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D18S535, D1S1656 and D10S2325: Three efficient short tandem repeats for
forensic genetics
Article in International Journal of Legal Medicine · October 1999
DOI: 10.1007/PL00007705 · Source: PubMed
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Int J Legal Med (1999) 112:360-363 ©Springer-Verlag 1999
ORIGINAL ARTICLE
P. Wiegand · M. V. Lareu · M. Schürenkamp ·
M. Kleiber · B. Brinkmann
D18S535, D1S1656 and D10S2325:
three efficient short tandem repeats for forensic genetics
Received: 10 August 1998 / Received in revised form: 5 November 1998
Abstract Three short tandem repeat (STR) polymor- D18S535 and D1S1656 are STRs containing 4 bp re-
phisms characterized by PCR product length < 175 bp were peat units as basic repeat motifs while D10S2325 shows a
investigated. D18S535 and D1S1656 contained a 4 bp unit pentameric repeat array.
as basic repeat motif, D10S2325 a 5 bp unit. The heterozy-
gosity rates were 0.76 (D18S535), 0.88 (D10S2325) and
0.90 (D1S1656), leading to a combined discrimination Material and methods
power of 0.9999. In contrast to D10S2325 and D18S535,
which showed a homogeneous repeat array without any Population sample: unrelated German Caucasians from the area
variation in the repeat motifs, repeat length and sequence around Halle/Saale. DNA extraction: Chelex-extracted DNA was
used as described previously (Wiegand et al. 1993a).
variation was found for D1S1656. Robust typing results
PCR reagents and thermocycler see Wiegand et al. (1998). PCR
could be observed for all three STRs using highly de-
primers and conditions:
graded DNA. D18S535 primer 1: 5¢ - TCA TGT GAC AAA AGC CAC AC,
primer 2: 5¢ - AGA CAG AAA TAT AGA TGA GAA TGC CA
(Lareu et al. 1998a); D1S1656 primer 1: 5¢ - GTG TTG CTC
Key words Short tandem repeats · Population genetics ·
AAG GGT CAA CT (Lareu et al. 1998b); primer 2: 5¢ - GAG
Sequencing data AAA TAG AAT CAC TAG GGA ACC; D10S2325 primer 1: 5¢ -
CTC ACG AAA GAA GCC TTC TG; primer 2: 5¢ - GAG CTG
AGA GAT CAC GCA CT (Lee et al. 1998); amplification proto-
Introduction col: 94°C - 1 min, 61°C - 1 min, 72°C - 1 min; 30 cycles.
Electrophoretical separation: the amplified alleles were re-
solved by high resolution polyacrylamide gel electrophoresis ac-
Robust typing results, high sensitivity and low suscepti- cording to Wiegand et al. (1993a, b) with the following modifica-
bility to degraded DNA are main criteria for the selection tions to improve the resolution of the PCR products:
of short tandem repeat (STR) systems, which may be thinner gels were prepared (0.45 mm instead of 0.75 mm) and
the polyacrylamide/crosslinker (piperazindiacrylamide; Biorad, Ger-
added to an established STR package. Based on these cri-
many) relation was changed to 7% C and 3.7% T respectively.
teria, systems with short fragment lengths (< 175 bp), low
STR sequencing: isolation of silver stained fragments, subse-
susceptibility to PCR slippage artifacts (also known as quent Taq-cycle-sequencing and sequence analysis was performed
stutter bands; Walsh et al. 1996) and highly reproducible as described previously (Brinkmann et al. 1998a).
Statistical analysis: test for heterogeneity between populations:
typing results were tested. A further relevant point of se-
R · C contingency table (G. Carmody, Ottawa, Canada) discrimi-
lection criteria was the heterozygosity rate, which should
nation power according to Jones (1972); Hardy-Weinberg equilib-
be > 75% leading to a substantial information content. rium: exact test (Guo and Thompson 1992, HWE 3.0 software
from C. Puers, Münster, Germany).
P. Wiegand (Y) · M. Kleiber
Institute of Legal Medicine, Results and discussion
Martin-Luther-Universität Halle-Wittenberg,
Franzosenweg 1, D-06112 Halle/Saale, Germany
Sequence data
M. V. Lareu
Institute of Legal Medicine, s/San Francisco s/n,
D18S535 (Lareu et al. 1998a) shows a conserved 4 bp and
E-15705 Santiago de Compostela, Spain
D10S2325 (Lee et al. 1998) a conserved 5 bp array (Table
M. Schürenkamp · B. Brinkmann 1). Two point mutations were found in the 3¢ flanking re-
Institute of Legal Medicine,
Westfälische Wilhelms-Universität Münster, gion of D10S2325. The sequence of alleles 7 and 8 is
Von-Esmarch-Str. 62, D-48129 Münster, Germany 5¢ FR - (TCTTA) TTG GGG GAG GCG GAC - 3¢ FR
n
P. Wiegand et al.: Three efficient short tandem repeats 361
Table 1 Sequencing data of
STR system Consensus sequence (FR = flanking region) Allele Fragment
the repeat array, allele defini-
nomen- length
tion and PCR product lengths
clature
of three STRs
D18S535 5¢FR - (GATA) - 3¢FR 9-16 130-158 bp
n
D1S1656 5¢FR - (TAGA) (TGA) (TAGA) (TAGG) (TG) - 3¢FR 9-19.3 125-168 bp
n 0-1 n 0-1 5
D10S2325 5¢FR - (TCTTA) - 3¢FR 6-17 113-168 bp
n
Table 2 STR allele frequency data from Halle area, North East area, Münster area) and a subpopulation from NW Spain
Germany. The nomenclature is given according to the number of showed no significant differences between the two Ger-
repeats. n = number of individuals. H = heterozygosity rate; MEC =
man subpopulations (p > 0.05) but significant differences
mean exclusion chance; DP = power of discrimination
between the German subpopulations and the Spanish pop-
STR D18S535 D1S1656 D10S2325 ulation data (Contingency test according to Carmody, Ot-
individuals n= 150 n= 150 n= 190 tawa, Canada; Wiegand et al. 1998). For D18S535 no sig-
nificant differences were found comparing German data
Allele
6 0.003 for Halle area and NW Spain (Fig.1b).
7 0.137 Considering studies with STRs of variant levels of
8 0.055 polymorphism among different ethnic groups (Brinkmann
9 0.086 0.003 0.113 et al. 1998a), one can explain the similarity of allele fre-
10 0.016 0.142 quency data in D18S535 according to a lower degree of
11 0.022 0.080 0.116 polymorphism caused by lower mutation rates in compar-
12 0.205 0.120 0.176 ison to D1S1656. Higher polymorphic systems with 4 bp
13 0.325 0.073 0.124 repeats as basic repeat motifs such as HumFIBRA and
14 0.203 0.102 0.082 HumACTBP2 show mutation rates > 0.4% leading to an
15 0.133 0.105 0.047
15.3 0.103
16 0.010 0.073 0.003
16.3 0.045
17 0.036 0.003
17.3 0.160
18 0.017
18.3 0.070
19.3 0.013
H 0.76 0.90 0.88
MEC 0.58 0.78 0.74
DP 0.91 0.97 0.96
whilefor the longer alleles 9 to 16 the following two tran-
Fig.1a D1S1656 allele frequency profiles: comparison of two
sitions could be detected: 5¢ FR - (TCTTA) n TTG GGG German (North East and North West Germany) and a Spanish
GAG ACG GGC - 3¢ FR. (North West Spain) subpopulation. n = number of individuals
D1S1656 (Lareu et al. 1998b) is characterized by a
more complex repeat structure containing variable num-
bers of 3 and 4 bp motifs leading to 1 bp differences for
alleles > 15 (Table 2).
D1S1656 and D10S2325 allele frequencies are below
0.2 while for D18S535 the dominant allele 13 exceeds
0.3. In relation to the allele distribution D1S1656 shows
the highest heterozygosity rate, followed by D10S2325
and D18S535. The combined power of discrimination (DP)
for the three STRs is 0.9999. No deviation from Hardy-
Weinberg equilibrium was found (p > 0.05).
Population genetic studies
Fig.1b D18S535 allele frequency profiles: comparison of a Ger-
Comparisons of population genetic data for D1S1656
man (North East Germany) and a Spanish (North West Spain) sub-
(Fig.1a) from two different German subpopulations (Halle population. n = number of individuals
362 P. Wiegand et al.: Three efficient short tandem repeats
Fig.2a PCR products of the STRs D10S2325, D1S1656 and There seem to be a correlation between the probability
D18S535 visualized after non-denaturing electrophoresis and sub- of polymerase slippage depending on the length of the re-
sequent silver staining. Lane 1,4 = allelic ladder for D10S2325 (al-
peat motifs, the homogeneity of the repeat stretch and in-
leles 7-15), D1S1656 (alleles 11,12,13,14,15,15.3,16.3,17.3) and
creasing number of repeats. For tetrameric STRs with
D18S5353 (alleles 9-16). Lane 2, 6, 10 = mixed experimental
bloodstain containing a 1:10 blood ratio of two different individu- longer and homogeneous repeat stretches, higher mutation
als (D10S2325 lane 2: alleles 7,11,12,13; D1S1656 lane 6: alleles rates were found compared to shorter stretches or com-
12,13,16,17; D18S535 lane 10: alleles 12,13,14). Lane 3, 7, 11 = posed STRs (Walsh et al. 1996; Brinkmann et al. 1998b).
highly degraded DNA extracted from a 10 years old femur
Pentameric repeat arrays (D10S2325) seem to have a
(D10S2325 lane 3: alleles 7,11; D1S1656 lane 7: alleles 12,15.3;
D18S535 lane 11: allele 12) clearly lower risk of slippage artifacts due to the increased
length of the repeat motif which is also well known for the
STR HumCD4 (Edwards et al. 1991).
To prove whether STRs which are characterized by rel-
atively short PCR products could improve the chance of
successful typing of highly degraded DNA, sceletal re-
mains were investigated. Compared to longer PCR prod-
ucts which failed to amplify highly degraded DNA such as
HumACTBP2 (Polymeropoulos et al. 1992), typing was
more robust and reliable using the present systems. Addi-
tionally, as expected, these STRs reached a typing sensi-
tivity < 100 pg template DNA and enabled successful typing
of experimental mixed stains using a 1:10 ratio (Fig.2a);
also typing of epithelial cells which were transferred from
the hands of the suspect onto the neck of the victim dur-
ing strangulation was successful for all three systems.
Fig.2b D18S535 - Lane 1: allelic ladder; lane 2: DNA extracted
from a 10-year-old femur using three times more DNA for PCR
compared to Fig. 2a, lane 11; the higher amount of DNA led to a
weak slippage band, 1 repeat shorter than the true homozygote pat- Conclusion
tern
PCR typing with these three STRs lead to reproducible and
robust results especially for highly degraded DNA. Using
increase in the evolution dynamic of ethnic diversity
non-denaturing electrophoretical conditions D10S2325
(Brinkmann et al. 1998b).
was the most suitable system for typing by side to side
comparision with allelic ladders.
Validity of typing
Using an improved electrophoretical separation method
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