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Genetic characterisation of six miniSTR loci in an Italian population sample
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Forensic Science International: Genetics Supplement Series 1 (2008) 356–357 www.elsevier.com/locate/FSIGSS
Research article
Genetic characterisation of six miniSTR loci in an Italian population sample Gabriella Peloso *, Pierangela Grignani, Carlo Previdere’ Department of Legal Medicine and Public Health, University of Pavia, Italy Received 6 September 2007; accepted 11 October 2007
Abstract Allele frequencies for the miniSTR loci D10S1248, D14S1434, D22S1045 (NC01) and D1S1677, D2S441, D4S2364 (NC02) were determined in a population sample from North-Western Italy. No significant deviations from Hardy–Weinberg equilibrium expectations were detected. The forensic usefulness of the selected miniSTRs was confirmed typing different aged samples and analysing 20 family trios with paternity confirmed with CODIS autosomic STRs. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: MiniSTRs; Population data; Italy
1. Introduction
2. Materials and methods
In the forensic practise, it is well known that DNA recovered from particular forensic specimens may sometimes show incomplete results in terms of partial genetic profiles with loss of the larger sized STR loci, PCR inhibition due to co-extracted contaminants or with amplification artefacts as allelic imbalance and allele drop-out [1,2]. These results are generally referred to the DNA degradation and chemical modification of the genetic material occurring in the forensic sample exposed to the environmental conditions [3]. In order to recover the genetic information from these degraded forensic samples, the most widely used approach was the reduction of the size of the PCR products, setting up multiplex PCR reactions with primers selected very close to the STR polymorphic regions [4]. These reduced STRs, defined miniSTRs, have been developed either redesigning CODIS STR loci primer sets [5] or selecting new STR candidates from public genome databases [6]. We selected six of these new miniSTR loci (namely D1S1677, D2S441, D4S2364, D10S1248, D14S1434 and D22S1045) in order to analyse a population sample from North-Western Italy.
DNA was extracted from blood and saliva samples collected from 100 unrelated North-Western Italians and 20 family trios with paternity confirmed with autosomic STRs, by phenol– chloroform or Chelex 100 purification. According to Coble [7], D10S1248, D14S1434, D22S1045 loci were amplified in a multiplex PCR reaction named NC01 and D1S1677, D2S441, D4S2364 loci a second multiplex named NC02. PCR conditions and primer sequences followed [7], with minor modifications. Primers were labelled with 6-Fam, Hex and Tet dyes. The amplified fragments were separated by capillary electrophoresis on an ABI PRISM 310 sequencer. Allele nomenclature was reviewed according to the latest changes as recommended in [7]. Evaluation of H–W equilibrium was performed using Arlequin 2000 software [8]. The forensic parameters were obtained by the PowerStats software (Promega).
* Corresponding author. E-mail address: [email protected] (G. Peloso). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.077
3. Results and discussion In this study, we analysed 100 unrelated individuals from North-Western Italy by PCR amplification of six miniSTR loci in two triplex PCRs named NC01 (D10S1248, D14S1434, D22S1045) and NC02 (D1S1677, D2S441, D4S2364). PCR products, in a range varying from 71 to 115 bp, were separated by capillary electrophoresis. The distributions of allele frequencies for each locus are represented in Table 1 together
G. Peloso et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 356–357 Table 1 Allele frequencies of the analysed miniSTRs Allele D10S1248 D14S1434 D22S1045 D1S1677 D2S441 D4S2364 8 9 10 11 11:03 12 13 14 15 16 17 18 N H PD
0.005 0.20 0.055 0.04 0.21 0.31 0.19 0.18 0.045 0.02
0.03 0.295 0.40 0.015
100 0.75 0.925
100 0.70 0.868
0.005
0.115 0.02 0.045 0.415 0.30 0.10 0.005 100 0.68 0.872
0.015 0.01 0.06 0.26 0.32 0.275 0.055 0.005 100 0.75 0.888
0.01 0.11 0.36 0.07 0.03 0.025 0.345 0.05
0.185 0.535 0.28
357
compared our data with other Italian and Caucasian population samples [6,9] by Fisher’s exact test on RXC contingency tables. The set of six miniSTRs was then evaluated analysing 20 family trios with paternity confirmed with CODIS autosomic STRs (W > 99.9999%) and no mutation was observed. The forensic usefulness of the selected miniSTRs was confirmed when reliable DNA profiles were produced analysing different aged samples such as bones, and bloodstains. Conflict of interest None.
100 100 0.74 0.57 0.863 0.772
with the relevant forensic parameters such as observed heterozigosity (H), matching probability, power of discrimination (PD) and power of exclusion (PE). No significant deviations from Hardy–Weinberg equilibrium expectations were detected performing exact test with Arlequin ver. 2000 [8], after applying the Bonferroni correction. The combined power of discrimination for the six miniSTRs was 0.9999. No statistically significant differences were seen when we
References [1] J.P. Whitaker, E.A. Cotton, P. Gill, Forensic Sci. Int. 123 (2001) 215–223. [2] L.A. Dixon, A.E. Dobbins, H.K. Pulker, et al. Forensic Sci. Int. 164 (2006) 33–44. [3] W. Bar, A. Kratzer, M. Machler, W. Schmid, Forensic Sci. Int. 39 (1988) 59–70. [4] P. Wiegand, M. Kleiber, Int. J. Legal Med. 114 (2001) 285–287. [5] C.R. Hill, M.C. Kline, J.J. Mulero, et al. J. Forensic Sci. 52 (2007) 870– 873. [6] M.D. Coble, J.M. Butler, J. Forensic Sci. 50 (2005) 43–53. [7] http://www.cstl.nist.gov/div831/strbase/miniSTR.htm. [8] http://lgb.unige.ch/arlequin/. [9] A. Rocchi, I. Spinetti, C. Toni, et al. Prog. Forensic Gen. 11 (2006) 377– 378.
Forensic Science International: Genetics Supplement Series 1 (2008) 356–357 www.elsevier.com/locate/FSIGSS
Research article
Genetic characterisation of six miniSTR loci in an Italian population sample Gabriella Peloso *, Pierangela Grignani, Carlo Previdere’ Department of Legal Medicine and Public Health, University of Pavia, Italy Received 6 September 2007; accepted 11 October 2007
Abstract Allele frequencies for the miniSTR loci D10S1248, D14S1434, D22S1045 (NC01) and D1S1677, D2S441, D4S2364 (NC02) were determined in a population sample from North-Western Italy. No significant deviations from Hardy–Weinberg equilibrium expectations were detected. The forensic usefulness of the selected miniSTRs was confirmed typing different aged samples and analysing 20 family trios with paternity confirmed with CODIS autosomic STRs. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: MiniSTRs; Population data; Italy
1. Introduction
2. Materials and methods
In the forensic practise, it is well known that DNA recovered from particular forensic specimens may sometimes show incomplete results in terms of partial genetic profiles with loss of the larger sized STR loci, PCR inhibition due to co-extracted contaminants or with amplification artefacts as allelic imbalance and allele drop-out [1,2]. These results are generally referred to the DNA degradation and chemical modification of the genetic material occurring in the forensic sample exposed to the environmental conditions [3]. In order to recover the genetic information from these degraded forensic samples, the most widely used approach was the reduction of the size of the PCR products, setting up multiplex PCR reactions with primers selected very close to the STR polymorphic regions [4]. These reduced STRs, defined miniSTRs, have been developed either redesigning CODIS STR loci primer sets [5] or selecting new STR candidates from public genome databases [6]. We selected six of these new miniSTR loci (namely D1S1677, D2S441, D4S2364, D10S1248, D14S1434 and D22S1045) in order to analyse a population sample from North-Western Italy.
DNA was extracted from blood and saliva samples collected from 100 unrelated North-Western Italians and 20 family trios with paternity confirmed with autosomic STRs, by phenol– chloroform or Chelex 100 purification. According to Coble [7], D10S1248, D14S1434, D22S1045 loci were amplified in a multiplex PCR reaction named NC01 and D1S1677, D2S441, D4S2364 loci a second multiplex named NC02. PCR conditions and primer sequences followed [7], with minor modifications. Primers were labelled with 6-Fam, Hex and Tet dyes. The amplified fragments were separated by capillary electrophoresis on an ABI PRISM 310 sequencer. Allele nomenclature was reviewed according to the latest changes as recommended in [7]. Evaluation of H–W equilibrium was performed using Arlequin 2000 software [8]. The forensic parameters were obtained by the PowerStats software (Promega).
* Corresponding author. E-mail address: [email protected] (G. Peloso). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.077
3. Results and discussion In this study, we analysed 100 unrelated individuals from North-Western Italy by PCR amplification of six miniSTR loci in two triplex PCRs named NC01 (D10S1248, D14S1434, D22S1045) and NC02 (D1S1677, D2S441, D4S2364). PCR products, in a range varying from 71 to 115 bp, were separated by capillary electrophoresis. The distributions of allele frequencies for each locus are represented in Table 1 together
G. Peloso et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 356–357 Table 1 Allele frequencies of the analysed miniSTRs Allele D10S1248 D14S1434 D22S1045 D1S1677 D2S441 D4S2364 8 9 10 11 11:03 12 13 14 15 16 17 18 N H PD
0.005 0.20 0.055 0.04 0.21 0.31 0.19 0.18 0.045 0.02
0.03 0.295 0.40 0.015
100 0.75 0.925
100 0.70 0.868
0.005
0.115 0.02 0.045 0.415 0.30 0.10 0.005 100 0.68 0.872
0.015 0.01 0.06 0.26 0.32 0.275 0.055 0.005 100 0.75 0.888
0.01 0.11 0.36 0.07 0.03 0.025 0.345 0.05
0.185 0.535 0.28
357
compared our data with other Italian and Caucasian population samples [6,9] by Fisher’s exact test on RXC contingency tables. The set of six miniSTRs was then evaluated analysing 20 family trios with paternity confirmed with CODIS autosomic STRs (W > 99.9999%) and no mutation was observed. The forensic usefulness of the selected miniSTRs was confirmed when reliable DNA profiles were produced analysing different aged samples such as bones, and bloodstains. Conflict of interest None.
100 100 0.74 0.57 0.863 0.772
with the relevant forensic parameters such as observed heterozigosity (H), matching probability, power of discrimination (PD) and power of exclusion (PE). No significant deviations from Hardy–Weinberg equilibrium expectations were detected performing exact test with Arlequin ver. 2000 [8], after applying the Bonferroni correction. The combined power of discrimination for the six miniSTRs was 0.9999. No statistically significant differences were seen when we
References [1] J.P. Whitaker, E.A. Cotton, P. Gill, Forensic Sci. Int. 123 (2001) 215–223. [2] L.A. Dixon, A.E. Dobbins, H.K. Pulker, et al. Forensic Sci. Int. 164 (2006) 33–44. [3] W. Bar, A. Kratzer, M. Machler, W. Schmid, Forensic Sci. Int. 39 (1988) 59–70. [4] P. Wiegand, M. Kleiber, Int. J. Legal Med. 114 (2001) 285–287. [5] C.R. Hill, M.C. Kline, J.J. Mulero, et al. J. Forensic Sci. 52 (2007) 870– 873. [6] M.D. Coble, J.M. Butler, J. Forensic Sci. 50 (2005) 43–53. [7] http://www.cstl.nist.gov/div831/strbase/miniSTR.htm. [8] http://lgb.unige.ch/arlequin/. [9] A. Rocchi, I. Spinetti, C. Toni, et al. Prog. Forensic Gen. 11 (2006) 377– 378.