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Forensic Science International: Genetics 2 (2008) 379–381 www.elsevier.com/locate/fsig
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 . 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 . 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 denaturation 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 1872-4973/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigen.2008.05.003
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  (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).
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 significance level used for six tests (one per locus) yields an actual significance of 0.008 . 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 equilibrium ( p > 0.05) (Table 2); however if Bonferroni correction 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, respectively). 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 , Caucasian, African American and Hispanic; Italy ; Spain ; three Singapore populations, Chinese, Malay and Indian ; Koreans ; Japan ; two ethnic populations in China , 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  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 . 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. References  P.S. Walsh, D.A. Metzer, Higuchi, Chelex-100 as a medium for simple extraction of DNA for PCR-based typing from forensic material, BioTechniques 10 (1991) 506–513.  M.D. Coble, J.M. Butler, Characterization of new miniSTR loci to aid analysis of degraded DNA, J. Forensic Sci. 50 (1) (2005) 43–53.  J.M. Butler, M.D. Coble, Authors’ response, J. Forensic Sci. 52 (2) (2007) 494.  B.S. Weir, Multiple Tests in Genetic Data Analysis II, Sinauer Associates, USA, 1996, p. 134.  A. Rocchi, I. Spinetti, C. Toni, S. Presciuttini, R. Domenici, Gene frequencies of six miniSTR in Tuscany (Italy), Int. Congr. Ser. 1288 (2006) 377–378.  P. Martı´n, O. Garcı´a, C. Albarra´n, P. Garcı´a, I. Yurrebaso, A. Alonso, Allele frequencies of six miniSTR loci (D10S1248, 14S1434, D22S1045, D4S2364, D2S441 and D1S1677) in a Spanish population, Forensic Sci. Int. 169 (2007) 252–254.  R.Y.Y. Yong, L.S.H. Gan, M.D. Coble, E.P.H. Yap, Allele frequencies of six miniSTR loci of three ethnic populations in Singapore, Forensic Sci. Int. 166 (2007) 240–243.  U. Chung, K.-J. Shin, M.J. Park, N.Y. Kim, W.I. Yang, S.-H. Cho, H.Y. Lee, Population data of nine miniSTR loci in Koreans, Forensic Sci. Int. 168 (2007) e51–e53.  H. Asamura, R. Uchida, K. Takayanagi, M. Ota, H. Fukushima, Allele frequencies of the six miniSTR loci in a population from Japan, Int. J. Legal Med. 120 (2006) 182–184.  R. Bai, M. Shi, X. Yu, J. Lv, Y. Tu, Allele frequencies for six miniSTR loci of two ethnic populations in China, Forensic Sci. Int. 168 (2007) e25–e28.  M.F. Pinheiro, L. Caine´, L. Pontes, D. Abrantes, G. Lima, M.J. Pereira, P. Rezende, Allele frequencies of sixteen STRs in the population of Northern Portugal, Forensic Sci. Int. 148 (2005) 221–223.  P. Lincoln, A. Carracedo, Publication of population data of human polymorphisms, Forensic Sci. Int. 110 (2000) 3–5.
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
Laura M. Caine´ National Institute of Legal Medicine, I.P., North Delegation, Portugal M. Fa´tima Pinheiroa,b,c National Institute of Legal Medicine, I.P., North Delegation, Portugal b Health Sciences Faculty, Fernando Pessoa University, Oporto, Portugal c Abel Salazar Biomedical Institute, Oporto University, Portugal a
*Correspondence address: Delegac¸a˜o do Norte do Instituto Nacional de Medicina Legal, Jardim Carrilho Videira, 4050-167 Porto, Portugal. Tel.: +351 22 207 38 50; fax: +351 22 332 59 31 E-mail address: [email protected]
(A.M. Lagoa) 13 December 2007