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Forensic validation and implementation of Y-chromosome SNP multiplexes
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Forensic Science International: Genetics Supplement Series 1 (2008) 181–183 www.elsevier.com/locate/FSIGSS
Research report
Forensic validation and implementation of Y-chromosome SNP multiplexes A. Blanco-Verea, M. Brio´n *, E. Ramos-Luis, M.V. Lareu, A. Carracedo Grupo de Medicina Xeno´mica, CEGEN-IML, University of Santiago de Compostela, Spain Received 4 September 2007; received in revised form 17 October 2007; accepted 25 October 2007
Abstract We try to show the utility and possibility to use Y-SNP in forensic genetics. We have selected 27 Y-SNPs distributed in 4 multiplexes. The first step was seeking the minimal DNA quantity to amplify multiplexes and the second step was comparing results from Y-SNP multiplexes with results from a registered trademark Y-STR multiplex using the same sexual assault samples. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Y-SNP; Y-STRs; Haplogroup; Forensic validation
1. Introduction Analysis of Y-chromosome haplogroups defined by binary polymorphisms has become a standard approach for studying the origin of modern human populations and for measuring the variability between them. However, their application in the forensic field, remain under research, principally due to two main features: the loss of forensic validation of the Ychromosome multiplexes described for SNP typing [1] and the difficulties in understanding the male lineage distribution and their frequencies among the different populations around the world. In order to solve this problem in the forensic casework of our laboratory we have performed the forensic validation of 4 different multiplexes with 27 Y-SNPs in total [2]. 2. Materials and methods In addition to a commercial control DNA sample (C + 1), we have used samples from sexual assault cases, degraded bones and dental pieces. See Table 1. In order to improve the multiplex results QIAGEN1 Multiplex PCR kit was used. Manufacturer recommendations were followed, and BSA was used in PCR with degraded samples. * Corresponding author. Tel.: +34 981582327; fax: +34 981580336. E-mail address: [email protected] (M. Brio´n). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.046
Multiplex single base extension reactions [3,4] were carried out following the Y-SNP amplifications by means of the SNaPshotTM Multiplex kit (Applied Biosystems). The products of Y-SNP single base extensions, were run on a ABI PRISM1 3130 Genetic Analyzer (Applied Biosystems) and analyzed using GeneMapper1 ID Analysis Software Version 3.2 (Applied Biosystems). Y-chromosome STRs were Table 1 Samples and concentrations Sample name
Sample (ng/ml)
ng in PCR
Type of sample
Degraded samples D1 D2 D3 D4 D5 D6 D7 C+1
0.533 0.406 0.0898 0.599 0.117 8.63 0.987 0.1
0.93 0.71 0.16 0.599 0.2 – 0.987 0.1
Femur Tooth Tooth Tooth Femur Female Femur Positive control
Sample name
Sample (ng/ml)
Sexual assault samples A1 0.0982 A2 0.021 A3 0.027 A4 0.0327 A5 0.0122 A6 0.00353 A7 0.283
ng in PCR
Y-STRs profile
0.42 0.09 0.115 0.14 0.05 0.015 0.85
Incomplete/mixture Incomplete/mixture negative Mixture Incomplete negative Complete
182
A. Blanco-Verea et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 181–183
Fig. 1. Multiplexes of C + 1 using 0.1 ng in each PCR.
Fig. 2. Sample A1. Example of mixture in multiplex 1 and in multiplex 3.
Fig. 3. A3 Multiplex 1 with Y-SNPs profile and A3 AmpliFlSTR Yfiler with Y-STRs profile.
A. Blanco-Verea et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 181–183
analyzed prior to this work with the PCR amplification kit AmpliFlSTR1 YFilerTM (Applied Biosystems). 3. Results and discussion The minimum concentration of male DNA used with our samples was 0.1 ng/ml, and the minimum quantity allowable to detect was 0.1 ng. Electropherograms for the 4 multiplexes of the C + 1 sample, using 0.1 ng for each PCR, are shown in Fig. 1. Mixture of at least 2 male DNAs can be observed in sample A1. Some SNPs like M173, M213, M269, M9, 92R7 in Multiplex 1 and M35 and M96 in Multiplex 3 showed ancient and mutated allelic state (see Fig. 2). Two different haplogroups can be detected: R1b1c and E3b1 in the same sample. The mixture was also detected when the Y-chromosome STRs were analysed. Another example of the high throughput of our multiplexes is the sample A3. We have achieved a Y-SNP profile in the
183
multiplex 1 while we have not had success with the PCR amplification kit AmpliFlSTR1 YFilerTM (Applied Biosystems). See Fig. 3. Haplogroup R1b1c can be determined in sample A3 showing again the necessity of considering the utility of SNPs as simple and powerful tools in forensic analysis.
Conflict of interest None.
References [1] M.A. Jobling, Forensic Sci. Int. 118 (2001) 158–162. [2] M. Brion, B. Sobrino, A. Blanco-Verea, M.V. Lareu, A. Carracedo, Int. J. Legal Med. 119 (2004) 10–15. [3] B. Sobrino, M. Brio´n, A. Carracedo, Forensic Sci. Int. 154 (2005) 181–194. [4] R. Lessig, et al. Forensic Sci. Int. 154 (2005) 128–136.
Forensic Science International: Genetics Supplement Series 1 (2008) 181–183 www.elsevier.com/locate/FSIGSS
Research report
Forensic validation and implementation of Y-chromosome SNP multiplexes A. Blanco-Verea, M. Brio´n *, E. Ramos-Luis, M.V. Lareu, A. Carracedo Grupo de Medicina Xeno´mica, CEGEN-IML, University of Santiago de Compostela, Spain Received 4 September 2007; received in revised form 17 October 2007; accepted 25 October 2007
Abstract We try to show the utility and possibility to use Y-SNP in forensic genetics. We have selected 27 Y-SNPs distributed in 4 multiplexes. The first step was seeking the minimal DNA quantity to amplify multiplexes and the second step was comparing results from Y-SNP multiplexes with results from a registered trademark Y-STR multiplex using the same sexual assault samples. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Y-SNP; Y-STRs; Haplogroup; Forensic validation
1. Introduction Analysis of Y-chromosome haplogroups defined by binary polymorphisms has become a standard approach for studying the origin of modern human populations and for measuring the variability between them. However, their application in the forensic field, remain under research, principally due to two main features: the loss of forensic validation of the Ychromosome multiplexes described for SNP typing [1] and the difficulties in understanding the male lineage distribution and their frequencies among the different populations around the world. In order to solve this problem in the forensic casework of our laboratory we have performed the forensic validation of 4 different multiplexes with 27 Y-SNPs in total [2]. 2. Materials and methods In addition to a commercial control DNA sample (C + 1), we have used samples from sexual assault cases, degraded bones and dental pieces. See Table 1. In order to improve the multiplex results QIAGEN1 Multiplex PCR kit was used. Manufacturer recommendations were followed, and BSA was used in PCR with degraded samples. * Corresponding author. Tel.: +34 981582327; fax: +34 981580336. E-mail address: [email protected] (M. Brio´n). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.046
Multiplex single base extension reactions [3,4] were carried out following the Y-SNP amplifications by means of the SNaPshotTM Multiplex kit (Applied Biosystems). The products of Y-SNP single base extensions, were run on a ABI PRISM1 3130 Genetic Analyzer (Applied Biosystems) and analyzed using GeneMapper1 ID Analysis Software Version 3.2 (Applied Biosystems). Y-chromosome STRs were Table 1 Samples and concentrations Sample name
Sample (ng/ml)
ng in PCR
Type of sample
Degraded samples D1 D2 D3 D4 D5 D6 D7 C+1
0.533 0.406 0.0898 0.599 0.117 8.63 0.987 0.1
0.93 0.71 0.16 0.599 0.2 – 0.987 0.1
Femur Tooth Tooth Tooth Femur Female Femur Positive control
Sample name
Sample (ng/ml)
Sexual assault samples A1 0.0982 A2 0.021 A3 0.027 A4 0.0327 A5 0.0122 A6 0.00353 A7 0.283
ng in PCR
Y-STRs profile
0.42 0.09 0.115 0.14 0.05 0.015 0.85
Incomplete/mixture Incomplete/mixture negative Mixture Incomplete negative Complete
182
A. Blanco-Verea et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 181–183
Fig. 1. Multiplexes of C + 1 using 0.1 ng in each PCR.
Fig. 2. Sample A1. Example of mixture in multiplex 1 and in multiplex 3.
Fig. 3. A3 Multiplex 1 with Y-SNPs profile and A3 AmpliFlSTR Yfiler with Y-STRs profile.
A. Blanco-Verea et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 181–183
analyzed prior to this work with the PCR amplification kit AmpliFlSTR1 YFilerTM (Applied Biosystems). 3. Results and discussion The minimum concentration of male DNA used with our samples was 0.1 ng/ml, and the minimum quantity allowable to detect was 0.1 ng. Electropherograms for the 4 multiplexes of the C + 1 sample, using 0.1 ng for each PCR, are shown in Fig. 1. Mixture of at least 2 male DNAs can be observed in sample A1. Some SNPs like M173, M213, M269, M9, 92R7 in Multiplex 1 and M35 and M96 in Multiplex 3 showed ancient and mutated allelic state (see Fig. 2). Two different haplogroups can be detected: R1b1c and E3b1 in the same sample. The mixture was also detected when the Y-chromosome STRs were analysed. Another example of the high throughput of our multiplexes is the sample A3. We have achieved a Y-SNP profile in the
183
multiplex 1 while we have not had success with the PCR amplification kit AmpliFlSTR1 YFilerTM (Applied Biosystems). See Fig. 3. Haplogroup R1b1c can be determined in sample A3 showing again the necessity of considering the utility of SNPs as simple and powerful tools in forensic analysis.
Conflict of interest None.
References [1] M.A. Jobling, Forensic Sci. Int. 118 (2001) 158–162. [2] M. Brion, B. Sobrino, A. Blanco-Verea, M.V. Lareu, A. Carracedo, Int. J. Legal Med. 119 (2004) 10–15. [3] B. Sobrino, M. Brio´n, A. Carracedo, Forensic Sci. Int. 154 (2005) 181–194. [4] R. Lessig, et al. Forensic Sci. Int. 154 (2005) 128–136.