.2
10,
11,
12
10,
11,
12
D7
10,11
10, 11
10, 11
10, 11, 12
11, 12, 13
10, 11, 12
CSF
11, 12
11, 12
14, 15, 16
14, 15, 16
14, 15, 16
D3
15, 16
15, 16
6, 7, 9.3
6, 7, 9.3
6, 7, 9.3
6, 7, 9.3
Tho
6, 7
12, 13, 14
9, 12, 13
9, 12
9, 12
D13
12
9, 11, 13
9, 11, 12
9, 11, 12
D16
9, 11
9, 11
17, 23, 25
20, 23, 25
19, 23, 24, 25
19, 23, 24, 25
D2
23, 25
10.2,
13,
14,
15
10.2,
14,
15,
15.2
D19
14, 15
14, 15
14, 15
15, 16, 17, 18
14, 15, 16, 18
14, 15, 16
14, 15, 16
vWA
15, 16
8, 10, 11
8, 9, 10
8, 10, 11
8, 10, 11
TPOX
8, 10
12, 16, 17, 18
13, 14, 16, 18
13, 16, 17, 18
13, 16, 17, 18
D18
16, 18
9, 11, 12
10, 11, 12
10, 11, 12
10, 11, 12
D5
11, 12
23, 24, 25
20, 23, 24, 25
20, 23, 25
20, 23, 25
FGA
23, 25
Ratio of endogenous
15:1
5:1
1.25:1
4:1
DNA: exogenous DNA
Exogenous Alleles
100
100
100
97
(% Called)
Mixture (% Loci)

88

100

59

81

100

Table B3. Autosomal STR profiles for various exogenous: endogenous DNA concentration ratios (listed below each sample), utilizing
a 15 min soak period and a 2 h digestion (n=3). Also includes the % of exogenous alleles called, as well as the % of loci that contain
mixture (alleles that do not match the known exogenous DNA profile).
Locus
Sample
Exogenous DNA
A
B
C
Amel
X, Y
X, Y
X, Y
X, Y
10, 12, 13, 14
10, 13, 14
D8
13, 14
13, 14
31, 31.2, 32.2
30.2, 31, 32.2
D21
31, 32.2
31, 32.2
10, 11, 12
8, 9, 10, 11
D7
10,11
10, 11
11, 12, 13
10, 11, 12
CSF
11, 12
11, 12
14, 15, 16
15, 16, 17
14, 15, 16
D3
15, 16
7, 9.3
6, 7, 9.3
Tho
6, 7
6, 7
9, 12, 13
9, 12
D13
12
12
9,
11,
12,
13
9,
11,
12
D16
9, 11
9, 11
20, 23, 24, 25
19, 23, 24, 25
D2
23, 25
23, 25
14, 15, 15.2
14, 15, 16
D19
14, 15
14, 15
15, 16, 18
15, 16, 18, 19
vWA
15, 16
15, 16
8, 9, 10
8, 10, 11
8, 10, 11
TPOX
8, 10
13, 14, 16, 18
12, 14, 16, 18
D18
16, 18
16, 18
10, 11, 12
D5
11, 12
11, 12
11, 12
20,
23,
24,
25
22,
23,
25
20,
23, 25
FGA
23, 25
Ratio of endogenous DNA:
6.7:1
20:1
1:1
exogenous DNA
Exogenous Alleles (% Called)
97
100
100
Mixture (% Loci)
100
69
50

60

Table B4. Autosomal STR profiles for various exogenous: endogenous DNA concentration ratios (listed below each sample), utilizing
a 30 min soak period and a 2 h digestion (n=4). Also includes the % of exogenous alleles called, as well as the % of loci that contain
mixture (alleles that do not match the known exogenous DNA profile).
Locus
Sample
Exogenous
A
B
C
D
DNA
Amel
X, Y
X, Y
X, Y
X, Y
X, Y
10, 13, 14
10, 12, 13, 14
10, 13, 14
10, 13, 14
D8
13, 14
30,
31,
32.2
30,
30.2,
31,
32.2
30,
30.2, 31, 32.2
D21
31, 32.2
31, 32.2
D7
10,11
10, 11
10, 11
10, 11
10, 11
CSF
11, 12
11, 12
11, 12
11, 12
11, 12
15, 16, 17
14, 15, 16
14, 15, 16
D3
15, 16
15, 16
6, 7, 9.3
6, 7, 9.3
Tho
6, 7
6, 7
6, 7
11, 12
9, 12
9, 12
9, 12
D13
12
9, 11, 12
9, 11, 12
9, 11, 12
D16
9, 11
9, 11
19,
23,
25
19,
23,
24,
25
19,
23, 24, 25
D2
23, 25
23, 25
12, 14, 15
D19
14, 15
14, 15
14, 15
14, 15
15, 16, 17
14, 15, 16
14, 15, 16
vWA
15, 16
15, 16
8, 10, 11
8, 10, 11
8, 10, 11
TPOX
8, 10
8, 10
13, 16, 17, 18
13, 16, 17, 18
13, 16, 17, 18
D18
16, 18
16, 18
10, 11, 12
10, 11, 12
D5
11, 12
11, 12
11, 12
23, 24, 25
20, 23, 25
20, 23, 25
20, 23, 25
FGA
23, 25
Ratio of endogenous
2:1
1:1
9:1
5:1
DNA: exogenous DNA
Exogenous Alleles
100
100
100
100
(% Called)
Mixture (% Loci)
44
56
81
81

61

Table B5. Autosomal STR profiles for various exogenous: endogenous DNA concentration ratios (listed below each sample), utilizing
a 60 min soak period and a 2 h digestion (n=4). Also includes the % of exogenous alleles called, as well as the % of loci that contain
mixture (alleles that do not match the known exogenous DNA profile).
Locus
Sample
Exogenous
A
B
C
D
DNA
Amel
X, Y
X, Y
X, Y
X, Y
X, Y
10, 13, 14, 15
D8
13, 14
13, 14
13, 14
13, 14
28,
31,
32.2
28,
30,
31,
32.2
30,
31,
31.2,
32.2
D21
31, 32.2
31, 32.2
10, 11, 12
D7
10,11
10, 11
10, 11
10, 11
10, 11, 12
10, 11, 12
CSF
11, 12
11, 12
11, 12
15, 16, 17
15, 16, 17, 18
D3
15, 16
15, 16
15, 16
6, 7, 9
6, 7, 9.3
6, 7, 9.3
6, 7, 9.3
Tho
6, 7
11, 12
12, 13, 14
12, 13
D13
12
12
9, 11, 12, 13
9, 11, 12
D16
9, 11
9, 11
9, 11
17,
22,
23,
25
22,
23,
25
17,
23,
24,
25
17,
23, 25
D2
23, 25
12, 14, 15
13, 14, 15
13, 14, 15
13, 14, 15
D19
14, 15
15, 16, 17
15, 16, 17
15, 16, 17, 18
15, 16, 17, 18
vWA
15, 16
8, 10, 11
8, 10, 12
8, 10, 11
TPOX
8, 10
8, 10
13, 15, 16, 18
15, 16, 18, 21
12, 16, 17, 18
12, 16, 17, 18
D18
16, 18
11, 12, 13
9, 10, 11, 12
9, 11, 12
D5
11, 12
11, 12
21, 22, 23, 25
18, 23, 25
23, 24, 25
23, 24, 25
FGA
23, 25
Ratio of endogenous
5:1
3:1
2:1
1:1
DNA: exogenous DNA
Exogenous Alleles
100
100
100
100
(% Called)
Mixture
88
81
69
63
(% Loci)

62

APPENDIX C
STR PROFILES FROM SCRATCHINGS
Autosomal and Y-STR profiles of epithelial cells from scratchings. Italicized red numbers indicate alleles consistent with the known
endogenous profile. Italicized green numbers indicate alleles not consistent with known endogenous or exogenous DNA. Numbers in
parentheses were present but fell below the 150 RFU threshold. An asterisk (*) indicates allelic dropout.

Table C1. Autosomal STR profiles from the 18 scratchings that contained exogenous DNA (n=11). Samples not containing
exogenous alleles were not included within the table. The % of exogenous alleles called, as well as the % of loci that contain mixture
(alleles that do not match the known exogenous DNA profile) are included. Each profile comes from exogenous DNA collected from
a separate fingernail.
Locus
1
5
6
8
9
10
11
13
15
16
17
Amel
X, Y
X, Y
X, Y
X, Y
X, Y
X, Y
X, Y
X, Y
X, Y
X, Y
X, Y
10, 12,
10, 12,
10, 13,
10, 12,
8, 13,
8, 13,
8, 13,
10, 13,
10, 14, 10, 13,
10, 13, 14
D8
14, 15 14, 15
14, 15
14
13
14
13, 14
13, 14
14
13, 14
30,
30,
30,
30, 30.2,
28, 29,
28, 29,
28, 29,
28, 29,
30,
30,
30.2,
D21
29, 31
33.2
33.2
30.2
31
30.2
31.2
30, 31.2
31.2
30, 31.2
31.2
9, 10,
8, 9, 11,
10, 12
10
10
10 (11)
10
8, 11, 12
8, 11
8,11, 12
D7
9, 12
12
12
9, 10,
10, 11,
10, 11,
10, 11,
9, 10
10, 11
CSF
10, 12
11, 12
11, 12
11, 12
11, 12
12, 13
12, 13
12
12
15, 16, 15, 16, 15, 16, 14, 15,
14, 15, 14, 15,
14, 15,
14, 15,
14, 15,
14, 15,
14, 15
D3
17, 18 17, 18
16, 17
18
16
17
16
16
16
16
5.3,
5.3,
5.3,
6, 9,
6, 9.3
6, 9.3
6, 9.3
6, 7, 9.3 6, 7, 9.3
6, 7, 9.3
Tho
6, 9.3
7.3, 10 7.3, 9.3
9.3
9.3
9, 10,
9, 10,
9, 10,
8, 9,
8, 9,
8, 9, 11, 8, 9, 12, 8, 9, 12,
8, 9, 12,
9, 11,
D13
11
11
11, 12
11, 12
11, 12
11, 12
12
12, 14
14
14
14
D16

9, 10,
12, 13

9, 10,
12

9, 13

11, 12

11, 12

11, 12

63

11, 12

11, 12,
13

11, 12,
13

11, 12,
13

11, 13

Table C1 (cont’d)

13, 14,
15, 16.2
16, 17,
18, 19
8, 11
12, 13,
14, 17

17, 20,
21, 23,
25
13, 14,
15, 16.2
17, 18,
19
8, 11
12, 13,
14, 17

17, 20,
21, 23,
25
13, 14,
15, 16.2
17, 18,
19
8, 11
12, 13,
14, 17

13, 14,
15, 16.2
17, 18,
19
8, 11
12, 13,
14, 17

10, 12

11, 12

9, 11, 12

11, 12

11, 12

20

20

20, 21,
22, 25

20, 21,
22, 25

20, 21,
22

20, 21,
22, 25

72

79

69

90

100

80

80

63

69

75

93

87

87

93

D2

17, 18

17, 18

20, 25

17, 19,
24

17, 19, 24

17, 19,
24

17, 19,
24

D19

13, 15

13, 15

13, 15

13, 14

13, 14

13, 14

14, 16

vWA

16

TPOX

8, 11

16, 17,
18
8, 11

16, 17,
18
8, 11

14, 16,
17, 18
8, 11

12

12

13, 15

D5

10, 11,
12

10, 11,
12, 13

10, 11,
12, 13

14, 16,
17, 18
8, 11
13, 17,
(18)
10, 12,
13

14, 16,
17
8, 11

D18

14, 16,
17, 18
8, 11
13, 17
(18)
10, 12,
13

FGA

20

20, 23

20, 21,
23

20

18.2,
20.2, 31,
32

52

68

97

79

88

88

50

75

Exogenous
Alleles
(% Called)
Mixture
(% Loci)

13, 17, 18
10, 12, 13

64

13, 17

17, 20,
23, 25

17, 20,
23, 25

Table C2. Y-STR profiles from the 18 scratchings (n=18). Sample number correlates with the sample number from Table 1C (above).
Includes profiles from scratchings that quantified at 0 pg/µL and samples that did not contain any exogenous alleles in the autosomal
profile. Includes the % of exogenous alleles called.
Locus
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
15,
15,
14,
456
*
*
*
16
16
16
15
15
15
15
*
15
*
*
*
16
16
16
13,
13
389I
13
*
*
13
13
13
14
14
13
14
13
*
13
*
13
13
14
22,
23,
20
390
24
*
24
24
*
23
*
23
23
23
*
23
*
23
*
24
24
29, 29,
29,
29
389II
30
*
30
30
30
30
29
29
29
*
29
29
29
*
31
30
30
17,
17
17
458
15
*
*
*
15
15
18
17
17
17
*
17
*
17
*
18
13, 13, 14,
14
19
15
*
*
15
15
15
*
13
14
14
14
14
*
14
14
14
15
11,
11,
11,
11,
11, 11, 13,
11, 11,
*,
*,
11, 13, 11,
11, 11, 11, 11, 11,
385
13,
14,
*, *
14
*
14
14
14
14,
13
*
15
14,
14
14
*
14
*
14
15
15
15
12,
13,
12,
393
*
*
*
13
13
13
13
13
13
13
*
13
*
13
*
13
14
13
10,
9,
9,
10,
10
391
11
11
11
11
11
9
11
*
11
11
11
11
*
10
11
10
11
10,
10,
12
439
11,
*
10
10
10
10
11
12
12
12
12
*
12
*
12
12
12
12
23,
21, 21, 21,
21,
26
635
*
*
*
25
25
*
23
*
23
23
23
*
26
25
26
26
26
11,
11, 11,
13
12
392
*
*
*
11
11
11
13,
13
*
*
13
*
13
*
13
12
14
65

Table C2 (cont’d)
12,
GATA H4
13

12

12

12

12

12

12,
13

8,
13

8,
13

12,
13
14

437

14

*

14

*

*

14

14

14

14

438

11

*

*

10

11

11

10,
12

10,
12

448

20

*

20

*

*

20

20

20

10,
12
19,
20,
21

Exogenous
Alleles
(% Called)

94

10

47

47

88

100

100

76

65

66

12

12
14,
15
10,
12

13

13

*

13

*

*

*

14

*

*

15

*

*

*

12

12

*

12

*

12

*

19

19

19

18

*

18

*

18

18

29

65

41

71

24

100

29

76

29

APPENDIX D
STR PROFILES OF TRANSPORTED SCRATCHINGS IN COIN ENVELOPES, BINDLE PAPERS, AND PLASTIC
MICROFUGE TUBES
Autosomal and Y-STR profiles of epithelial cells from scratchings. Italicized red numbers indicate alleles consistent with the known
endogenous profile. Italicized green numbers indicate alleles not consistent with known endogenous and exogenous DNA. An asterisk
(*) indicates allelic dropout. Dashes indicate there was not sufficient exogenous DNA quantifications, so samples were not analyzed.
OL indicates that there were off-ladder alleles present.

Table D1. Autosomal STR profiles from fingernail clippings transported in coin envelopes (n=15). Includes the % of exogenous
alleles called, as well as the % of loci that contain mixture (alleles that are not consistent with the exogenous DNA profile). Dashes
indicate there were not sufficient exogenous DNA quantifications, so samples were not analyzed.
Locus
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15
-- -- -- -- -- -- -- -- -- -- -- -Amel
X, Y
X, Y
X, Y
12, 13, 15
12, 13, 15
8, 10, 13, 15
-- -- -- -- -- -- -- -- -- -- -- -D8
29, 30, 32.2
28, 29, 30, 32.2 28, 29, 31, 33.2 -- -- -- -- -- -- -- -- -- -- -- -D21
8, 10, 12
8, 10, 11, 12
9, 10, 12
-- -- -- -- -- -- -- -- -- -- -- -D7
10, 12
10, 12
-- -- -- -- -- -- -- -- -- -- -- -CSF
10, 12
15, 16, 17
15, 16, 17
14, 16, 18
-- -- -- -- -- -- -- -- -- -- -- -D3
6, 8, 9.3
-- -- -- -- -- -- -- -- -- -- -- -Tho
7, 9.3
7, 9.3
8, 10, 13, 14
8, 10, 13, 14
-- -- -- -- -- -- -- -- -- -- -- -D13
10, 12
9, 11, 13
-- -- -- -- -- -- -- -- -- -- -- -D16
11
11
17, 25, 26
17, 25, 26
18, 20, 22, 25
-- -- -- -- -- -- -- -- -- -- -- -D2
13, 15.2
13, 14, 15.2
14, 15, 16
-- -- -- -- -- -- -- -- -- -- -- -D19
16, 17, 18
16, 17, 18
16, 17, 18
-- -- -- -- -- -- -- -- -- -- -- -vWA
8, 12
-- -- -- -- -- -- -- -- -- -- -- -TPOX
8, 11
8, 11
12, 14, 17, 18
12, 14, 17, 18
12, 13, 15
-- -- -- -- -- -- -- -- -- -- -- -D18
9, 12, 13
9, 12, 13
11, 12, 13
-- -- -- -- -- -- -- -- -- -- -- -D5
19, 21, 23, 24
19, 21, 23, 24
-- -- -- -- -- -- -- -- -- -- -- -FGA
21, 23
67

Table D1 (cont’d)
Exogenous
Alleles
(% Called)
Mixture
(% Loci)

90

97

94

--

--

--

--

--

--

--

--

--

--

--

--

75

75

75

--

--

--

--

--

--

--

--

--

--

--

--

68

Table D2. Y- STR profiles from fingernail clippings transported in coin envelopes (n=15). Includes the % of exogenous alleles called.
Locus
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16,
*, 17
456
16
16
16
16
*
16
16
16
*
16
16
16
16
18
389I
13
13
13
*
12
*
13
13
*
13
13
13
13
13
13
*, 25
390
24
24
24
*
*
24
24
24
*
24
24
24
*
25
389II
29
29
30
*
*
30
30
*
*
30
30
30
30
*
29
458
16
16
15
*
*
15
15
15
*
15
15
15
15
*
15
13,
19
14
14
15
*
*
15
15
15
*
*
15
15
*
15
15
11,
11,
11,
11,
11,
11,
11,
11,
11,
11,
14,
385
*,*
*,*
*,*
11, *
*,*
14
14
14
14
14
18
14
14
14
18
393
13
13
13
*
*
*
13
*
*
13
13
13
13
*
13
10,
10, *
391
11
10
11
*
*
11
11
11
*
11
11
11
10
11
10,
439
*
12
10
*
*
10
10
10
*
10
10
*
*
10
11
635
23
23
25
*
*
*
25
*
*
21
25
25
*
*
23
11,
392
13
11
*
*
*
11
*
*
11
11
11
*
*
11
13
*, 13
GATA H4
12
12
12
*
*
12
12
*
12
12
*
12
12
13
437
*
15
14
*
*
*
14
*
*
*
14
14
*
*
14
10,
438
12
11
11
*
11
11
*
*
10
11
11
11
*
11
12
448
*
19
20
*
*
20
20
*
*
20
20
20
20
*
20
Exogenous
Alleles
82
100
100
12
100
71
100
53
6
94
82
100
59
18
100
(% Called)

69

Table D3. Autosomal STR profiles from fingernail clippings transported in bindle papers (n=15). The % of exogenous alleles called,
as well as the % of loci that contain mixture (alleles that do not match the known exogenous DNA profile) are included. Dashes
indicate there were not sufficient exogenous DNA quantifications, so samples were not analyzed.
Locus
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
---------------Amel
---------------D8
---------------D21
---------------D7
---------------CSF
---------------D3
---------------Tho
---------------D13
---------------D16
---------------D2
---------------D19
---------------vWA
---------------TPOX
---------------D18
---------------D5
---------------FGA
Exogenous
---------------Alleles
(% Called)
Mixture
---------------(% Loci)

70

Table D4. Y- STR profiles from fingernail clippings transported in bindle papers (n=15). Includes the % of exogenous alleles called.
Locus
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
456
*
*
16
*
*
16
16
16
*
*
16
*
16
16
16
389I
*
*
*
*
*
13
13
*
*
*
*
13
13
13
13
390
*
*
24
*
*
24
24
*
24
24
*
*
24
*
25
389II
*
*
*
*
*
30
30
*
*
*
*
30
30
30
29
458
*
*
*
*
*
15
15
*
*
*
15
*
15
15
15
19
14
*
*
*
*
15
15
*
*
*
*
15
*
*
15
11,
11,
11,
11, *, 12, 11,
11,
11,
385
*,*
*,*
14,
*, 14
*,*
11, *
*,*
*,*
14
14
14
14
14
14
14
18
393
*
*
*
*
*
13
13
*
*
*
13
*
13
13
13
391
10
*
11
11
*
11
11
10
11
*
*
11
11
11
10
439
*
*
*
*
*
10
10
*
*
11
10
10
10
10
10
635
*
*
*
*
*
25
25
*
*
*
*
*
25
25
23
392
13
*
11
*
*
11
11
11
11
*
*
11
11
*
11
GATA H4
12
*
*
*
*
12
12
*
*
12
*
12
12
12
13
437
*
*
*
*
*
14
14
*
*
*
14
14
14
14
14
9, *
438
*
*
*
*
*
11
11
*
*
*
*
11
*
11
448
*
*
*
*
*
20
20
*
*
*
20
*
20
20
20
Exogenous
Alleles
24
0
35
12
0
100
100
24
18
18
47
59
88
76
100
(% Called)

71

Table D5. Autosomal STR profiles from fingernail clippings transported in microfuge tubes, with fingernail removed before
processing (n=15). Includes the % of exogenous alleles called, as well as the % of loci that contain mixture (alleles not consistent with
the exogenous DNA profile). Dashes indicate there were not sufficient exogenous DNA quantifications, so samples were not analyzed.
Locus
1
2 3
4
5
6
7
8
9
10 11 12 13 14 15
- -- -- -- -- -- -Amel
X, Y
X, Y
X, Y
X, Y
X, Y
X, Y
- - 12, 13, 15
12, 15
10, 14, 15 10, 14, 15
10, 15
10, 14, 15 -- -- -- -- -- -D8
- 28, 29, 30, - 27.2,
28, 30.2,
28, 30.2,
28, 33.2
-- -- -- -- -- -D21
28, 30.2
- 29.2
33.2
33.2
32.2
8, 10, 11, - 9.2, OL
9, 10
9, 10
9, 10
-- -- -- -- -- -D7
9
12
- - 10, 12
OL
10, 12
8, 10, 12
-- -- -- -- -- -CSF
8, 10
8, 10
- - - 14, 15, 16, 14, 15, 16,
14, 15, 16,
15, 16, 17
15, 16
14, 16
-- -- -- -- -- -D3
- 17, 18
18
18
- 6.3, 9.3
6, 7, 9.3
6, 7, 9.3
6, 9.3
6, 7, 9.3
-- -- -- -- -- -Tho
7, 9.3
- 8, 10, 13, - 8, 10, 12,
8, 10, 12,
13, 14
8, 12, 13
10, 12
-- -- -- -- -- -D13
14
- 13
13
- 9, 11, 12,
9, 11, 12,
10
9, 11, 13
-- -- -- -- -- -D16
11
9, 12
- 13
13
- 17, 18, 19,
17, 25, 26
17, 25
18, 22
-- -- -- -- -- -D2
17, 19
17, 19
- 22
13, 14,
- 12,
14, 14.2,
14, 14.2,
14, 14.2,
14, 15
-- -- -- -- -- -D19
15.2
- 14.2
15
15
15
- 16, 17, 18
17, 18
-- -- -- -- -- -vWA
17
17
17
17
- - 8, 12
8, 12
8, 12
-- -- -- -- -- -TPOX
8, 11
8, 11
8
- D18

12, 14, 17,
18

-

-

12,
17.2

-

17, 18

17, 18

72

12

12, 17, 18

--

--

--

--

--

--

Table D5 (cont’d)
-

-

100

-

-

33

80

-

-

87

D5

9, 12, 13

FGA

19, 21, 23,
24

Exogenous
Alleles
(% Called)
Mixture
(% Loci)

9, 12
23.2,
24.2

-

9, 12

9, 12

9, 12, 13

9, 11, 12,
13

--

--

--

--

--

--

23, 24

23, 24

21, 23

21, 23, 24

--

--

--

--

--

--

-

100

100

48

100

--

--

--

--

--

--

-

33

60

93

93

--

--

--

--

--

--

73

Table D6. Y- STR profiles from fingernail clippings transported in microfuge tubes (n=15). Includes the % of exogenous alleles
called.
Locus
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
456
16
16
*
*
*
16
16
16
16
*
*
16
16
16
16
389I
13
13
*
*
*
13
13
13
13
*
*
*
13
13
*
390
24
24
*
*
*
24
24
24
24
*
*
24
24
24
*
389II
29
29
*
*
*
29
29
29
29
*
*
30
30
30
*
458
16
16
*
*
17
17
17
17
17
*
*
15
15
15
*
19
14
14
*
*
*
14
14
14
14
*
*
15
15
15
*
11,
11,
11,
11,
11,
11,
11,
11,
11,
385
*,*
*,*
*,*
*,*
*,*
*,*
14
14
14
14
14
14
14
14
14
393
13
13
*
*
*
13
13
13
13
*
*
13
13
13
*
391
10
10
*
*
*
11
11
11
11
*
11
11
11
11
*
10,
439
12
*
*
*
12
12
12
12
*
10
10
10
10
*
12
635
23
23
*
*
*
23
23
23
23
*
*
25
25
25
*
392
13
13
*
*
*
13
13
13
13
*
*
11
11
11
*
GATA H4
12
12
*
*
*
13
13
13
13
*
*
12
12
12
*
437
15
15
*
15
*
15
15
15
15
*
14
*
14
14
*
438
12
*
*
*
*
12
12
12
12
*
*
11
11
11
*
*, 23
448
19
*
*
*
*
18
18
18
18
*
*
*
*
20
Exogenous
Alleles
100
88
0
6
6
100
100
100
100
0
18
82
94
100
6
(% called)

74

APPENDIX E
Y-STR PROFILES FOR DNA STORAGE AND DEGRADATION

Table E1. Y-STR profiles of exogenous DNA recovered from fingernail clippings containing
blood after storage at room temperature for various time periods (n=7). Exogenous alleles were
present for all samples.
0
2
1
2
1
3
6
Locus Exogenous
days
days
week
weeks
month months months
16
16
16
16
16
16
16
16
456
13
13
13
13
13
13
13
13
389I
25
25
25
25
25
25
25
25
390
29
29
29
29
29
29
29
29
389II
15
15
15
15
15
15
15
15
458
15
15
15
15
15
15
15
15
19
11, 14
11, 14
11, 14
11, 14
11, 14
11, 14
11, 14
11, 14
385
13
13
13
13
13
13
13
13
393
10
10
10
10
10
10
10
10
391
10
10
10
10
10
10
10
10
439
23
23
23
23
23
23
23
23
635
11
11
11
11
11
11
11
11
392
GATA
13
13
13
13
13
13
13
13
H4
14
14
14
14
14
14
14
14
437
11
11
11
11
11
11
11
11
438
20
20
20
20
20
20
20
20
448

75

REFERENCES

76

REFERENCES

Alaska Scientific Crime Detection Laboratory. Forensic biology procedures manual. 2012.
Allouche M, Hamdoum M, Mangin P, Castella V. 2008. Genetic identification of decomposed
cadavers using nails as DNA source. Forensic Sci Int-Gen. 3: 46 – 49.
Ballantyne KN, Goedbloed M, Fang R, Schaap O, Lao O, Wollstein A, Choi Y, van Duijn K,
Vermeulen M, Brauer S, Decorte R, Poetsch M, von Wurmb-Schwark N, de Knijff P,
Labuda D, Vézina H, Knoblauch H, Lessig R, Roewer L, Ploski R, Dobosz T, Henke L,
Henke J, Furtado MR, Kayser M. 2010. Mutability of Y-chromosomal microsatellites:
rates, characteristics, molecular bases, and forensic implications. Am J Hum Genet. 87:
341 – 353.
Bengtsson CF, Olsen ME, Ørsted Brandt L, Bertelsen MF, Willerslev E, Tobin DJ, Wilson AS,
Gilbert MTP. 2011. DNA from keratinous tissue. Part I: hair and nail. Ann Ant. 194: 17 –
25.
Bille TW, Cromartie C, Farr M. 2009. Effects of cyanoacrylate fuming, time after recovery, and
location of biological material on the recovery and analysis of DNA from post-blast pipe
bomb fragments, J. Forensic Sci. 54: 1059 – 1067.
Budowle B, Moretti TR, Niezgoda SJ, Brown BL. (1998, June). CODIS and PCR-based short
tandem repeat loci: law enforcement tools. Proceedings of the Second European
Symposium on Human Identification, Innsbruck, Austria.
Cline RE, Laurent NM, Foran DR. 2003. The fingernails of Mary Sullivan: developing reliable
methods for selectively isolating endogenous and exogenous DNA from evidence. J
Forensic Sci. 48(2): 1 – 6.
Comey CT, Koons BW, Presley KW, Smerick JB, Sobieralski CA, Stanley DM, Baechtel FS.
1994. DNA extraction strategies for amplified fragment length polymorphism analysis. J
Forensic Sci. 39(5): 1254 – 1269.
Cook O, Dixon L. 2006. The prevalence of mixed DNA profiles in fingernail samples taken from
individuals in the general population. Forensic Sci Int-Gen. 1: 62 – 68.
Doran AE and Foran DR. 2014. Assessment and mitigation of DNA loss utilizing centrifugal
filtration devices. Forensic Sci Int-Gen. 13: 187 – 190.
Edson SM, Ross JP, Coble MD, Parsons TJ, Barritt SM. 2004. Naming the dead—confronting
the realities of rapid identification of degraded skeletal remains. Forensic Sci. Rev. 16: 64
– 90.

77

Federal Bureau of Investigation (FBI). Forensic science communications: trace evidence
recovery guidelines. 1999. 1(3).
Federal Bureau of Investigation (FBI). 2012 Uniform Crime Report. Website:
http://www.fbi.gov/about-us/cjis/ucr. Accessed March 6, 2014.
Greenspoon SA, Scarpetta MA, Drayton ML, Turek SA. 1998. QIAamp spin columns as a
method of DNA isolation for forensic casework. J Forensic Sci. 43 (5): 1024 – 1030.
Gross AM, Liberty AA, Ulland MM, Kuriger JK. 2008. Internal validation of the AmpFlSTR
Yfiler™ amplification kit for use in forensic casework. J Forensic Sci. 53 (1): 125 – 134.
Harbison SA, Petricevic SF, Vintiner SK. 2003. The persistence of DNA under fingernails
following submersion in water. Int Congr Ser. 1239: 809 – 813.
Hebda LM, Doran AE, Foran DR. 2014. Collecting and analyzing DNA evidence from
fingernails: a comparative study. J Forensic Sci. 59 (5): 1343 – 1350.
Kayser M, de Knijff P. 2011. Improving human forensics through advances in genetics,
genomics, and molecular biology. Nat Rev Genet. 12: 179 – 192.
Kuman AM. 2013. Bio-Resource. http://1.bp.blogspot.com/y2ZyenfQow0/UZpShbDEJQI/AAAAAAAAB8/jxO4F5M7e2I/s640/TaqMan+GX+cartoon.jpg
Life Technologies Corporation. 2012. AmpFlSTR® Identifiler® PCR Amplification Kit User
Guide. Carlsbad, CA.
Life Technologies Corporation. 2012. Quantifiler Kits User’s Manual. Carlsbad, CA.
Matte M, Williams L, Frappier R, Newman J. 2011. Prevalence and persistence of foreign DNA
beneath fingernails. Forensic Sci Int-Gen. 6: 236 – 243.
Melzak KA, Sherwood CS, Turner RFB, Haynes CA. 1996. Driving forces for DNA adsorption
to silica in perchlorate solutions. J Colloid Interf Sci. 181 (0421): 635 – 644.
Mohan Banik R, Prakash M. 2004. Laundry detergent compatibility of the alkaline protease from
Bacillus cereus. Microbiol Res. 159: 135 – 140.
Mótyán JA, Tóth F, Tözsér J. 2013. Research applications of proteolytic enzymes in molecular
biology. Biomolecules. 3: 923 – 942.
Prinz M, Boll K, Baum H, Shaler R. 1997. Multiplexing of Y-chromosome specific STRs and
performance for mixed samples. Forensic Sci Int. 85: 209 – 218.
QIAGEN. 2012. QIAamp DNA Investigator Handbook. Germany.
78

Roewer L. 2009. Y chromosome STR typing in crime casework. Forensic Sci Med Pathol. 5: 77
– 84.
Seidel A. 2013. Ask Ana: See through nails—can I fix it? http://www.nailcarehq.com/seethrough-nails/
U.S. Department of Justice. Federal Bureau of Investigation: Laboratory Division. Handbook of
forensic services. 2011.
U.S. Department of Justice. A national protocol for sexual assault medical forensic examinations.
2013.
Wallin JM, Buonchristiani M, Lazaruk KD, Fildes N, Holt CL, Walsh PS. 1998. TWGDAM
validation of the AmpFISTR Blue PCR amplification kit for forensic casework. J
Forensic Sci. 43: 854 – 870.
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 – 450.
Yoshida-Yamamato S, Nishimura S, Okuno T, Rakuman M, Takii Y. 2010. Efficient DNA
extraction from nail clippings using the protease solution from Cucumis melo. Mol
Biotechnol. 46: 41 – 48.

79