- Email: [email protected]
The analysis of UAE populations using AmpFℓSTR® Y Filer™: Identification of novel and null alleles
Available online at www.sciencedirect.com
Forensic Science International: Genetics Supplement Series 1 (2008) 255–256 www.elsevier.com/locate/FSIGSS
Research article
The analysis of UAE populations using AmpF‘STR1 Y FilerTM: Identification of novel and null alleles N. Zahra, L.A.A. Sallam, S. Hadi, W. Goodwin * School of Forensic and Investigative Sciences, University of Central Lancashire, Preston PR1 2HE, UK Received 24 August 2007; accepted 10 October 2007
Abstract The analysis of Y chromosome polymorphisms has become common place for the identification of male component in forensic cases. During the development of a Y STR population database for three major populations in the United Arab Emirates (Arab, Indian and Pakistani) using the AmpF‘STR1 Y FilerTM kit, a number of novel and null alleles were discovered. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Y STRs; AmpFlSTR1 Y FilerTM kit; United Arab Emirates; Novel alleles; Null alleles
1. Introduction
2. Methodology
The analysis of STRs on the 60 Mb Y chromosomes has been used to complement the application of standard autosomal STRs markers. Being haploid and passed through the paternal lineage, the Y markers can be used for human identification in cases such as sexual assaults, paternity testing and missing person investigations [1,2]. The AmpF‘STR1 Y FilerTM kit was developed and validated to amplify 17 loci in a multiplex reaction [3]. It includes the nine loci that make up the minimal haplotype (DYS19, DYS389 I/II, DYS390, DYS391, DYS392, DYS393 and DYS385 a/b), additional loci suggested by the Scientific Working Group on DNA Analysis Methods (DYS438 and DYS439) and a further six loci added to increase the discriminating power of the system (DYS456, DYS458, DYS635, YGATA H4, DYS437 and DYS448). In order to support the Abu Dhabi Police Forensic Science Department in forensic casework, the Y FilerTM kit was used to create a Y STR database for the three major populations in UAE (Arab, Indian and Pakistani). During the study novel and null alleles were observed.
Blood samples were collected from 414 unrelated healthy male individuals from the three UAE populations: Arab (199), Indian (106) and Pakistani (109). DNA was extracted using a standard phenol–chloroform procedure. Extracts were quantified by gel electrophoresis and diluted accordingly to a concentration of 0.1–1 ng/ml. Amplification of DNA with AmpF‘STR1 Y FilerTM kit was carried out according to manufacture’s instruction [4] in a reduced volume of 8.3 ml. PCR products were run on an ABI Prism1 310. Alleles were genotyped using GenoTyper1 software V3.7. Samples showing off-ladder alleles and drop outs were amplified in singleplex reactions and sequenced.
* Corresponding author. Tel.: +44 1772 894254; fax: +44 1772 894981. E-mail address: [email protected] (W. Goodwin). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.137
3. Results and discussion From the 414 individuals genotyped, 10 alleles in six different loci were observed to be off-ladder. All the new alleles differ by at least 4 bp from the nearest allele in the allelic ladder. The sequences of the novel alleles are given in Table 1. DYS635 showed the highest number of novel alleles—all the new alleles displayed a deletion of two repeat units from the reference sequence. Also, variation in allele 18 was observed with the two variants originating from different populations. In addition to the new alleles, allelic dropouts were detected at four different loci (DYS438, DYS448, DYS389 II and DYS458) in six individuals (Table 2). The sample showing a null allele at DYS438 (Fig. 1) was successfully amplified with PowerPlex1 Y System kit. Although the main cause of allelic
256
N. Zahra et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 255–256
Table 1 The novel alleles for six different loci observed in the UAE population Locus
DYS635
DYS458
Reference sequence
Allele
New allele sequence
(TCTA)4(TGTA)2 (TCTA)2 (TGTA)2(TCTA)2 (TGTA)0,2(TCTA)
19 18 18 17
(TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)9 (TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)8 (TCTA)4(TGTA)2(TCTA)2(TGTA)3(TCTA)7 (TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)7
(GAAA)n
21 13
(GAAA)21 (GAAA)13
Number of samples exhibiting new allele Arab
Pakistani
1
1
Indian 2
1 1 5 1
DYS19
(TAGA)3TAGG(TAGA)n
16
(TAGA)3TAGG(TAGA)13
DYS448
(AGAGAT)nN42(AGAGAT)n
16
(AGAGAT)8N(AGAGAT)8
1
DYS389 I
(TCTG)3(TCTA)n
13/16
(TCTG)3(TCTA)13 + (TCTG)3(TCTA)10
1
DYS456
(AGAT)n
12
(AGAT)12
5
1
Fig. 1. Electropherogram showing drop-out at DYS438 when amplified with Y FilerTM. Table 2 The number of Indian and Pakistani individuals exhibiting null alleles at four loci Population
Locus
Number of individuals
Pakistani
DYS438 DYS448 DYS389 II
1 2 1
DYS458
2
Indian
drop-out is primer binding site mutation, further investigation is required to determine the occurrence of null alleles. Acknowledgments Thanks go to the Abu Dhabi Police for supplying the samples for this study and to Dr. David Ballard from the Institute of Cell and Molecular Science (ICMS) at the Queen Mary’s University, London for his help in the sequencing of the samples.
Conflict of interest None.
References [1] J.M. Butler, Recent developments in Y-short tandem repeat and Y-single nucleotide polymorphism analysis, Forensic. Sci. Rev. 15 (2003) 91– 111. [2] P. Gill, C. Brenner, B. Brinkmann, B. Budowle, A. Carracedo, M.A. Jobling, P. de Knijff, M. Kayser, M. Krawczak, W.R. Mayr, N. Morling, B. Olaisen, A. Sajantila, C. Tyler-Smith, DNA commission of the International Society of forensic genetics: recommendations on forensic analysis using Y-chromosome STRs, Forensic. Sci. Int. 124 (2001) 5– 10. [3] R.E. Romero, R. Lizarazo, Validation of the AmpF‘STR1 Y FilerTM kit, Int. Congr. Ser. 1288 (2006) 280–282. [4] Applied Biosystem, AmpF‘STR1 Y FilerTM PCR amplification kit. User’s Manual, 2004.
Forensic Science International: Genetics Supplement Series 1 (2008) 255–256 www.elsevier.com/locate/FSIGSS
Research article
The analysis of UAE populations using AmpF‘STR1 Y FilerTM: Identification of novel and null alleles N. Zahra, L.A.A. Sallam, S. Hadi, W. Goodwin * School of Forensic and Investigative Sciences, University of Central Lancashire, Preston PR1 2HE, UK Received 24 August 2007; accepted 10 October 2007
Abstract The analysis of Y chromosome polymorphisms has become common place for the identification of male component in forensic cases. During the development of a Y STR population database for three major populations in the United Arab Emirates (Arab, Indian and Pakistani) using the AmpF‘STR1 Y FilerTM kit, a number of novel and null alleles were discovered. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Y STRs; AmpFlSTR1 Y FilerTM kit; United Arab Emirates; Novel alleles; Null alleles
1. Introduction
2. Methodology
The analysis of STRs on the 60 Mb Y chromosomes has been used to complement the application of standard autosomal STRs markers. Being haploid and passed through the paternal lineage, the Y markers can be used for human identification in cases such as sexual assaults, paternity testing and missing person investigations [1,2]. The AmpF‘STR1 Y FilerTM kit was developed and validated to amplify 17 loci in a multiplex reaction [3]. It includes the nine loci that make up the minimal haplotype (DYS19, DYS389 I/II, DYS390, DYS391, DYS392, DYS393 and DYS385 a/b), additional loci suggested by the Scientific Working Group on DNA Analysis Methods (DYS438 and DYS439) and a further six loci added to increase the discriminating power of the system (DYS456, DYS458, DYS635, YGATA H4, DYS437 and DYS448). In order to support the Abu Dhabi Police Forensic Science Department in forensic casework, the Y FilerTM kit was used to create a Y STR database for the three major populations in UAE (Arab, Indian and Pakistani). During the study novel and null alleles were observed.
Blood samples were collected from 414 unrelated healthy male individuals from the three UAE populations: Arab (199), Indian (106) and Pakistani (109). DNA was extracted using a standard phenol–chloroform procedure. Extracts were quantified by gel electrophoresis and diluted accordingly to a concentration of 0.1–1 ng/ml. Amplification of DNA with AmpF‘STR1 Y FilerTM kit was carried out according to manufacture’s instruction [4] in a reduced volume of 8.3 ml. PCR products were run on an ABI Prism1 310. Alleles were genotyped using GenoTyper1 software V3.7. Samples showing off-ladder alleles and drop outs were amplified in singleplex reactions and sequenced.
* Corresponding author. Tel.: +44 1772 894254; fax: +44 1772 894981. E-mail address: [email protected] (W. Goodwin). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.137
3. Results and discussion From the 414 individuals genotyped, 10 alleles in six different loci were observed to be off-ladder. All the new alleles differ by at least 4 bp from the nearest allele in the allelic ladder. The sequences of the novel alleles are given in Table 1. DYS635 showed the highest number of novel alleles—all the new alleles displayed a deletion of two repeat units from the reference sequence. Also, variation in allele 18 was observed with the two variants originating from different populations. In addition to the new alleles, allelic dropouts were detected at four different loci (DYS438, DYS448, DYS389 II and DYS458) in six individuals (Table 2). The sample showing a null allele at DYS438 (Fig. 1) was successfully amplified with PowerPlex1 Y System kit. Although the main cause of allelic
256
N. Zahra et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 255–256
Table 1 The novel alleles for six different loci observed in the UAE population Locus
DYS635
DYS458
Reference sequence
Allele
New allele sequence
(TCTA)4(TGTA)2 (TCTA)2 (TGTA)2(TCTA)2 (TGTA)0,2(TCTA)
19 18 18 17
(TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)9 (TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)8 (TCTA)4(TGTA)2(TCTA)2(TGTA)3(TCTA)7 (TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)7
(GAAA)n
21 13
(GAAA)21 (GAAA)13
Number of samples exhibiting new allele Arab
Pakistani
1
1
Indian 2
1 1 5 1
DYS19
(TAGA)3TAGG(TAGA)n
16
(TAGA)3TAGG(TAGA)13
DYS448
(AGAGAT)nN42(AGAGAT)n
16
(AGAGAT)8N(AGAGAT)8
1
DYS389 I
(TCTG)3(TCTA)n
13/16
(TCTG)3(TCTA)13 + (TCTG)3(TCTA)10
1
DYS456
(AGAT)n
12
(AGAT)12
5
1
Fig. 1. Electropherogram showing drop-out at DYS438 when amplified with Y FilerTM. Table 2 The number of Indian and Pakistani individuals exhibiting null alleles at four loci Population
Locus
Number of individuals
Pakistani
DYS438 DYS448 DYS389 II
1 2 1
DYS458
2
Indian
drop-out is primer binding site mutation, further investigation is required to determine the occurrence of null alleles. Acknowledgments Thanks go to the Abu Dhabi Police for supplying the samples for this study and to Dr. David Ballard from the Institute of Cell and Molecular Science (ICMS) at the Queen Mary’s University, London for his help in the sequencing of the samples.
Conflict of interest None.
References [1] J.M. Butler, Recent developments in Y-short tandem repeat and Y-single nucleotide polymorphism analysis, Forensic. Sci. Rev. 15 (2003) 91– 111. [2] P. Gill, C. Brenner, B. Brinkmann, B. Budowle, A. Carracedo, M.A. Jobling, P. de Knijff, M. Kayser, M. Krawczak, W.R. Mayr, N. Morling, B. Olaisen, A. Sajantila, C. Tyler-Smith, DNA commission of the International Society of forensic genetics: recommendations on forensic analysis using Y-chromosome STRs, Forensic. Sci. Int. 124 (2001) 5– 10. [3] R.E. Romero, R. Lizarazo, Validation of the AmpF‘STR1 Y FilerTM kit, Int. Congr. Ser. 1288 (2006) 280–282. [4] Applied Biosystem, AmpF‘STR1 Y FilerTM PCR amplification kit. User’s Manual, 2004.