C H A P T E R

5
STR Profiles
Multiplex PCR, Tri-Alleles, Amelogenin,
and Partial Profiles

“Knowledge becomes serviceable only when it is used.”

John Widtsoe

INTRODUCTION
Up to this point in the book we have been focusing on information derived from a single locus.
Multiplex polymerase chain reaction (PCR) enables examination of many loci simultaneously, and
this multi-locus genotyping information can increase interpretation capabilities beyond what is avail-
able from a single locus. Short tandem repeat (STR) profiles can be illustrated with simple triangles,
line drawings, or more sophisticated tools for illustrating electropherograms (D.N.A. Box 5.1).
Copyright © 2014. Elsevier Science & Technology. All rights reserved.

D.N.A. BOX 5.1

TOOLS TO CREATE EXAMPLE

ELECTROPHEROGRAM DATA
To create example electropherograms for that enable DNA electropherograms to be
teaching purposes, triangles have been used to created with specific alleles and peak heights (e.g.
illustrate allele, genotype, or profile peaks. Simple Figure 1.5).
line drawings can also be made using PowerPoint These programs are freely available on the
or other graphical tools as have been done in this NIST STRBase website: EPG Maker program
book and previous editions of Forensic DNA from Steven Myers: http://www.cstl.nist.gov/
Typing. However, new tools have been developed strbase/tools/EPG-Maker(SPMv.3,Dec2-2011).xlt
to create more realistic electropherogram data. (13 Mb Excel file)
Steven Myers (California Department of Justice, EPG Maker program from Jo Bright: http://
Richmond, CA) and Jo Bright (ESR, Auckland, www.cstl.nist.gov/strbase/tools/Bright-scaler.
New Zealand) have prepared some Excel macros xlsx (113 kb Excel worksheet)

Advanced Topics in Forensic DNA Typing: Interpretation

http://dx.doi.org/10.1016/B978-0-12-405213-0.00005-1 109 2015 Published by Elsevier Inc.
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 110 5. STR PROFILES

This chapter addresses single-source samples. An understanding of expected behavior of

single-source DNA samples is important prior to attempting DNA mixtures containing multiple
contributors, which are covered in Chapter 6.
Much can be learned during data interpretation by looking at the full profile. Did signal bleed-
through lead to pull-up between dye channels? Viewing data as an overlap of dye colors can help
assess this situation. Is there loss of signal with longer-sized PCR products due to DNA degradation
or PCR inhibition? A comparison of peak heights between the shortest- and longest-size amplicons
within a color channel will provide helpful clues. A “ski slope” with the smallest loci on the left side
of the electropherogram producing significantly higher peak heights compared to the larger loci on
the right side of the electropherogram suggests that PCR amplification has been compromised due to
lack of target molecules from DNA degradation or lack of polymerase power due to PCR inhibition.

MULTIPLEX PCR CHALLENGES

There are a number of advantages of multiplex PCR, where more than one location is targeted and
copied during the polymerase chain reaction. Multiplex PCR enables multiple regions of DNA to be
examined from a small amount of original starting material.
For amplicons to be obtained from the targeted regions of DNA, PCR primer pairs need to be
compatible with similar annealing temperatures. In addition, excessive regions of complementarity
among the primers in the multiplex PCR reaction need to be avoided to prevent the formation of
primer dimers that will reduce or prevent amplification of the desired target region. Sometimes
mobility modifiers are added to specific primers and used to adjust overall PCR product sizes in
order to maintain PCR primer positions with loci under different configurations in various STR
kits (see Chapter 4 in Butler 2012).
Having primers that possess similar annealing temperatures and little-to-no complementarity
among primers in the PCR reaction mix is only a starting point in generating effective STR profiles.
In order to produce a balanced STR profile (such as illustrated in Figure 1.5), primer concentrations
Copyright © 2014. Elsevier Science & Technology. All rights reserved.

need to be optimized. A balanced profile enables comparison across loci, which can be particularly
helpful in efforts to deconvolute a DNA mixture containing two or more contributors.
STR kit manufacturers spend a great deal of effort during kit development working on inter-locus
balance within and across dye channels. This balance, which is demonstrated through developmental
validation, is primarily performed with pristine samples using a range of DNA amounts. PCR ampli-
fication of evidentiary material, which is not pristine and may be limited in quantity, can yield partial
profiles. PCR inhibitors in evidentiary samples may also result in uneven amplification across the
tested STR loci.

STR PROFILE EVALUATION

STR data interpretation typically begins by looking at a DNA profile in its entirety. Off-scale data
and the resultant pull-up peaks can suggest that too much DNA template was present in the multi-
plex PCR reaction. The loss of signal from longer-sized STR loci is an indicator of PCR inhibitors or
degraded DNA. The presence of more than two alleles at a locus can suggest a potential mixture e
although it is important to not focus on a single locus due to the possibility of tri-allelic patterns.

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 STR PROFILE EVALUATION 111
The ratio of X and Y alleles from the amelogenin sex-typing primer pair can also aid in DNA
mixture interpretation. Inter-locus balance within a dye channel can help inform an analyst on poten-
tial PCR inhibition or DNA degradation. Inter-locus balance between dye channels can inform an
STR kit developer on dye sensitivity and PCR primer balance.
Table 5.1 lists the fluorescent dyes employed in a number of STR kits and dye sets used with
capillary electrophoresis (CE) to detect the STR kit amplicons. Newer STR kits typically involve
five or six dyes with one dye channel reserved for an internal size standard (see Chapter 3).
There is a fairly narrow input range of DNA template quantities that produce optimal profiles.
This is one of the reasons that human DNA quantitation is required on forensic samples by the
FBI Quality Assurance Standards (FBI QAS 2011).
Because multiple dye colors are used to create DNA profiles, fluorescent can bleed through into
adjacent color channels if data peaks are off-scale or the spectral calibration is not properly calibrated.
These pull-up peaks from off-scale data can fall into allele size bins for STR loci in another color and
need to be edited out of a properly interpreted profile. Alternatively, the DNA sample could be re-
amplified with less DNA, or the original PCR products could be re-injected with a lower electroki-
netic injection into the CE instrument to achieve on-scale data. Thus, pull-up peaks complicate
STR profile interpretation.
Figure 5.1 illustrates some pull-up peaks that bleed through into adjacent dye channels from the
off-scale TPOX 8,8 homozygote. Expert-systems software or a human performing manual data re-
view must decide that the pull-up peaks are not real STR alleles based on their shape and position
in the electropherogram. Other complications in STR data interpretation that can arise include the
presence of tri-allelic patterns, amelogenin deletions, and STR alleles extending beyond their
expected size ranges (see Chapter 3).
The European Network of Forensic Science Institutes (ENFSI) published principles related to
testing STR multiplexes (Gill et al. 2000). Most developmental validation studies follow these princi-
ples, many of which are spelled out in the SWGDAM validation guidelines (SWGDAM 2012). An
important part of validation is assessing stutter product formation, heterozygote balance, and
inter-locus balance with various ranges of DNA amounts to define limits of reliability with low-
Copyright © 2014. Elsevier Science & Technology. All rights reserved.

template DNA samples. Stochastic effects with low amounts of DNA can cause STR profile
inter-locus balance to deteriorate.

TABLE 5.1 STR Kits and Dye Sets Used

Example STR Kit Dye Labels Dye Set

Profiler Plus, SGM Plus, COfiler, Profiler 5-FAM, JOE, NED, ROX F
Identifiler, MiniFiler, NGM, NGM SElect 6-FAM, VIC, NED, PET, LIZ G5
GlobalFiler 6-FAM, VIC, NED, TAZ, SID, LIZ J6 (3500)

PowerPlex 16, 16HS FL, JOE, TMR, CXR F

PowerPlex ESI 16/17, ESX 16/17, 18D, 21, Fusion FL, JOE, TMR-ET, CXR-ET, CC5 G5
Qiagen Investigator Kits B, G, Y, R, O G5
Research assays 6-FAM, TET, HEX, ROX C

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 112 5. STR PROFILES

D8S1179 D21S11 D7S820 CSF1PO

90 110 130 150 170 190 210 230 250 270 290 310 330 350 370
8000
6000 6-FAM label
4000 (blue channel)
2000
0
13 30 10 10 12
13 30 11

D3S1358 TH01 D13S317 D16S539 D2S1338

90 110 130 150 170 190 210 230 250 270 290 310 330 350 370
8000
6000 VIC label
4000 (green channel)
2000
0
14 8 9.3 11 11 19 23
15 11 12

D19S433 vWA TPOX D18S51

90 110 130 150 170 190 210 230 250 270 290 310 330 350 370
8000
6000 NED label
4000 (yellow channel)
2000
0
14 17 8 15 19
15 18 8

A... D5S818 FGA

90 110 130 150 170 190 210 230 250 270 290 310 330 350 370
8000
6000 PET label
4000 (red channel)
2000
0
X 11 23
X 11 24

FIGURE 5.1 Identifiler kit STR profile from NIST SRM 2391b component 9 (9947A). Off-scale data leads to pull-up across
dye channels (indicated by arrows). PCR conditions include 1-ng DNA template run at half-reaction and 28 cycles.
Figure courtesy of Becky Hill, NIST.
Copyright © 2014. Elsevier Science & Technology. All rights reserved.

TRI-ALLELIC PATTERNS
Occasionally three alleles can be observed at a locus in a single-source DNA profile (Figure 5.2).
These tri-alleles or tri-allelic patterns are copy number variants (CNVs). They result from extra chro-
mosome fragments being present in a sample that produce an additional PCR product when sub-
jected to PCR primers for that locus. In some cases, an entire extra chromosome may be present,
such as in trisomy 21 or Down syndrome. In fact, the detection of three alleles at D21S11 and other
STR markers on chromosome 21 has been used as a diagnostic screen for Down syndrome detection
(Pertl et al. 1994, Samura et al. 2001, Yoon et al. 2002).
Expanding efforts of human genome sequencing have discovered that CNVs are more common
than originally thought (Freeman et al. 2006, Sjödin & Jakobsson 2012). Tri-allelic patterns are typi-
cally rare at a specific locus, but are likely to occur within a 15-locus STR profile about once every
1,000 samples (D.N.A. Box 5.2). An examination of 5,964 DNA profiles from Belgium found three
cases of tri-allelic patterns, one with D8S1179 and two with D18S51 (Mertens et al. 2009).

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 TRI-ALLELIC PATTERNS 113
AM... D3S1358 D1S1656 D2S441 D10S1248 D13S317 Penta E
100 200 300 400 500
1200

800

400

0
X 15 12 14 11 13 12 14 16
632 604 682 494 1185 471 503 471 467
Y 16 11 14 13
606 490 1185 477 559

D16S539 D18S51 D2S1338 CSF1PO Penta D

100 200 300 400 500
1200

800

400

0
9 11 18 21 23 11 13 10 13
423 465 491 557 717 523 617 526 419
24
698

TH01 vWA D21S11 D7S820 D5S818 TPOX DYS391

100 200 300 400 500
1200

800

400

0
8 15 17 27 30.2 10 12 9 11 10
302 334 239 342 339 254 529 257 303 298
9 11 12 10 10
376 234 529 298 298

D8S1179 D12S391 D19S433 FGA D22S1045

100 200 300 400 500
1200

800

400

0
9 13 16 21 13.2 25 11
322 341 394 386 451 664 753
15 25 11
404 664 753

FIGURE 5.2 STR profile from a single-source DNA sample amplified with the PowerPlex Fusion kit that exhibits a tri-
allelic pattern at TPOX (see arrow). Figure courtesy of Becky Hill, NIST.
Copyright © 2014. Elsevier Science & Technology. All rights reserved.

Chromosomal duplication and triplication have also been observed in Y-chromosome STRs (Butler
et al. 2005) and X-chromosome STRs (Lim et al. 2009).

Type 1 and Type 2 Tri-Alleles

Tim Clayton and colleagues at the UK Forensic Science Service developed a classification scheme
for tri-allelic patterns (Clayton et al. 2004). Type 1, which is more common, involves triallelic patterns
where the sum of peak heights from two of the alleles is similar to the peak height of the third allele.
Type 2 triallelic patterns involve a fairly balanced set of three alleles (Figure 5.3). In 15 tri-allelic
patterns reported by a study of 32,800 PowerPlex 16 profiles, 12 were Type 1 and 3 Type 2 with these
tri-alleles seen in 10 of the 15 STRs examined (Huel et al. 2007).
More than 240 different tri-allelic patterns have been reported at all 13 CODIS STR loci with most
of them being seen at TPOX, D18S51, D21S11, VWA, and FGA. A frequently updated listing of tri-
allelic patterns may be found on the NIST STRBase web site (STRBase 2014). While only 7 different
D18S51 tri-allelic patterns had been reported to STRBase through April 2005 (Butler 2006), as of

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 114 5. STR PROFILES

D.N.A. BOX 5.2

ESTIMATION OF AVERAGE FREQUENCY FOR

TRI-ALLELIC PATTERNS IN STR PROFILES
Several years ago, DNA analyst Malena Noting a total of 68 tri-alleles observed across
Jimenez from the Missouri State Highway Patrol these 69,600 samples, a tri-allelic pattern is ex-
supplied the NIST STRBase website with infor- pected to occur about once every one thousand
mation on tri-alleles observed during analysis of samples on average. However, as can be seen
69,600 convicted offender samples. Below is a with this data, the distribution of tri-allelic pat-
summary of the number of reported tri-alleles for terns is not even across loci.
each locus examined with their PowerPlex 16
single-source profiles. Source: Steven Myers, California Department of Justice pre-
sentation provided to the author based on data collected from the
NIST STRBase website at http://www.cstl.nist.gov/strbase/tri_
tab.htm.

STR Locus # Tri-Alleles STR Locus # Tri-Alleles STR Locus # Tri-Alleles

CSF1PO 1 D3S1358 2 D16S539 3
FGA 11 D5S818 1 D18S51 3
TH01 0 D7S820 0 D21S11 9

TPOX 9 D8S1179 2 Penta D 3

vWA 10 D13S317 4 Penta E 10
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(a) (b)
Type 1 Type 2
3
1 2 3

1
2

(1+2≈3) (1≈2≈3)

FIGURE 5.3 Illustration of tri-allelic patterns that are sometimes observed at a single locus in a multiplex STR profile.
They may be classified into one of two different groups based on relative peak heights: (a) “Type 1” where the sum of
two peak heights is almost equal to the third (1 þ 2 z 3) or (b) “Type 2” where fairly balanced peak heights are observed
(1 z 2 z 3).

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 TRI-ALLELIC PATTERNS 115
TABLE 5.2 A Summary of 35 D18S51 Tri-Allelic Patterns Reported on STRBase (as of
Jan 2014) Observed in Five Different Countries and Nine U.S. States

D18S51 Reported Frequency D18S51 Reported Frequency

Tri-Alleles (at time observed) Tri-Alleles (at time observed)

8, 11, 15 e 14, 15, 16 1 in 11,000

11, 13, 17 e 14, 15, 17 1 in 14,245

12, 13, 14 1 in 503 14, 15, 22 e
12, 13, 15 (2) 1 in 15,000; 14, 16, 17 e
other not reported
12, 13, 18 1 in 69,600 14, 16, 18 e
12, 14, 15 1 in 10,613 14, 16, 22 e
12, 14, 18 1 in 69,600 14, 18, 19 e
12, 15, 19 1 in 15,000 14, 19, 20 e
12, 16, 17 1 in 39,000 15, 16, 20 1 in 1120

12, 17, 18 e 15, 17, 18 e

12, 18, 19 e 15, 19, 20 e
13, 14, 15 e 16, 17, 18 (3) 2 in 16,046;
1 in 11,000
13, 14, 16 e 16, 17, 19 1 in 28,252
13, 14, 17 e 16, 17, 20 1 in 12,115
13, 14, 21 1 in 11,000 16, 19, 20 e
13, 15, 16 (2) 1 in 11,500; 17, 18, 19 (3) e
1 in 69,600
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13,15,19 e 19, 20, 21 e

19, 22.2, 23.2 e

The tri-alleles listed in bold font were recognized as Type 1 (imbalanced) tri-allelic patterns. Most did not have
reported frequencies (e).

January 2014 the number of different D18S51 tri-alleles had grown to 35 (Table 5.2). Thus, collection
of more STR profiles is resulting in more observations of tri-allelic patterns.

Inheritance Studies
In order to better understand the origin of tri-allelic patterns, several studies have examined inher-
itance patterns (Rolf et al. 2002, Zamir et al. 2002, Lukka et al. 2006, Lane 2008, Vidal & Cassar 2008).
These studies suggest that tri-alleles arise from inheritance of two chromosomes or chromosomal
regions (e.g. a CNV duplication) from one parent.
A tri-allelic pattern at D3S1358 was observed in a tested child (17/18/19) during routine parentage
testing (Vidal & Cassar 2008). Follow-up analysis found that the alleged paternal grandmother was

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 116 5. STR PROFILES

also tri-allelic (15/18/19) while the mother exhibited a normal diploid genotype (15/17). Thus, the
child likely inherited the 18/19 from her father and the 17 from her mother. Unfortunately, the father
was unavailable for testing. DNA samples from the mother, child, and alleged paternal grandmother
were further examined at 11 additional loci surrounding D3S1358 on chromosome 3. Only D3S1358
showed a duplication suggesting that the CNV duplication in this case was confined to a small
portion of chromosome 3 (Vidal & Cassar 2008).
In another study (Rolf et al. 2002), in which the mother exhibited three alleles (18/19/33.2) at the
SE33 (ACTBP2) locus, these three alleles segretated separately to her five children who appear to
receive only one maternal allele each. With a father possessing a 29.2/31.2 SE33 genotype, the chil-
dren had normal genotypes of 29.2/33.2, 29.2/33.2, 31.2/33.2, 19/31.2, and 18/31.2, where underlin-
ing is used to illustrate the maternal allele received. The authors of this study concluded that their
data can best be explained by assuming somatic mosaicism, which is present in the tissues investi-
gated as well as the maternal germ cells (Rolf et al. 2002). The 18 and 19 alleles are likely the mosaics
and they may have arisen from an early mutation event during embryogenesis. This study concludes
that because the frequency of somatic mosaicism is related to the mutation rate, loci with higher
mutation rates will exhibit tri-allelic patterns more often.
The inheritance of a TPOX tri-allelic pattern was observed in a paternity trio where the child was 8/
10/11, the mother was 8/8/10, and the father was 11/11 (Lukka et al. 2006). Analysis of neighboring
STR loci to TPOX on the short arm of chromosome 2 support the assumption that the mother in this
case supplied two alleles to the child while the father supplied one. Noting the TPOX tri-alleles are usu-
ally type 2 (Figure 5.3), the authors of this study suggest a relative high frequency of chromosomal rear-
rangements in the TPOX region near the telomere of chromosome 2 (Lukka et al. 2006). However, there
may be another reason for the type 2 tri-allelic patterns frequently observed at TPOX (Lane 2008).

TPOX Tri-Alleles and “Allele 10”

A 2008 study reported that approximately 2.4% of indigenous South Africans have three rather
than two TPOX alleles (Lane 2008). Data collected during routine paternity testing revealed that
Copyright © 2014. Elsevier Science & Technology. All rights reserved.

the extra allele is almost always allele 10 and that it appears to segregate independently of the other
alleles at the main TPOX locus. Approximately twice as many females as males have TPOX tri-allelic
genotypes, which suggests that the extra “allele 10” is on the X chromosome.
An analysis of STRBase submissions patterns provides support for the observation that allele 10
is extremely common in TPOX tri-allelic patterns. In 113 STRBase submissions involving 18 different
TPOX tri-allelic patterns (reported as of January 2014), only 9 times (8%) is allele 10 not present. There
are five 8/9/11, three 8/11/12, and one 9/11/12 tri-alleles. Likewise, 30 reports of the 113 submis-
sions are 8/10/11. This 23% of reported TPOX tri-alleles correlates fairly well with the observed 8,11
genotype frequency of 27% found in the NIST 1036 dataset (Butler et al. 2012, Hill et al. 2013).
A set of 20 tri-allelic TPOX samples from the Dominican Republic containing either a tri-allele in a
mother or her child found in all but two instances the presence of the “10” allele (Díaz et al. 2009). One
of these instances was an 8/9/11 mother with no parental TPOX data available. In the other instance,
the child (a son) was a TPOX 6/8/11 with an 8,8 mother. Thus, the father, who was not tested, likely
transmitted a 6/11 to his son. Since a man will not pass on an X chromosome to his son, the absence of
a TPOX “10” in the child agrees with the hypothesis that the duplicated “allele 10” is on the X chro-
mosome (Lane 2008).

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 TRI-ALLELIC PATTERNS 117
Frequency Estimates
It is challenging to determine an accurate frequency estimate for a specific tri-allele. While some
STRBase submissions contain frequency information, it only pertains to a specific data set at the
time submitted. For example, a CSF1PO 10,12,13 tri-allele was reported as 1 in 11,000 samples exam-
ined by the International Commission on Missing Persons and 1 in 31,330 samples tested by a labo-
ratory in Korea. However, these values only reflect how many samples were tested by their lab at the
time the information was submitted to STRBase. Thus, we only have limited information at best with
which to estimate a frequency. If there are 12 million U.S. profiles with D18S51 results and likely more
than 30 million profiles world-wide with D18S51, then perhaps these values could be used as the de-
nominator in an estimate. However, not all observed tri-allelic patterns are submitted to STRBase,
and therefore the numerator of such a frequency estimation would not be accurate due to incomplete
information.
In some ways, tri-alleles are like heteroplasmy in mtDNA (Melton 2004) or duplication/deletions
in Y-STR results (Butler et al. 2005). As can be seen in Table 5.2, the frequency of occurrence of each
D18S51 tri-allele, when it was reported, ranges from 1 in 503 to 1 in 69,600.
Unfortunately, there is not a commonly accepted method to incorporate frequencies of tri-allelic
patterns into the statistical calculation for STR profile rarity. My current recommendation would be
to leave the tri-allele out of the statistical comparison because of the uncertainty with a proper
numerator or denominator to use in the specific tri-allelic frequency estimate. However, the report
should mention whether or not the question (Q) and known (K) samples qualitatively match at the
locus containing the tri-allelic pattern. If they match, then you are strengthening the match statistic e
we just do not know by how much.
Some authors have suggested not including tri-allelic information in case reports due to the pos-
sibility of clinically relevant data being inadvertently revealed if a chromosomal duplication, such as
trisomy 21, is present (Lukka et al. 2006).

Tri-Allelic Anomalies
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False tri-alleles may occur if an extreme off-ladder allele from an adjacent locus in the STR
multiplex falls into the range of a neighboring locus e and sometimes even into a legiti-
mate allele bin (see examples in Chapter 3). Thus, using different STR kits, with locus in dif-
ferent size and dye color confirmations, can be helpful in confirming tri-allelic patterns (e.g.
Figure 3.8).
Abnormal DNA profiles may arise from various biological processes, including copy number
variants either as duplications or deletions, somatic mutations (which could vary between different
tissues tested), full chromosomal duplication (such as trisomy 21), and mosaicism.
Some true Type 1 tri-allelic patterns may be masked by what appears to be high stutter at a spe-
cific allele. Rather than being a DNA mixture of two individuals where the minor component is only
being “observed” at a single locus, a sample possessing high stutter at a single allele may in fact be
exhibiting mosaicism (a mixture of two cell types) within a single individual (Zimmer 2013). Scien-
tists from the Washington State Patrol Crime Laboratory reported this phenomenon with an anom-
alous result at D3S1358 encountered in a sexual assault-homicide case (Shutler & Roy 2012) (D.N.A.
Box 5.3).

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 118 5. STR PROFILES

D.N.A. BOX 5.3

ELEVATED STUTTER AT A SINGLE STR LOCUS: A DNA

MIXTURE OR TRI-ALLELIC MOSAICISM?
Occasionally in what appears to be an other- elevated stutter? For example, is this result from
wise single-source sample, the signal in the a low-template DNA sample amplified with an
stutter position at a single STR locus will appear increased number of PCR cycles where elevated
higher than expected. In the figure shown here, stutter is a definite possibility due to stochastic
the second stutter peak is expected to be in the effects?
range of 12% to 15% rather than the 28% shown. In a letter to the editor of Forensic Science
The percentages shown are based on relative peak International: Genetics, two scientists from the
heights compared to the tallest allele peaks with Washington State Patrol (WSP) Crime Laboratory
relative fluorescence units of 414, 3863, 926, and reported some genetic anomalies that they believe
3,258 (for the peaks from left to right, respectively). are consistent with gonadal mosaicism (Shutler &
Roy 2012). Mosaicism is the presence of genetically
4500 different tissues within a single individual. These
Relative fluorescence units (RFU)

4000 different cells could potentially have different

genotypes due to post-zygotic mutation either in
3500
somatic (non-germline) or gonadal (germline)
3000 cells. The figure shown here is a representation of
2500 D3S1358 results of a semen stain recovered from
the suspect’s pants in the WSP case study (their
2000
Supplemental Figure 4). The D3S1358 allele posi-
1500 tions are 16, 17, 18, and 19. An oral reference
28%
1000 sample from the suspect matched at all other loci
11% with the exception of 11% stutter being seen in
500
the 18 position, which is 28% in the semen stain
Copyright © 2014. Elsevier Science & Technology. All rights reserved.

0 result shown. The WSP authors conclude that the

120 125 130 135 140 145 semen-derived profile could be explained as being
DNA size (nt) consistent with a Type 1 tri-allelic pattern since
the sum of the 18 and 19 allele peak heights
Assuming that other STR loci in the full pro-
(926 þ 3258 ¼ 4184 RFU) is approximately the
file do not show any other signs of a second
same size as the allele 17 peak height (3863 RFU).
contributor (i.e. all loci exhibit only one or two
This tri-allele (17/18/19) could have arisen
alleles and their accompanying stutter products)
“during mitosis leading to mosaicism and could be
and that bleedthrough from another dye channel
explained as being due to a sperm precursor cell
is not likely, then the reporting analyst is left
somatic mutation” (Shutler & Roy 2012).
with a difficult decision. Is this elevated stutter
the only surviving evidence of a “mystery man” Source: Shutler, G., & Roy, T. (2012). Genetic anomalies
minor component where other alleles have consistent with gonadal mosaicism encountered in a sexual
dropped out or are being masked by the major assault-homicide. Forensic Science International: Genetics, 6,
e159ee160. Electropherogram illustration of results from this
component alleles and their stutter products? Or study drawn with Jo Bright’s EPG Marker available at http://
is there some other potential explanation for the www.cstl.nist.gov/strbase/tools/Bright-scaler.xlsx.

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 GENDER IDENTIFICATION WITH AMELOGENIN 119
GENDER IDENTIFICATION WITH AMELOGENIN
The ability to designate whether a sample originated from a male or a female source is useful in
sexual assault cases, where distinguishing between the victim and the perpetrator’s evidence is
important. Likewise, missing persons and mass disaster investigations can benefit from gender iden-
tification of the remains. Over the years a number of gender identification assays have been demon-
strated using PCR methods (Sullivan et al. 1993, Eng et al. 1994, Reynolds & Varlaro 1996). By far the
most popular method for sex-typing today is the amelogenin system as it can be performed in
conjunction with STR analysis.
Amelogenin is a gene that codes for proteins found in tooth enamel. The British Forensic Science
Service was the first to describe the particular PCR primer sets that are used so prevalently in forensic
DNA laboratories today (Sullivan et al. 1993). These primers flank a 6-bp deletion within intron 1 of
the amelogenin gene on the X homolog (Figure 5.4). PCR amplification of this area with their primers
results in 106-bp and 112-bp amplicons from the X and Y chromosomes, respectively.
An advantage in using a single primer set to amplify both chromosomes is that the X chromosome
product itself plays a role as a positive control. This PCR-based assay is extremely sensitive.
Mannucci and co-workers were able to detect as little as 20 pg (z3 diploid copies) as well as sample
mixtures where female DNA was in 100-fold excess of male DNA (Mannucci et al. 1994).
Other regions of the amelogenin gene have size differences between the X and Y homologs and
may be exploited for sex-typing purposes. A careful study found that 19 regions of absolute homol-
ogy, ranging in size from 22 bp to 80 bp, exist between the human amelogenin X and Y genes that can
be used to design a variety of primer sets (Haas-Rochholz & Weiler 1997). Thus, by spanning various
deletions of the X and/or Y chromosome, it is possible to generate PCR products from the X and Y

FIGURE 5.4 Schematic of the amelogenin

6 bp
sex-typing assay. The X and Y chromosomes
deletion
contain a high degree of sequence homology
X at the amelogenin (AMEL) locus and primers,
Copyright © 2014. Elsevier Science & Technology. All rights reserved.

as depicted by the arrows, can target a 6 bp

deletion that is present only on the X chro-
Y mosome (Sullivan et al. 1993). In most cir-
cumstances, the presence of a single X peak
indicates that the sample comes from a fe-
Normal X Y male, while two peaks identifies the sample’s
Male: source as male. Both AMEL Y and AMEL X
X,Y null alleles have been reported.

X
Normal X
Female: Male
X,X (AMEL Y null)

Y
Male
(AMEL X null)

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 120 5. STR PROFILES

homologs that differ in size and contain size ranges that can be integrated into future multiplex STR
amplifications. For example, an 80-bp amplicon from the X-chromosome and an 83-bp amplicon from
Y-chromosome were used in the NIST 26plex assay (Hill et al. 2009).
While amelogenin is an effective method for sex-typing biological samples in most cases, the
results are not foolproof either due to primer binding sites that lead to null alleles or chromosomal
deletions. Amelogenin Y allele drop-outs have been observed due to loss of portions of the Y chro-
mosome in some population groups. Amelogenin X allele drop-outs have been seen primarily due
to primer binding site mutations.

Amelogenin Y Allele Drop-Out

Portions of the Y-chromosome can be deleted in normal males. It has been noted that a rare
deletion of the amelogenin gene on the Y-chromosome can cause the Y-chromosome amplicon to
be absent (Santos et al. 1998). In such a case, a male sample would falsely appear as a female with
only the amelogenin X allele being amplified. This deletion of the Y chromosome amelogenin region
appears to be more common in Indian populations (Thangaraj et al. 2002) than those of European or
African origins. A study of almost 30,000 males in the Austrian National DNA database revealed that
only six individuals lacked the amelogenin Y-amplicon (Steinlechner et al. 2002). These individuals
were verified to be male with Y-STRs and amplification of the SRY region.
More recent studies have attempted to map the Y deletions in detail and to track the specific
biogeographic ancestry of these interesting variants (Cadenas et al. 2007, Jobling et al. 2007). Through
examining adjacent STR markers and sequence-specific tag sites, the extent of the Y chromosome
deletion can be mapped (Takayama et al. 2009). When amplifying Y-STR loci, the locus DYS458 in
Yfiler or PowerPlex Y23 is the most likely one to be lost with amelogenin Y deletions due to its close
proximity to the AMEL Y.
A 2008 survey of paternity testing laboratories using the Applied Biosystems primers (i.e. Identi-
filer or Profiler Plus kits) noted an overall rate of 0.033% males (from 144,391 samples) that lacked the
amelogenin Y allele (AABB, 2008). However, 1.8% East Asian males (6 of 505) exhibited a missing
Copyright © 2014. Elsevier Science & Technology. All rights reserved.

amelogenin Y allele demonstrating the population specificity with this particular Y-chromosome
deletion.

Amelogenin X Allele Drop-Out

Amelogenin X allele drop-out has also been observed in males. In this case only the amelogenin
Y-amplicon is present (Shewale et al. 2000, Alves et al. 2006, Maciejewska & Paw1owski 2009). In
one study, this phenomenon was observed only three times out of almost 7,000 males examined
(Shewale et al. 2000). The authors of this study felt that the AMEL X null was most likely a result
of a rare polymorphism in the primer binding sites for the amelogenin primers used in commercial
STR kits. A different set of amelogenin primers targeting the same 6-bp deletion on the X chromo-
some amplified both the X and Y alleles of amelogenin (Shewale et al. 2000). However, in some pop-
ulations, this loss of the AMEL X allele is more common. In a study of 503 individuals from São Tomé
Island (West Africa), 10 male individuals displayed only the Y allele from amelogenin amplification
due to a primer binding site mutation in the AMEL X allele (Alves et al. 2006). A different mutation
caused one male out of 5534 Polish males tested to display only the AMEL Y allele (Maciejewska &
Paw1owski 2009).

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 PARTIAL DNA PROFILES 121
A report of males examined in paternity testing labs using Applied Biosystems STR typing kits
found that there were a higher number of African American males compared to other U.S. population
groups showing only the AMEL Y allele (i.e. the AMEL X allele dropped out). Still, this AMEL X null
is fairly rare, seen only 48 times in 144,391 males tested, or 0.03% of the time (AABB 2008).
In a 2011 U.S. Patent application, Life Technologies scientists reported a C/T SNP in the AMEL X
sequence that impacts their reverse amelogenin primer (Mulero et al. 2011). Since it is located at the
30 -end of their reverse primer, they report using a universal base to overcome possible sequence
variants at this position.

Additional Male Confirmation Markers to Confirm Gender

Given the possibility of amelogenin Y being deleted and thus providing a false negative for male
samples, additional Y-chromosome markers have sometimes been included in assays to confirm
gender assignments. A group from the Israel Police DNA Laboratory proposed the addition of a
Y-STR to help confirm male samples possessing an AMEL Y null allele (Oz et al. 2008).
In the announcement of the proposal for the U.S. core expansion (Hares 2012), DYS391 was
included as a core locus for this purpose e to aid in male confirmation when deletions spanning
AMEL Y occur. The megaplexes built to meet the proposed expanded U.S. core, AmpFlSTR
GlobalFiler and PowerPlex Fusion, both contain DYS391 (see Chapter 1). Amplification of a portion
of SRY, the sex-determining region of the Y-chromsome that is located at 2.65 Mb, has been used as
well for a male confirmation marker (Kastelic et al. 2009, Giuliodori et al. 2011).
The GlobalFiler STR kit from Life Technologies (South San Francisco, CA) includes a Y insertion-
deletion (InDel) that can be either “1” (deletion) or “2” (insertion). In a U.S. patent application laying
out the GlobalFiler design, this Y-InDel is cited as rs2032678 (Hennessy & Wang 2012). Small ampli-
con sizes of 81 bp or 86 bp enable successful results with degraded DNA samples.
Provided the GlobalFiler Y-InDel is rs2032678, it is a genetic marker originally known as M175
because it was the 175th marker discovered by Peter Underhill of Stanford University using dena-
turing high-performance liquid chromatography (Underhill et al. 2007). M175 exhibits deletion of
a five-base sequence “TTCTC” with Y-SNP haplogroup O individuals who typically are East or
Copyright © 2014. Elsevier Science & Technology. All rights reserved.

Southeast Asians (van Oven et al. 2012). Most samples will be “2,” which is the ancestral “insertion”
form, unless they are Asian in origin. M175 is located at 15.5 Mb on the reference Y-chromosome
sequence, which is on the long arm of the Y-chromosome over 7 Mb from amelogenin that is at
6.74 Mb (Figure 5.5). Thus, a deletion around the amelogenin Y region is not expected to impact
the rs2032678 Y-InDel or DYS391.

PARTIAL DNA PROFILES

A partial DNA profile is by definition one that is missing information that was sought as part of
the initial genetic testing performed on a biological sample. For example, if a particular DNA test is
performed that should amplify 16 loci, yet only 13 loci produced results, then the result is a partial
profile that is missing information at three loci.
Allele drop-out is when only a single allele is missing at a tested locus, while locus drop-out
involves missing both alleles in a single-source sample. Allele drop-out is most commonly due to
stochastic effects when amplifying a DNA sample of low quantity or low quality (e.g. possesses

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 122 5. STR PROFILES

Mb
PAR1
Y
SRY
5
p
AMEL Y
centromere 10

DYS391
15
q Y-InDel (M175, rs2032678)

20

heterochromatin
25

30

PAR2

FIGURE 5.5 Relative positions of amelogenin (AMEL Y) and male confirmation markers found on the Y-chromosome. Red
shading covers area that may be lost with AMEL Y null alleles due to deletion of that portion of the short arm of the
Y-chromosome.

DNA size (bp) relative to an internal size standard (not shown)

(a)
Full Profile (Good Quality)
Relative fluorescence units (RFUs)
Copyright © 2014. Elsevier Science & Technology. All rights reserved.

(b)
Partial Profile (Poor Quality)

FIGURE 5.6 A comparison of DNA profiles originating from the same biological source but of different qualities. (a) Intact,
good-quality DNA yields a full profile. (b) Degraded, poor-quality DNA yields a partial profile with only the shorter-size PCR
products producing detectable signal. With the degraded DNA sample shown in (b), information is lost at the longer-sized STR loci.
Also note the lower relative fluorescence units with the poor-quality partial profile in (b). Figure courtesy of Margaret Kline, NIST.

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 READING LIST AND INTERNET RESOURCES 123
PCR inhibitors). Figure 5.6 illustrates a full profile versus a partial profile obtained from the same bio-
logical source. Chapter 7 will contain more on the interpretation challenges that arise with partial
profiles. Chapter 13 covers statistical approaches attempting to account for allele drop-out in partial
profiles, especially those arising with complex DNA mixtures.

Reading List and Internet Resources

Multiplex PCR Challenges
Butler, J. M. (2012). Advanced Topics in Forensic DNA Typing: Methodology. San Diego: Elsevier Academic Press, 167e211.
Edwards, M. C., & Gibbs, R. A. (1994). Multiplex PCR: advantages, development, and applications. PCR Methods and
Applications, 3, S65eS75.
Gill, P., et al. (2005). A graphical simulation model of the entire DNA process associated with the analysis of short tandem
repeat loci. Nucleic Acids Research, 33, 632e643.
Kimpton, C., et al. (1993). Automated DNA profiling employing ‘multiplex’ amplification of short tandem repeat loci. PCR
Methods and Applications, 3, 13e22.
Kimpton, C. P., et al. (1996). Validation of highly discriminating multiplex short tandem repeat amplification systems for
individual identification. Electrophoresis, 17, 1283e1293.
Wallin, J. M., et al. (2002). Constructing universal multiplex PCR systems for comparative genotyping. Journal of Forensic
Sciences, 47, 52e65.
Walsh, P. S., et al. (1992). Preferential PCR amplification of alleles: mechanisms and solutions. PCR Methods and Applications, 1,
241e250.

STR Profile Evaluation

Bright, J. A., et al. (2010). Examination of the variability in mixed DNA profile parameters for the Identifiler multiplex. Forensic
Science International: Genetics, 4, 111e114.
Bright, J. A., et al. (2011). Determination of the variables affecting mixed MiniFiler DNA profiles. Forensic Science International:
Genetics, 5, 381e385.
Debernardi, A., et al. (2011). One year variability of peak heights, heterozygous balance and inter-locus balance for the DNA
positive control of AmpFlSTR(c) Identifiler(c) STR kit. Forensic Science International: Genetics, 5, 43e49.
FBI QAS. (2011). http://www.fbi.gov/about-us/lab/biometric-analysis/codis/qas-standards-for-forensic-dna-testing-
laboratories-effective-9-1-2011. Accessed March 23, 2014.
Copyright © 2014. Elsevier Science & Technology. All rights reserved.

Gill, P., et al. (1997). Development of guidelines to designate alleles using an STR multiplex system. Forensic Science Inter-
national, 89, 185e197.
Gill, P., et al. (2000). Report of the European Network of Forensic Science Institutes (ENFSI): formulation and testing of
principles to evaluate STR multiplexes. Forensic Science International, 108, 1e29.
Hill, C. R., et al. (2011). Concordance and population studies along with stutter and peak height ratio analysis for the
PowerPlexÒ ESX 17 and ESI 17 Systems. Forensic Science International: Genetics, 5(4), 269e275.
Kirkham, A., et al. (2013). High-throughput analysis using AmpFlSTRÒ IdentifilerÒ with the Applied Biosystems 3500xl
Genetic Analyser. Forensic Science International: Genetics, 7, 92e97.
Muller, K., et al. (2010). Casework testing of the multiplex kits AmpFlSTR SEfiler Plus PCR amplification kit (AB), PowerPlex S5
System (Promega) and AmpFlSTR MiniFiler PCR amplification kit (AB). Forensic Science International: Genetics, 4, 200e205.
Myers, B. A., et al. (2012). Evaluation and comparative analysis of direct amplification of STRs using PowerPlexÒ 18D and
IdentifilerÒ Direct systems. Forensic Science International: Genetics, 6, 640e645.
Petricevic, S., et al. (2010). Validation and development of interpretation guidelines for low copy number (LCN)
DNA profiling in New Zealand using the AmpFlSTR SGM Plus multiplex. Forensic Science International: Genetics, 4,
305e310.
Poetsch, M., et al. (2011). PowerplexÒ ES versus PowerplexÒ S5 e casework testing of the new screening kit. Forensic Science
International: Genetics, 5, 57e63.
Tvedebrink, T., et al. (2012). Performance of two 17 locus forensic identification STR kits e Applied Biosystems’s AmpFlSTRÒ
NGMSElect and Promega’s PowerPlexÒ ESI17 kits. Forensic Science International: Genetics, 6, 523e531.

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Butler, J. M. (2014). Advanced topics in forensic dna typing : Interpretation. Retrieved from http://ebookcentral.proquest.com
Created from uam-ebooks on 2019-10-17 17:00:06.
 124 5. STR PROFILES

STR KIT Developmental Validation Studies

Butler, J. M. (2012a). Quality assurance and validation. Chapter 7. In Advanced Topics in Forensic DNA Typing: Methodology (pp.
167e211). San Diego: Elsevier Academic Press.
Collins, P. J., et al. (2004). Developmental validation of a single-tube amplification of the 13 CODIS STR loci, D2S1338,
D19S433, and amelogenin: the AmpFlSTR Identifiler PCR amplification kit. Journal of Forensic Sciences, 49(6), 1265e1277.
Davis, C., et al. (2013). Prototype PowerPlexÒ Y23 System: a concordance study. Forensic Science International: Genetics, 7(1),
204e208.
Ensenberger, M. G., et al. (2010). Developmental validation of the PowerPlex 16 HS System: an improved 16-locus fluorescent
STR multiplex. Forensic Science International: Genetics, 4, 257e264.
Ensenberger, M. G., et al. (2014). Developmental validation of the PowerPlexÒ 21 System. Forensic Science International:
Genetics, 9, 169e178.
Green, R. L., et al. (2013). Developmental validation of the AmpFlSTRÒ NGM SElect PCR Amplification Kit: A next-
generation STR multiplex with the SE33 locus. Forensic Science International: Genetics, 7, 41e51.
Greenspoon, S. A., et al. (2004). Validation and implementation of the PowerPlex 16 BIO System STR multiplex for forensic
casework. Journal of Forensic Sciences, 49, 71e80.
Holt, C. L., et al. (2002). TWGDAM validation of AmpFlSTR PCR amplification kits for forensic DNA casework. Journal of
Forensic Sciences, 47, 66e96.
Krenke, B. E., et al. (2002). Validation of a 16-locus fluorescent multiplex system. Journal of Forensic Sciences, 47, 773e785.
Krenke, B. E., et al. (2005). Validation of male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex. Forensic
Science International, 151, 111e124.
McLaren, R. S., et al. (2013). Developmental validation of the PowerPlexÒ ESI 17 Pro System. Forensic Science International:
Genetics, 7(3), e69ee73.
Mulero, J. J., et al. (2006). Development and validation of the AmpFlSTRÒ Yfiler PCR amplification kit: a male specific, single
amplification 17 Y-STR multiplex system. Journal of Forensic Sciences, 51, 64e75.
Mulero, J. J., et al. (2008). Development and validation of the AmpFlSTRÒ MiniFiler PCR Amplification Kit: a miniSTR
multiplex for the analysis of degraded and/or PCR inhibited DNA. Journal of Forensic Sciences, 53, 838e852.
Mulero, J. J., et al. (2008). Developmental validation of the AmpFlSTRÒ SEfiler PlusÔ PCR amplification kit: an improved
multiplex with enhanced performance for inhibited samples. Forensic Science International: Genetics Supplement Series, 1,
121e122.
Oostdik, K., et al. (2013). Developmental validation of the PowerPlexÒ 18D System, a rapid STR multiplex for analysis of
reference samples. Forensic Science International: Genetics, 7, 129e135.
SWGDAM.. (2012). Scientific Working Group on DNA Analysis Methods: Validation guidelines for DNA analysis methods.
Available at http://swgdam.org/SWGDAM_Validation_Guidelines_APPROVED_Dec_2012.pdf. Accessed March 23, 2014.
Copyright © 2014. Elsevier Science & Technology. All rights reserved.

Thompson, J. M., et al. (2013). Developmental validation of the PowerPlexÒ Y23 System: a single multiplex Y-STR analysis
system for casework and database samples. Forensic Science International: Genetics, 7(2), 240e250.
Tucker, V. C., et al. (2011). Developmental validation of the PowerPlexÒ ESI 16 and PowerPlexÒ ESI 17 Systems: STR
multiplexes for the new European standard. Forensic Science International: Genetics, 5, 436e448.
Tucker, V. C., et al. (2012). Developmental validation of the PowerPlexÒ ESX 16 and PowerPlexÒ ESX 17 Systems. Forensic
Science International: Genetics, 6, 124e131.
Wallin, J. M., et al. (1998). TWGDAM validation of the AmpFlSTR Blue PCR amplification kit for forensic casework analysis.
Journal of Forensic Sciences, 43, 854e870.
Wang, D. Y., et al. (2012). Developmental validation of the AmpFlSTRÒ IdentifilerÒ Plus PCR Amplification Kit: an estab-
lished multiplex assay with improved performance. Journal of Forensic Sciences, 57, 453e465.

Tri-Allelic Patterns
Butler, J. M. (2006). Genetics and genomics of core STR loci used in human identity testing. Journal of Forensic Sciences., 51(2),
253e265.
Butler, J. M., et al. (2005). Chromosomal duplications along the Y-chromosome and their potential impact on Y-STR inter-
pretation. Journal of Forensic Sciences, 50, 853e859.
Clayton, T. M., et al. (2004). A genetic basis for anomalous band patterns encountered during DNA STR profiling. Journal of
Forensic Sciences, 49, 1207e1214.

I. DATA INTERPRETATION
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 READING LIST AND INTERNET RESOURCES 125
Crouse, C. A., et al. (1999). Analysis and interpretation of short tandem repeat microvariants and three banded patterns using
multiple allele detection systems. Journal of Forensic Sciences, 44, 87e94.
Díaz, V., et al. (2009). The presence of tri-allelic TPOX genotypes in Dominican population. Forensic Science International:
Genetics Supplement Series, 2, 371e372.
Freeman, J. L., et al. (2006). Copy number variation: new insights in genome diversity. Genome Research, 16, 949e961.
Hill, C. R., et al. (2013). U.S. population data for 29 autosomal STR loci. Forensic Science International: Genetics, 7, e82ee83.
Huel, R. L., et al. (2007). Variant alleles, triallelic patterns, and point mutations observed in nuclear short tandem repeat typing
of populations in Bosnia and Serbia. Croatian Medical Journal, 48, 494e502.
Lane, A. B. (2008). The nature of tri-allelic TPOX genotypes in African populations. Forensic Science International: Genetics, 2,
134e137.
Lim, E. J., et al. (2009). Genetic polymorphism and haplotype analysis of 4 tightly linked X-STR duos in Koreans. Croatian
Medical Journal, 50, 305e312.
Lukka, M., et al. (2006). Triallelic patterns in STR loci used for paternity analysis: evidence for a duplication in chromosome 2
containing the TPOX STR locus. Forensic Science International, 164, 3e9.
Melton, T. (2004). Mitochondrial DNA heteroplasmy. Forensic Science Reviews, 16, 1e20.
Mertens, G., et al. (2009). Observation of tri-allelic patterns in autosomal STRs during routine casework. Forensic Science
International: Genetics Supplement Series, 2, 38e40.
Pertl, B., et al. (1994). Rapid molecular method for prenatal detection of Down’s syndrome. Lancet, 343, 1197e1198.
Rolf, B., et al. (2002). Somatic mutations at STR loci e a reason for three-allele pattern and mosaicism. Forensic Science In-
ternational, 126, 200e202.
Samura, O., et al. (2001). Diagnosis of trisomy 21 in fetal nucleated erythrocytes from maternal blood by use of short tandem
repeat sequences. Clinical Chemistry, 47, 1622e1626.
Shutler, G., & Roy, T. (2012). Genetic anomalies consistent with gonadal mosaicism encountered in a sexual assault-homicide.
Forensic Science International: Genetics, 6, e159ee160.
Sjödin, P., & Jakobsson, M. (2012). Population genetic nature of copy number variation. Genomic Structural Variants: Methods in
Molecular Biology, 838, 209e223.
Tri-Allelic Patterns on NIST STRBase website. (2014). http://www.cstl.nist.gov/strbase/tri_tab.htm. Accessed March 23, 2014.
Vidal, C., & Cassar, M. (2008). A case of tri-allelic pattern at locus D3S1358 on chromosome 3p21 inherited from paternal
grandmother. Forensic Science International: Genetics, 2, 372e375.
Yoon, H. R., et al. (2002). Rapid prenatal detection of Down and Edwards Syndromes by fluorescent polymerase chain
reaction with short tandem repeat markers. Yonsei Medical Journal, 43, 557e566.
Zamir, A., et al. (2002). Presentation of a three-banded pattern e analysis and interpretation. Journal of Forensic Sciences, 47,
824e826.
Zimmer, C. (2013). DNA double take. New York Times (Sept 16, 2013). Available at http://www.nytimes.com/2013/09/17/
Copyright © 2014. Elsevier Science & Technology. All rights reserved.

science/dna-double-take.html. Accessed March 23, 2014.

Gender Identification with Amelogenin

Budowle, B., et al. (1996). Multiplex amplification and typing procedure for the loci D1S80 and amelogenin. Journal of Forensic
Sciences, 41, 660e663.
Eng, B., et al. (1994). Anomalous migration of PCR products using nondenaturing polyacrylamide gel electrophoresis: the
amelogenin sex-typing system. Journal of Forensic Sciences, 39, 1356e1359.
Francès, F., et al. (2007). Amelogenin test: From forensics to quality control in clinical and biochemical genomics. Clinica
Chimica Acta, 386, 53e56.
Haas-Rochholz, H., & Weiler, G. (1997). Additional primer sets for an amelogenin gene PCR-based DNA-sex test. International
Journal of Legal Medicine, 110, 312e315.
Hill, C. R., et al. (2009). A 26plex autosomal STR assay to aid human identity testing. Journal of Forensic Sciences, 54(5), 1008e1015.
Mannucci, A., et al. (1994). Forensic application of a rapid and quantitative DNA sex test by amplification of the X-Y
homologous gene amelogenin. International Journal of Legal Medicine, 106, 190e193.
Reynolds, R., & Varlaro, J. (1996). Gender determination of forensic samples using PCR amplification of ZFX/ZFY gene
sequences. Journal of Forensic Sciences, 41, 279e286.
Sullivan, K. M., et al. (1993). A rapid and quantitative DNA sex test: fluorescence-based PCR analysis of X-Y homologous gene
amelogenin. BioTechniques, 15, 637e641.

I. DATA INTERPRETATION
Butler, J. M. (2014). Advanced topics in forensic dna typing : Interpretation. Retrieved from http://ebookcentral.proquest.com
Created from uam-ebooks on 2019-10-17 17:00:06.
 126 5. STR PROFILES

Tschentscher, F., et al. (2008). Amelogenin sex determination by pyrosequencing of short PCR products. International Journal of
Legal Medicine, 122, 333e335.

Amelogenin Anomalies and Null Alleles

AABB. (2008). Annual Report Summary for Testing in 2008. Available at http://www.aabb.org/sa/facilities/Documents/
rtannrpt08.pdf. Accessed March 23, 2014.
Alves, C., et al. (2006). The amelogenin locus displays a high frequency of X homologue failures in Sao Tome Island (West
Africa). In Progress in Forensic Genetics 11, ICS 1288 (pp. 271e273).
Cadenas, A. M., et al. (2007). Male amelogenin dropouts: phylogenetic context, origins and implications. Forensic Science
International, 166, 155e163.
Chang, Y. M., et al. (2007). A distinct Y-STR haplotype for Amelogenin negative males characterized by a large Y(p)11.2
(DYS458-MSY1-AMEL-Y) deletion. Forensic Science International, 166, 115e120.
Chen, W., et al. (2014). Detection of the deletion on Yp11.2 in a Chinese population. Forensic Science International: Genetics, 8,
73e79.
Davis, C., et al. (2012). A case of amelogenin Y-null: a simple primer binding site mutation or unusual genetic anomaly? Legal
Medicine, 14, 320e323.
Jobling, M. A., et al. (2007). Structural variation on the short arm of the human Y chromosome: recurrent multigene deletions
encompassing Amelogenin Y. Human Molecular Genetics, 16(3), 307e316.
Kumagai, R., et al. (2008). DNA analysis of family members with deletion in Yp11.2 region containing amelogenin locus. Legal
Medicine, 10, 39e42.
Lattanzi, W., et al. (2005). A large interstitial deletion encompassing the amelogenin gene on the short arm of the Y chro-
mosome. Human Genetics, 116, 395e401.
Maciejewska, A., & Pawlowski, R. (2009). A rare mutation in the primer binding region of the Amelogenin X homologue gene.
Forensic Science International: Genetics, 3, 265e267.
Mitchell, R. J., et al. (2006). Amelogenin Y negative males: multiple origins. Progress in Forensic Genetics 11, ICS, 1288,
274e276.
Mulero, J., et al. (2011). Amelogenin SNP on chromosome X. U.S. Patent Application, US 2011/0237443 A1.
Murphy, K. M., et al. (2007). Constitutional duplication of a region of chromosome Yp encoding AMELY, PRKY, and TBL1Y:
implications for sex chromosome analysis and bone marrow engraftment analysis. Journal of Molecular Diagnostics, 9,
408e413.
Ou, X., et al. (2012). Null alleles of the X and Y chromosomal amelogenin gene in a Chinese population. International Journal of
Legal Medicine, 126, 513e518.
Raina, A., et al. (2010). Misinterpretation of results in medico-legal cases due to microdeletion in the Y-chromosome. Molecular
Copyright © 2014. Elsevier Science & Technology. All rights reserved.

and Cellular Probes, 24, 418e420.

Santos, F. R., et al. (1998). Reliability of DNA-based sex tests. Nature Genetics, 18(2), 103e103.
Shadrach, B., et al. (2004). A rare mutation in the primer binding region of the Amelogenin gene can interfere with gender
identification. Journal of Molecular Diagnostics, 6(4), 401e405.
Shewale, J. G., et al. (2000). Anomalous amplification of the amelogenin locus typed by AmpFlSTR Profiler Plus
amplification kit. Forensic Science Communications, 2(4). Available at http://www.fbi.gov/about-us/lab/forensic-
science-communications/fsc/oct2000/index.htm/shewale.htm. Accessed March 23, 2014.
Steinlechner, M., et al. (2002). Rare failures in the amelogenin sex test. International Journal of Legal Medicine, 116(2),
117e120.
Takayama, T., et al. (2009). Determination of deletion regions from Yp11.2 of an amelogenin negative male. Legal Medicine, 11,
S578eS580.
Thangaraj, K., et al. (2002). Is the amelogenin gene reliable for gender identification in forensic casework and prenatal
diagnosis? International Journal of Legal Medicine, 116(2), 121e123.
Turrina, S., et al. (2009). Evaluation of deleted region from Yp11.2 of two amelogenin negative related males. Forensic Science
International: Genetics Supplement Series, 2, 240e241.
Zehethofer, K., & Rolf, B. (2011). A molecular analysis of three amelogenin negative males in two routine paternity tests.
Forensic Science International: Genetics, 5, 550e551.

I. DATA INTERPRETATION
Butler, J. M. (2014). Advanced topics in forensic dna typing : Interpretation. Retrieved from http://ebookcentral.proquest.com
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Male Confirmation Markers
Giuliodori, A., et al. (2011). Rapid analysis for confirmation of amelogenin negative males characterized by a Yp11.2 deletion.
Forensic Science International: Genetics Supplement Series, 3, e285ee286.
Hares, D. R. (2012). Expanding the CODIS core loci in the United States. Forensic Science International: Genetics, 6, e52ee54.
Hennessy, L., & Wang, D. (2012). Methods and kits for multiplex amplification of short tandem repeat loci. US Patent
Application, US 2012/0122093 A1.
Kastelic, V., et al. (2009). Validation of SRY marker for forensic casework analysis. Journal of Forensic Sciences, 54, 551e555.
Oz, C., et al. (2008). A Y-chromosome STR marker should be added to commercial multiplex STR kits. Journal of Forensic
Sciences, 53, 858e861.
Tozzo, P., et al. (2013). Deletion of amelogenin Y-locus in forensics: literature revision and description of a novel method for
sex confirmation. Journal of Forensic and Legal Medicine, 20, 387e391.
Underhill, P. A., et al. (1997). Detection of numerous Y chromosome biallelic polymorphisms by denaturing high-performance
liquid chromatography. Genome Research, 7, 996e1005.
van Oven, M., et al. (2012). A multiplex SNP assay for the dissection of human Y-chromosome haplogroup O representing the
major paternal lineage in East and Southeast Asia. Journal of Human Genetics, 57, 65e69.
Copyright © 2014. Elsevier Science & Technology. All rights reserved.

I. DATA INTERPRETATION
Butler, J. M. (2014). Advanced topics in forensic dna typing : Interpretation. Retrieved from http://ebookcentral.proquest.com
Created from uam-ebooks on 2019-10-17 17:00:06.
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Butler, J. M. (2014). Advanced topics in forensic dna typing : Interpretation. Retrieved from http://ebookcentral.proquest.com
Created from uam-ebooks on 2019-10-17 17:00:06.