Development of multiplex PCRs for evolutionary and forensic applications of 37 human Y chromosome SNPs

Abstract 

This work describes an efficient and rapid test for typing 37 single nucleotide polymorphisms (SNPs) of the non-recombining region of Y chromosome (NRY) from a minimal amount of DNA using six PCR multiplexes. Markers were drawn following a hierarchical strategy based on the phylogenetic tree of Y chromosome proposed by the Y Chromosome Consortium [The Y Chromosome Consortium, A nomenclature system for the tree of human Y-chromosomal binary haplogroups, Genome Res. 12 (2002) 339–348]. Two multiplexes—arbitrarily named MY1 and MY2—were developed to explore the basal branches of the tree encompassing all the major clades A-R: MY1 for markers M35, M89, M172, M170, M9, M173, M45 and MY2 for markers M52, M216, M174, M181, M201, M91, M96, M214. Four multiplexes able of typing the more superficial branches typical of most frequent European haplogroups E3b, J2, R1 and I, were also developed and named MY-E3b (M78, M107, M224, M165, M148, M81), MY-J2 (M158, M68, M47, M102, M137, M67), MY-R1 (M17, M269, M18, P25, SRY10831.2) and MY-I (M72, M223, M26, M21, M161). SNP genotyping was carried out by hot-start PCR amplification with primers yielding fragments between 63 and 210 nucleotides, followed by minisequencing reaction based on dideoxy single-base extension and capillary electrophoresis of extension products. The sequential application of these multiplexes is a robust and effective resource for typing the most frequent European Y-SNP haplogroups, and appears to be suitable for forensic purposes and evolutionary studies.

Keywords: Y-SNPs, Multiplex PCR, Minisequencing, Capillary electrophoresis, Phylogeography, Forensic genetics