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Forensic Science International: Genetics 2 (2008) 379–381

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Letter to the Editor

Allele frequencies of six miniSTR loci in the population of Northern Portugal

Abstract
A possible approach to try to recover information from degraded DNA is to reduce the size of the PCR products by designing primers that bind
as close as possible to the STR repeat region, known as miniSTRs. Allele frequencies and forensic parameters for the six miniSTRs loci D1S1677,
D2S441, D4S2364, D10S1248, D14S1434 and D22S1045 were investigated in a sample group consisting of 228 anonymous apparently healthy
unrelated individuals living in North of Portugal. The results show that all loci were in Hardy–Weinberg equilibrium. The combined power of
discrimination and power of exclusion for the six loci were 0.99999 and 0.9789, respectively. All but one (D4S2364) loci showed a moderate degree
of polymorphism (observed heterozygosity >0.6). The allele sizes ranged between 66 and 118 bp in our population, which is beneficial for typing
degraded samples than those of a commercial STR kit.
# 2008 Elsevier Ireland Ltd. All rights reserved.

Keywords: North of Portugal; Population genetics; MiniSTRs

Population: 228 anonymous apparently healthy unrelated
individuals living in North of Portugal. The vast majority of
individuals are Caucasian. The samples were obtained from
individuals involved in paternity testing. The collection took
place after informed consent was acquired.
DNA extraction: Buccal swabs were collected and air dried.
DNA was extracted using Chelex1 100 resin [1].
PCR: Two triplex PCRs – miniplex 01 (D10S1248,
D14S1434, D22S1045) and miniplex 02 (D1S1677,
D2S441, D4S2364) – were performed with the primer sets
designed by Coble and Butler [2]. Fluorescently labeled
primers were purchased from Applied Biosystems and
unlabeled primers from Operon (Germany). PCR reactions
were carried out with the Qiagen1 Multiplex PCR kit in a total
volume of 12.5 mL containing 6.25 mL of Qiagen1 Multiplex
PCR master mix, 1.25 mL of Q-Solution, 1.25 mL of 10
primer mix (for example, for miniplex 01 all the primers were
at 2.0 mM for the 10 solution) and 1 mL (1 ng/mL) of
genomic DNA. The first triplex PCR mixture contained
0.2 mM primer set of each marker D10S1248, D14S1434 and
D22S1045 labeled with fluorescent dye 6-FAM, PETand NED,
respectively. The other triplex PCR contained 0.3 mM primer
set of D1S1677 (NED), 0.15 mM of D2S441 (VIC) and 0.2 mM
of D4S2364 (6-FAM). Amplification was done with the
GeneAmp1 9700 (Applied Biosystems). Pre-PCR denatura-
tion was performed at 95 8C for 15 min followed by 30 cycles
of denaturing at 94 8C for 60 s, annealing at 55 8C for 90 s,
extension at 72 8C for 60 s and a final extension at 60 8C for
45 min.
Typing: The amplified products were separated and detected
by capillary electrophoresis on an ABI PRISM1 3100 Genetic
Analyzer (Applied Biosystems), using 1 mL of multiplex PCR
product mixed with 13.55 mL of Hi-Di formamide (Applied
Biosystems) and 0.45 mL of GeneScan-500LIZ size standard
(Applied Biosystems). The results were analysed with
GeneScan1 3.7 software (Applied Biosystems) and allele
designations were determined by comparison with a homemade
allelic ladder.
Allelic ladder generation and sequencing: Allelic ladder
was created by mixing and amplifying samples previously
typed so as to include all observed alleles in our sample group.
Briefly, a 1:1000 dilution of the amplified mixed samples was
prepared and then 2 mL of this dilution were amplified
individually for each set of primers using the thermocycling
parameters outlined above for the PCR. Allelic ladders were
amplified for 19 cycles instead of the standard 30 cycles. Allelic
designations were determined by sequencing two homozygote
samples of the allelic ladder to calibrate repeat number.
Commercial DNA standard 9947 (Applied Biosystems), was
genotyped and sequenced as standard reference. Nomenclature
was according to the new recommendations of Butler and
Coble [3] (also available at www.cstl.nist.gov/div831/strbase/
miniSTR).
Results: Allele frequencies are shown in Table 1. Forensic
statistical parameters are summarised in Table 2. Exact
test of population differentiation between this sample of
Portugal and other populations is summarised in Table 3.
Comparison of observed heterozygosity with other 15
common forensic STRs in similar population is shown in
Table 4.
Quality control: Proficiency testing of the GEP-ISFG WG
(http://www.gep-isfg.com).

1872-4973/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.fsigen.2008.05.003
380 Letter to the Editor / Forensic Science International: Genetics 2 (2008) 379–381
Analysis of data: Allele frequencies were calculated and
the Hardy–Weinberg equilibrium was tested using the exact
test, both involving the GENEPOP (Version 3.4) software
package. Bonferroni correction assumes that a 0.05 sig-
nificance level used for six tests (one per locus) yields an
actual significance of 0.008 [4]. The potential usefulness of
the considered loci was assessed by calculating statistical
parameters of forensic interest using homemade software.
Exact test of population differentiation based on allele
frequencies was carried out with the program Arlequin Ver
3.1.
Access to the data: See electronic supplementary data.
Other remarks: The observed allele sizes ranged between
66 and 118 bp in our population. The results show that all
but one (D1S1677) loci were in Hardy–Weinberg equili-
brium ( p > 0.05) (Table 2); however if Bonferroni correc-
tion is used the departures observed at this locus are
not significant. The independence of loci was also
verified.
The power of discrimination and power of exclusion were
the lowest for D4S2364 (0.7285 and 0.3197, respectively)
and the highest for D2S441 (0.9057 and 0.5571, respec-
tively). The combined power of discrimination and power of
exclusion for the six loci were 0.99999 and 0.9789,
respectively.
A comparison of the allele frequencies in the population
under study has been performed with other studies (Table 3):
three U.S. populations [2], Caucasian, African American and
Hispanic; Italy [5]; Spain [6]; three Singapore populations,
Chinese, Malay and Indian [7]; Koreans [8]; Japan [9]; two
ethnic populations in China [10], Han ethnic and Korean
ethnic. The most statistically significant differences were
verified between Portuguese and African American, and
between Portuguese and Asian populations ( p < 0.05). As
expected, the Portuguese versus Spanish pair has no
significant differentiation for all markers, probably due to
the existence of a same group of ancestral individuals
and geographical proximity that allow a non-negligible
genetic flow between the two populations over the last
generations. The pairs Portuguese versus U.S. Caucasians
and Portuguese versus Italy have no significant differences
also due to the existence of a same group of ancestral
individuals.
Except for D4S2364, all loci exhibited an observed
heterozygosity greater than 0.6, which indicated a moderate
degree of polymorphism. A comparison of the observed
heterozygosity values with 15 other STRs obtained from
earlier study genotyping [11] for similar population is
summarised in Table 4. Two of the miniSTRs have
comparatively medium level of heterozygosity, while
the other four miniSTRs have lower heterozygosity
values.
In conclusion, a Northern Portugal population database has
been established for the six miniSTR systems studied;
therefore, these markers can now be used for personal
identification purposes in this population.
This paper follows the guidelines for publication of
population data requested by the journal [12].
Acknowledgement
We want to express our gratitude to Dr. Peter Vallone
(Biochemical Science Division, National Institute of Standards
and Technology, USA) for reviewing this paper.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.fsigen.2008.05.003.
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Arlindo M. Lagoa*
Medical Faculty, Oporto University, Portugal
Teresa V. Martins
Master Degree Student at National Institute
of Legal Medicine, I.P., North Delegation, Portugal
 Letter to the Editor / Forensic Science International: Genetics 2 (2008) 379–381 381

Laura M. Cainé *Correspondence address:

National Institute of Legal Medicine, I.P., Delegação do Norte do Instituto Nacional
North Delegation, Portugal de Medicina Legal, Jardim Carrilho Videira,
4050-167 Porto, Portugal.
M. Fátima Pinheiroa,b,c
a
National Institute of Legal Medicine, I.P., Tel.: +351 22 207 38 50; fax: +351 22 332 59 31
North Delegation, Portugal E-mail address: arlindolagoa@gmail.com
b
Health Sciences Faculty, (A.M. Lagoa)
Fernando Pessoa University, Oporto, Portugal
c
Abel Salazar Biomedical Institute, 13 December 2007
Oporto University, Portugal