Forensic Science International: Genetics 12 (2014) 136143

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Forensic Science International: Genetics

journal homepage: www.elsevier.com/locate/fsig

Construction of a library of cloned short tandem repeat (STR)

alleles as universal templates for allelic ladder preparation
Le Wang a,*, Xing-Chun Zhao a,*, Jian Ye a,*, Jin-Jie Liu b, Ting Chen b, Xue Bai a, Jian Zhang a,
Yuan Ou a, Lan Hu a, Bo-Wei Jiang c, Feng Wang a
a
b
c

Key Laboratory of Forensic Genetics, Institute of Forensic Science, Ministry of Public Security, Beijing 100038, PR China
School of Forensic Medicine, Shanxi Medical University, Taiyuan 030001, PR China
The First Research Institute of the Ministry of Public Security, Beijing 100048, PR China

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 23 February 2014
Received in revised form 7 June 2014
Accepted 11 June 2014

Short tandem repeat (STR) genotyping methods are widely used for human identity testing applications,
including forensic DNA analysis. Samples of DNA containing the length-variant STR alleles are typically
separated and genotyped by comparison to an allelic ladder. Here, we describe a newly devised library of
cloned STR alleles. The library covers alleles X and Y for the sex-determining locus Amelogenin and 259
other alleles for 22 autosomal STR loci (TPOX, D3S1358, FGA, D5S818, CSF1PO, D7S820, D8S1179, TH01,
vWA, D13S317, D16S539, D18S51, D21S11, D2S1338, D6S1043, D12S391, Penta E, D19S433, D11S4463,
D17S974, D3S4529 and D12ATA63). New primers were designed for all these loci to construct
recombinant plasmids so that the library retains core repeat elements of STR as well as 50 - and 30 anking sequences of 500 base pairs. Since amplicons of commercial STR genotyping kits and systems
developed in laboratories are usually distributed from 50 to <500 base pairs, this library could provide
universal templates for allelic ladder preparation. We prepared three different sets of allelic ladders for
this locus TH01 and an updated version of an allelic ladder for the DNATyper119 multiplex system using
these plasmids to conrm the suitability of the library as a good source for allelic ladder preparation.
Importantly, the authenticity of each construct was conrmed by bidirectional nucleotide sequencing
and we report the repeat structures of the 259 STR alleles. The sequencing results showed all repeat
structures we obtained for TPOX, CSF1PO, D7S820, TH01, D16S539, D18S51 and Penta E were the same as
reported. However, we identied 102 unreported repeat structures from the other 15 STR loci,
supplementing our current knowledge of repeat structures and leading to further understanding of these
widely used loci.
2014 Elsevier Ireland Ltd. All rights reserved.

Keywords:
Forensic science
Short tandem repeat (STR)
Allelic ladder
Recombinant plasmid
Repeat structure

1. Introduction
During the past two decades, short tandem repeat (STR)
genotyping has emerged as the dominant technique for human
identity determination and paternity testing [14]. Allelic ladders,
which serve as standards in STR analysis, are necessary for
adjusting for different sizing measurements obtained from
different instruments and under different conditions used by
various laboratories, which makes allelic ladders an indispensable
component of commercial kits and newly developed STR analysis
systems.

* Corresponding authors. Tel.: +86 10 66269184; fax: +86 10 63267051.

E-mail addresses: wangle_02@163.com (L. Wang), zhaoxchun@sina.com
(X.-C. Zhao), yejian77@126.com (J. Ye).
http://dx.doi.org/10.1016/j.fsigen.2014.06.005
1872-4973/ 2014 Elsevier Ireland Ltd. All rights reserved.

Allelic ladders are prepared on the basis of the polymerase

chain reaction (PCR) for which the templates can be obtained by
three optional strategies. The rst strategy is to cut gels after
polyacrylamide gel electrophoresis (PAGE) to collect separated
fragments of target alleles [5]. The DNA template thus prepared is
not suitable for long-term storage because it is inclined to degrade.
Another disadvantage of this strategy is the amount of DNA
dissolved from gels is usually very limited. Once the entire DNA
template is used, the demanding procedures of PAGE and gel
cutting have to be repeated. Additionally, the concentration and
purity of prepared DNA templates from different batches of
experiments can vary signicantly. The second strategy is based on
plasmid construction. Each allele is amplied by PCR and inserted
into plasmids that can be transformed easily into Escherichia coli
[6]. Thus, large amounts of genetically engineered plasmids
bearing STR fragments, which are ideal PCR templates for allelic

L. Wang et al. / Forensic Science International: Genetics 12 (2014) 136143

ladder preparation, can be prepared conveniently from bacterial

cultures. The amount of DNA obtained as PCR templates from one
batch of plasmid preparation is more than sufcient for long-term
allelic ladder preparation. Prepared DNA plasmids are usually of
high quality and can be stored stably and conveniently. The third
strategy is to use prepared allelic ladders as PCR templates.
Obviously, this strategy would be invalid if we did not have
prepared allelic ladders, which is an unavoidable situation when
we develop allelic ladders for new STR analysis systems. Further,
this strategy tends to introduce contamination and the intra-locus
and inter-locus imbalances are easily magnied.
New STR analysis systems are being reported each year. The
STR loci used by different systems normally overlap, whereas
the primer sets used for the same locus can be different. Allelic
ladders for the same locus cannot be shared across different
systems. In this study, we modied the second strategy and
constructed a library of STR alleles. A total of 261 alleles of 22
autosomal STR loci and the sex-determining locus Amelogenin
were inserted into plasmids. The inserted fragment included the
repeat sequences as well as 50 - and 30 -anking regions long
enough to cover all the primer binding sites reported for each
locus. Thus, the library can supply universal templates for allelic
ladder preparation.
2. Materials and methods

amplitude threshold set at 50 relative uorescence units (RFUs)

for all colors.
2.4. Plasmid construction
Target fragments (including core repeats and anking regions)
were amplied directly from blood samples kept on lter paper
using the primers given in Table 1. Homozygous blood samples
were used as PCR templates with higher priority, whereas the
heterozygous samples could be the choice for some alleles with
relatively low frequency. For heterozygous samples, different
alleles could be separated when single bacterial clones were picked
and cultured to yield plasmids and the allele of interest selected via
genotyping experiments. PCR was done in a total volume of 50 ml
containing 20 mM TrisHCl pH 8.3, 50 mM KCl, 1.6 mM MgCl2,
0.8 mg/ml bovine serum albumin, 0.2% Tween-20, 3.2% glycerol,
0.02% NaN3, 200 mM each dNTP, 0.5 mM each primer, 5 U of
TaqDNA polymerase (Roche) and two 1.2 mm diameter circles of
storage paper. Thermal cycling parameters were similar to those
described above, except the denaturingannealingextension
cycle was repeated 40 times. Amplied DNA was subjected to
Table 1
Primer sequences used for molecular cloning.
Locus

Primer

Length of
anking
sequence (bp)

TPOX

F: CACATACCCAGCACACACCTG
R: CAGGTGTGTGCTGGGTATGTG
F: AAATGCTATCTGGCTGAACGT
R: GCTGCTGATTTGAATGGGTC
F: CATTAGGGTTAGGAAACATTG
R: GGCTGAGGGCTCAGAGGTGTG
F: TTCACTGTGACCTAAGACAA
R: TGGGCCTCAGCTTGCTTTGC
F: CCAACCCAGGCTTCGAGAAG
R: CAGGATTAGAAGCCGTAACTGAA
F: TCAATCTGGGAATCGAGAAC
R: AGGGTTCATGGACCCTAATG
F: CGTGCCCAGCTAGCTAAT
R: AGACCGTGTCTTGCTCTGT
F: TACCTGGAAATGACACTGCTACAACT
R: TCAGGGTCCATGCAAACC
F: CCTTTGTCCCAGTCCTGTCC
R: GGAGACAGAGATTACATGGGTTAGG
F: CAGGAAATAGATGGGATC
R: CTGCATATGAAGTTACAGTAAG
F: CATGTCAGCTTCATCCTCTTC
R: GAGCACGACTCTCCTTACAAT
F: AGGCGTGGGGGAACAGGCATTA
R: CTTAATTACATGTGTCAAGA
F: TGATTGTTTTAGTAGTTATAGG
R: CAGATCTGTACAGCTGTCCT
F: GATCACTGGAGAATAAATGTG
R: ACACAGGCTTGAGGCCAA
F: AGTAGTGGTGAAGGGGACAA
R: ATGATTCCCAAGGGGCTGTC
F: AGTGATCTCATGAAAAGTTC
R: CCGCATTAATATGTTGACCTC
F: AGCCTGGCAACAGAGCCA
R: CATAGCAGTATCACTAGACTTG
F: GAAATGATTGAGAACTCTAC
R: TTCTGTCGTCTCTCTCATTC
F: TGCAATGTATAGGTCGTTT
R: CCATCTTGAAATTCTTAATA
F: AGGATGATTTATCCATTAGGCATTG
R: CTTGACTCCTGGAAGAACC
F: CAGCACTGGGGCTTTAGG
R: AAACCCCAATCTTCTCCC
F: AAGAAAACTGAAACTGCA
R: TAAATGTGCTCAGTCCA
F: CTGCCTCCTGGGTTT
R: AACTTCCCTGACCTCCTA

500
522
465
759
500
500
500
500
500
525
500
500
536
587
226
479
500
607
514
558
500
564
500
500
500
500

500
500
519
500
501
577
545
500
450
500
620
816
613
550
482
395
519
482

2.1. Samples
Blood samples were collected from healthy Chinese individuals, mainly from six provinces of China (Beijing, Hebei,
Shandong, Henan, Chongqing and Guangdong), and kept on
lter paper. Written informed consent was given by blood
donors and this work was approved by the Ethical Review
Board of the Institute of Forensic Science, Ministry of Public
Security of China.
2.2. Amplication for genotyping
Supplementary Table 1 gives the primer sequences used for STR
typing for D11S4463, D17S974, D3S4529 and D12ATA63. PCRs
were performed in a total volume of 10 ml containing 20 mM Tris
HCl pH 8.3, 50 mM KCl, 1.6 mM MgCl2, 0.8 mg/ml bovine serum
albumin, 0.2% (v/v) Tween-20, 3.2% (v/v) glycerol, 0.02% (w/v)
NaN3, 200 mM each dNTP, 0.32 mM each primer, 1 U of TaqDNA
polymerase (Roche) and a 1.0 mm diameter circle of storage paper.
For the other 19 loci, genotyping was done with the DNATyper119
multiplex system (developed by our institute and available
commercially in China; see Supplementary Table 2 for further
information). Amplication was done with the GeneAmp1 9700
thermal cycler (Applied Biosystems, USA). Pre-PCR denaturation
occurred at 95 8C for 11 min. This was followed by 28 cycles of
denaturing at 94 8C for 30 s, annealing at 59 8C for 2 min, extension
at 72 8C for 1 min and a nal extension step at 60 8C for 60 min.
2.3. Electrophoresis, detection and analysis
A 1 ml sample of PCR products was added to 10 ml of
deionized formamide containing an internal size standard.
Samples were denatured for 3 min at 95 8C followed by cooling
on ice for 10 min. All samples were separated on the 3100
Genetic Analyzer (Applied Biosystems) using POPTM-7 polymer
(Applied Biosystems) and a 36 cm capillaryTM (Applied Biosystems). All samples were injected for 10 s at 3 kV. The PCR
products were separated at 15 kV at a run temperature of 60 8C.
Initial fragment sizing and allele calling were done with
GeneMappler1ID v3.2 (Applied Biosystems) with the peak

137

D3S1358
FGA
D5S818
CSF1PO
D7S820
D8S1179
TH01
vWA
D13S317
D16S539
D18S51
D21S11
Amelogenin
D2S1338
D6S1043
D12S391
Penta E
D19S433
D11S4463
D17S974
D3S4529
D12ATA63

138

L. Wang et al. / Forensic Science International: Genetics 12 (2014) 136143

agarose electrophoresis, recovered from gels, inserted into the

pMD-18T vector (TaKaRa) and transformed into JM109 competent
cells. Positive clones were conrmed by STR genotyping and
bidirectional nucleotide sequencing.
2.5. Preparation of allelic ladders
Puried plasmids were digested with SalI (TaKaRa) and EcoRI
(TaKaRa) for 1 h then incubated at 95 8C for 5 min to denature and
permanently inactivate the restriction enzymes. For each locus,
digested DNA for single alleles was mixed, diluted, amplied, reanalyzed and balanced to produce a single ladder for each locus,
which were mixed, balanced and puried while concentrated as
the nal allelic ladder for a multiplex STR system.
3. Results
3.1. Molecular cloning of STR alleles
We collected 259 alleles for 22 autosomal STR loci and cloned
them into the pMD-18T vector (Fig. 1). Similarly, we constructed
recombinant plasmids for the X and Y alleles for the sexdetermining locus Amelogenin. Primers used for cloning are given
in Table 1. Instead of using any reported primer sequence or the
primers we used for genotyping in this work, we redesigned
primers for all 23 loci. For each STR locus, both forward and reverse
primer binding sites were selected far away (500 base pairs) from
repeat sequences. Thus, the amplied fragments preserved longer
anking sequences on both sides of the core repeating elements
(Fig. 1). Smaller PCR products are usually pursued in developing
STR typing systems. For example, Mini-STR systems are good
solutions for degraded DNA sample typing [6]. By contrast,
amplication of longer fragments would suffer technical difculties in several aspects of genotyping; e.g. amplication of larger

Fig. 1. Diagram for molecular cloning of STR alleles. Core repeat elements are
represented by small green boxes and the 50 - and 30 -anking sequences are shown
in light blue. In all, 259 STR fragments were inserted into the pMD18-T vector.

fragments tends to be inhibited by PCR inhibitors. To our

knowledge, almost all STR analysis systems, including commercial
kits and those described in the literature, constrain the size of PCR
products to <500 base pairs. The fragments we cloned should
cover all primer binding sites for those STR analysis systems. In
other words, the recombinant plasmids we constructed could be a
library of universal templates for allelic ladder preparation.
The sex-determining locus Amelogenin is not an STR. The X
chromosome version of the Amelogenin gene contains a 6 bp
deletion compared to the Y chromosome version. We also cloned a
539 bp conserved fragment of the Y version and its counterpart of
the X chromosome into the pMD18-T vector (Supplementary Fig. 1).
3.2. Preparation of allelic ladders
To conrm that the recombinant plasmids we constructed in
this work were universal templates for allelic ladder preparation,
we took TH01 as an example, and prepared three different sets
of allelic ladders for this locus using the same recombinant
plasmids as PCR template (Fig. 2). In detail, the three sets of

Fig. 2. Allelic ladders for the TH01 locus. Nine recombinant plasmids were used as PCR templates. Primer sequences were adopted from (A) the DNATyper119 system, (B) the
PowerPlex 16 kit [7] and (C) published work [8].

L. Wang et al. / Forensic Science International: Genetics 12 (2014) 136143

139

Fig. 3. Allelic ladder for the DNATyper119 system prepared using the recombinant plasmids as template.

TH01 primer sequences were adopted from the DNATyper119

system, the PowerPlex 16 kit [7] and published work [8],
respectively. For the convenience of comparison, all three sets of
primers in Fig. 2 were labeled with the same uorescent dye
carboxytetramethylrhodamine (TAMRA), but this allelic library
is suitable for preparing ladders labeled with various uorescent
dyes (data not shown).
In addition, we used 217 of the 261 plasmids and prepared an
updated allelic ladder for the DNATyper119 multiplex system
(Fig. 3). Compared to the old version (Supplementary Fig. 2)
prepared by the traditional gel-cutting strategy, the background
of the updated allelic ladder is clear. Thanks to the convenience
of plasmid concentration determination, balances among alleles
(both intra-locus and inter-locus balance) can be achieved
easily.
3.3. Repeat structure analysis of cloned STR alleles
We sequenced all 261 recombinant plasmids bidirectionally to
study the repeat structures of each allele. The sequencing results
showed that all repeat structures we obtained for TPOX, CSF1PO,
D7S820, TH01, D16S539, D18S51 and Penta E were the same as
reported; however, we identied 102 unreported repeat structures
from 15 STR loci. Representative results of the repeat structures for
the alleles cloned are summarized in Table 2. See Supplementary
Table 3 for the entire summary of repeat structures.
Although alleles 12 and 20 of D3S1358 are present in
commercial allelic ladders [9], their repeat structures have not
been reported previously. We identied a new repeat structure
for allele 19, which contains one less [TCTG] unit but one more
[TCTA] unit compared to the previously reported repeat
structure.
For the FGA locus, we identied a repeat structure for allele 13,
which is a rare allele and is absent from commercial allelic ladders.
New variations for alleles 29 and 30 are given in Table 2.

The core sequence for D5S818 is strictly [AGAT] repeats

according to sequenced alleles [10]. We conrmed the repeat
structure of allele 16 is [AGAT]16 as expected.
The core sequence for D8S1179 is composed of [TCTA] and
[TCTG] repeats. We discovered new arrangements of [TCTA] and
[TCTG] units for alleles 12, 15 and 17.
The repeat structure of allele 13 for the vWA locus we
identied was different from all three reported structures
[11,12], but it resembled the repeat structure of allele 14, which
was rst reported by Brinkmann et al. [12] and conrmed by
our work. The only difference was that allele 13 possessed one
less [TCTA] unit compared to allele 14. The repeat structure for
allele 21 described here is new, which is the rst case of a vWA
allele containing as many as ve consecutive [TCTG] repeats in
its core sequence.
The repeat structure of the D13S317 locus was intriguing.
According to Lins et al. [10], the D13S317 allele N contains N
[TATC] repeats. However, our sequencing results led to a deeper
understanding of this locus and showed D13S317 allele N might
contain N, N + 1 or N + 2 [TATC] repeats, depending on whether
2, 1 or 0 [AATC] units followed, which we named the [AATC] tail.
Fig. 4 shows the 50 - and 30 -anking sequences are highly
conserved and the core repeats at positions 114 are composed
of variable number of [TATC] repeats. However, the sequence
elements at positions 15 and 16 could be [TATC] or [AATC]; as a
result, some D13S317 alleles might contain one or two more
[TATC] repeats than expected. Genotyping of all the alleles was
done with the DNATyper119 system and conrmed with the
IdentilerTM kit.
The repeat structure of D21S11 can be summarized as:
[TCTA]m[TCTG]n[TCTA]3TA[TCTA]3TCA[TCTA]2TCCATA[TCTA]p
where m, n and p are integrals. Different combinations of m, n and p
with the same sum are indistinguishable in DNA proling and are
considered as the same allele. Owing to this complexity, more than
one repeat structure has been reported for each allele of D21S11.

L. Wang et al. / Forensic Science International: Genetics 12 (2014) 136143

140

Table 2
Representative results of the repeat structures for the alleles cloned.
Locus

Allele

Repeat structure in this work

Repeat structure reported earlier

Reference

D3S1358

12
19
20

TCTA[TCTG]2[TCTA]9
TCTA[TCTG]2[TCTA]16
TCTA[TCTG]3[TCTA]16

No report
TCTA[TCTG]3[TCTA]15
No report

[16]

13
29
30

[TTTC]3TTTTTTCT[CTTT]5CTCC[TTCC]2
[TTTC]3TTTTTTCT[CTTT]21CTCC[TTCC]2
[TTTC]3TTTTTTCT[CTTT]1CTCT[CTTT]20CTCC[TTCC]2

No report
[TTTC]3TTTTTTCT[CTTT]15CCTT[CTTT]5CTCC[TTCC]2
[TTTC]3TTTTTTCT[CTTT]16CCTT[CTTT]5CTCC[TTCC]2

D5S818

16

[AGAT]16

No report

D8S1179

12
15
17

[TCTA]1[TCTG]1[TCTA]10
[TCTA]2[TCTG]1[TCTA]12
[TCTA]2[TCTG]1 [TCTA]14

[TCTA]12
[TCTA]1[TCTG]1 [TCTA]13
[TCTA]2[TCTG]2 [TCTA]13

[18]
[18]
[18]

vWA

13

[TCTA]1[TCTG]1[TCTA]1[TCTG]4[TCTA]2
TCCA[TCTA]3TCCATCCA

21

[TCTA]1[TCTG]5[TCTA]15TCCATCTA

[TCTA]2[TCTG]4[TCTA]3TCCA[TCTA]3
[TCTA]1[TCTG]4[TCTA]8TCCATCTA
[TCTA]1[TCTG]4[TCTA]10
[TCTA]1[TCTG]4[TCTA]16TCCATCTA

[11]
[12]
[12]
[12]

D13S317

5
8
9
10

[TATC]5AATC AATC
[TATC]8AATC AATC
[TATC]9AATC AATC
[TATC]12

11
12
13
14

[TATC]12AATC
[TATC]13AATC
[TATC]14AATC
[TATC]15AATC

No report
[TATC]8
[TATC]9
[TATC]10
[TATC]10AATC
[TATC]11
[TATC]12
[TATC]13
[TATC]14

[10]
[10]
[10]
[10]
[10]
[10]
[10]
[10]

26

[TCTA]6[TCTG]5[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]7
[TCTA]5[TCTG]5[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]10

FGA

D21S11

28

30.3
31

31.3
33
34

35

36

37

D6S1043

9
10
14
16
17
18
18.2
19
20
20.3

[TCTA]6[TCTG]5[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]5TCA[TCTA]6
[TCTA]4[TCTG]6[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]13

[TCTA]7[TCTG]5[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]5TCA[TCTA]6
[TCTA]9[TCTG]5[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]11
[TCTA]8[TCTG]5[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]13

[TCTA]9[TCTG]5[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]13

[TCTA]9[TCTG]5[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]14

[TCTA]9[TCTG]5[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]15

[AGAT]9
[AGAT]10
[AGAT]14
[AGAT]10[ACAT]1[AGAT]5
[AGAT]11[ACAT]1[AGAT]5
[AGAT]12[ACAT]1[AGAT]5
[AGAT]12AT[ACAT]1[AGAT]5
[AGAT]13[ACAT]1[AGAT]5
[AGAT]13GGAT[ACAT]1[AGAT]5
[AGAT]12GAT[AGAT]2[ACAT]1[AGAT]5

[17]
[17]

[TCTA]4[TCTG]6[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]8
[TCTA]4[TCTG]6[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]10
[TCTA]5[TCTG]6[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]9
No report

[19]

[TCTA]5[TCTG]6[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]12
[TCTA]6[TCTG]5[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]12
[TCTA]6[TCTG]6[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]11
[TCTA]7[TCTG]5[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]11
No report

[11]

[TCTA]5[TCTG]6[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA [TCTA]14
[TCTA]10[TCTG]5[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]11
[TCTA]5[TCTG]6[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]15
[TCTA]11[TCTG]5[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]11
[TCTA]10[TCTG]5[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]12
[TCTA]10[TCTG]5[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]13
[TCTA]10[TCTG]6[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]12
[TCTA]11[TCTG]5[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]12
[TCTA]9[TCTG]11[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]12
[TCTA]11[TCTG]5[TCTA]3TA[TCTA]3TCA
[TCTA]2TCCATA[TCTA]13

[20]

No report
No report
No report
No report
No report
[AGAT]6[ACAT]1[AGAT]11
No report
No report
[AGAT]6[ACAT]1[AGAT]1[ACAT]1[AGAT]11
No report

[19]
[20]

[19]
[20]
[21]

[22]
[20]
[22]
[11]
[22]
[22]
[11]
[22]
[11]

[13]

[13]

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141

Table 2 (Continued )
Allele

Repeat structure in this work

Repeat structure reported earlier

Reference

21
21.3
23

[AGAT]15[ACAT]1[AGAT]5
[AGAT]13GAT[AGAT]2[ACAT]1[AGAT]5
[AGAT]17[ACAT]1[AGAT]5

No report
[AGAT]6[ACAT]1[AGAT]2AGT[AGAT]12
No report

[13]

14
16
19
19.3
22

[AGAT]7[AGAC]6[AGAT]1
[AGAT]8[AGAC]7[AGAT]1
[AGAT]11[AGAC]8
[AGAT]2GAT[AGAT]9[AGAC]7[AGAT]1
[AGAT]13[AGAC]8[AGAT]1

26

[AGAT]16[AGAC]10

[14]
[14]
[23]
[14]
[14]
[14]
[14]

27

[AGAT]18[AGAC]8[AGAT]1

No report
[AGAT]9[AGAC]6[AGAT]1
[AGAT]12[AGAC]6[AGAT]1
[AGAT]5GAT[AGAT]7[AGAC]6[AGAT]1
[AGAT]15[AGAC]6[AGAT]1
[AGAT]12[AGAC]10
[AGAT]17[AGAC]8[AGAT]1
[AGAT]17[AGAC]9
No report

D19S433

4
6.2
8
11.2
12.2
13.2
14.2
15.2
16.2
17.2
18.2

[AAGG]1[AAAG]1[AAGG]1[TAGG]1[AAGG]2
[AAGG]1AA[AAGG]1[TAGG]1[AAGG]5
[AAGG]1[AAAG]1[AAGG]1[TAGG]1[AAGG]6
[AAGG]1AA[AAGG]1[TAGG]1[AAGG]10
[AAGG]1AA[AAGG]1[TAGG]1[AAGG]11
[AAGG]1AA[AAGG]1[TAGG]1[AAGG]12
[AAGG]1AA[AAGG]1[TAGG]1[AAGG]13
[AAGG]1AA[AAGG]1[TAGG]1[AAGG]14
[AAGG]1AA[AAGG]1[TAGG]1[AAGG]15
[AAGG]1AA[AAGG]1[TAGG]1[AAGG]16
[AAGG]1AA[AAGG]1[TAGG]1[AAGG]17

No
No
No
No
No
No
No
No
No
No
No

D17S974

13

[CTAT]13

No report

Locus

D12S391

report
report
report
report
report
report
report
report
report
report
report

Fig. 4. Sequence alignment of the D13S317 alleles. The [TATC] repeat units are highlighted with an alternating yellow and green background, whereas [AATC] tails have a
magenta background. The highly conserved 50 - and 30 -anking sequences have a blue background. Repeat units are indicated with red numbers.

142

L. Wang et al. / Forensic Science International: Genetics 12 (2014) 136143

Our sequencing results provide new repeat structures for alleles

26, 28, 31, 33, 34, 35, 36 and 37, extending our basic knowledge of
this locus. We also provide sequence details for microvariant
alleles 30.3 and 31.3, in which a repeat structure of a TCA
trinucleotide is inserted into the [TCTA]p unit at the end of the
repeat core and separate this [TCTA]p unit into two nonneighboring [TCTA] repeats.
Repeat structures for some alleles of D6S1043 were reported
by Phillips et al. [13]. Our sequencing results agree well with
their work for alleles 11, 12, 13 and 15, but differ signicantly for
alleles 18, 20 and 21.3. We repeated and checked our DNA
proling and sequencing results, further conrming the data
given in Table 2. This discordance might be owing to the
collection of samples from different areas of the world. Further
work, perhaps by a third research group, is required to fully
address this issue.
For D12S391, repeat structures for alleles from 1526 have
been reported [14]. Our sequencing results identied new repeat
structures for alleles 16, 19, 19.3, 22 and 26, and released repeat
structures for rare alleles 14 and 27, which exhibit patterns similar
to other alleles.
Among all the loci analyzed in this study, D19S433 has
the greatest number of x.2 microvariant alleles. We discovered
that all these alleles contain an [AA] dinucleotide insertion after
the rst [AAGG] repeat. Surprisingly, we discovered a rare allele
with the repeat [AAGG]1[AAAG]1[AAGG]1[TAGG]1[AAGG]2,
which should be named allele 4 according to the DNA proling
result and the naming recommendations of the International
Society of Forensic Genetics [15]. The most condensed
allele discovered before this study was allele 5.2. This result
expanded our understanding of this locus signicantly,
and should be taken into consideration in DNA proling
practices.
For loci D2S1338, D3S4529, D12ATA63 and D17S974, the repeat
structures
are
generally
accepted
to
consist
of
[TGCC][TTCC](GTCC[TTCC]), [ATCT]ATTT[ATCT], [TAA][CAA] and
[CTAT] repeats, respectively. However, detailed sequences for
every allele except those among the GenBank sequences remain to
be elucidated (see Table 2). Further, we identied allele 13 as a new
member of the D17S974 allelic family.
4. Discussion
This work demonstrated a modied method of allelic ladder
preparation dependent on a novel library of STR alleles. Although
the library covers the X and Y alleles for Amelogenin and 259
alleles of 22 widely used autosomal loci, further expansion of the
library for other STR loci is currently undergoing. The plasmids
constructed here bear long anking sequences on both sides of core
repeats, so the library is not tailored for a single primer set but is
suitable for various primers in different multiplex systems. More
than 100 new repeat structures from 15 STR loci were identied in
this work, extending our knowledge of these loci substantially.
Specically, we discovered the repeat structure for D13S317 alleles
was more complicated than expected. One or two [AATC] units,
named here as [AATC] tail, might be present or absent following
[TATC] repeats (Fig. 4). Some rare alleles were identied, including
alleles 14 and 27 of D12S391, allele 4 of D19S433 and allele 13 of
D17S974. These rare alleles would contribute to accurate
genotyping. It would be highly informative and helpful for
individual identication if arrestees from criminal cases were
genotyped and found to bear such rare alleles.
Next generation sequencing (NGS) technologies are rapidly
evolving and slowly being adopted by forensic laboratories.
As such, it is likely that STR typing will be performed by NGS in
the future [24]. The repeat structures reported in this work

could be used as reference sequences to assist NGS-based STR

data analysis, and our recombinant plasmids have the potential
of being used as reference materials to verify concordance
between capillary electrophoresis generated and NGS generated
STR data.
Acknowledgements
The authors thank Dr. John M. Butler for critical discussion and
reading of the manuscript and Dr. Wanli Bi for technical assistance.
This study was supported, in part, by grants 2012JB001,
2013JBYY009 and 2014JB001 to the Institute of Forensic Science,
Ministry of Public Security of China and, in part, by grant
2013GABJC035 to the Ministry of Public Security of China.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.fsigen.2014.06.005.
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