Rapid screening for Native American mitochondrial and Y-chromosome haplogroups detection in routine DNA analysis Gala Zuccarelli 1 , Evguenia Alechine 1 , Mariela Caputo, Cecilia Bobillo, Daniel Corach, Andrea Sala * Servicio de Huellas Digitales Gene ´ticas y Ca ´tedra de Gene ´tica y Biologı´a Molecular, School of Pharmacy and Biochemistry, Buenos Aires University, Argentina 2 1. Introduction The population of Argentina is the result of intercontinental admixture events. It has been recently demonstrated that the composition of the Argentinean extant population exhibit a differential Native American matri and patrilineages contribution. The four Native American mitochondrial haplogroups A2, B2, C1 and D1 are represented in average 54% of the general population, whereas Native American Y-chromosome specific haplogroup (M3-Q3) is represented in 4.9% of the population [1]. Aiming to refine the Argentinean population admixture levels and improve the sampling efficiency of diverse Native American matri and patrilineages, a rapid screening approach has been developed based on specific Single Nucleotide Polymorphisms (SNP) typing located within the coding region of the mtDNA genome and in the male-specific region of the Y-chromosome (MSY) [2], by means of Multiplex Real Time PCR [3] followed by High Resolution Melting (HRM) analysis [4]. SNPs located within haploid genetic systems, such as mitochon- drial DNA (mtDNA) and MSY, are useful markers for studying migrations, allowing determination of phylogenetic divergence between lineages without the ambiguities caused by meiotic shuffling [5,6]. Based on binary markers located in the Y-chromo- some more than 300 Y chromosomal haplogroups have been defined in human populations [7]. However, in our development the focus was oriented to the SNP associated with the most frequent ancestral Native American patrilineage: M3, for identifying and collecting samples with Native American Y-chromosomes for further analysis. Mitochondrial haplogroups have firstly been defined in human population genetic studies by restriction fragment length poly- morphism analysis [8,9]. At present, sequencing of the entire mtDNA molecule is the ‘‘gold standard’’ for haplogroup determi- nation [10]. Alternatively, other novel strategies have also been developed, such as SNaPshot minisequencing and microarrays analysis [11,12]. The strategy here described allows discerning the major Native American mitochondrial and Y-chromosome haplogroups to which the donor of a sample might belong in a highly accurate, fast and cost effective way. 2. Materials and methods 2.1. DNA samples All analyzed samples were collected from unrelated donors who signed informed consent statements. The study was approved by the Ethical Committee of the School of Pharmacy and Biochemistry, University of Buenos Aires. Forensic Science International: Genetics 5 (2011) 105–108 ARTICLE INFO Keywords: Multiplex Real Time PCR High Resolution Melting mtDNA Y-SNPs Native American haplogroups Argentinean population ABSTRACT Aiming to detect individuals of Native American maternal or paternal ancestry a rapid screening approach has been developed. Its strategy was based on SNP typing by Real Time PCR (rt-PCR) followed by High Resolution Melting analysis (HRM). After extraction, DNA was quantitated by rt-PCR using commercial kits; samples were then submitted to two multiplex reactions in order to determine the major Native American mtDNA and Y-chromosome haplogroups by HRM. One cocktail included primers flanking nucleotide substitutions that define mtDNA haplogroup C and sub-haplogroups A2, B2, and D1. The other included primers flanking Y-SNPs M3, M269 and U179 that allowed discriminating Q and non- Q haplogroups. In all cases amplicons were <125 nucleotides long in order to increase the peak resolution. The accuracy of the results obtained was established by means of sequencing analysis of the amplicons. The new working-flow here proposed facilitates and speeds-up the screening process that may preclude a detailed sequencing analysis of particular samples, or for further molecular epidemiological investigations in which continental origin influences might be relevant. ß 2010 Elsevier Ireland Ltd. All rights reserved. * Corresponding author. E-mail address: asala@ffyb.uba.ar (A. Sala). 1 Both authors contributed equally to the work. 2 shdg@ffyb.uba.ar. Contents lists available at ScienceDirect Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig 1872-4973/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigen.2010.08.018