A single multiplex SNaPshot reaction of 42 SNPs to classify admixture populations into mitochondrial DNA haplogroups

SNaPshot minisequencing reaction is in increasing use because of its fast detection of many polymorphisms in a single assay. In this work we described a highly sensitive single nucleotide polymorphisms (SNPs) typing method with detection of 42 mitochondrial DNA (mtDNA) SNPs in a single PCR and SNaPshot multiplex reaction in order to allow haplogroup classification in Latin American admixture population. We validated the panel typing 160 Brazilian individuals. DNA was extracted from blood spotted on filter paper using Chelex protocol. Forty SNPs were selected targeting haplogroup-specific mutations in Europeans, Africans and Asians (only precursors of Native Americans haplogroups A2, B2, C1, and D1) and two non-coding SNPs were chosen to increase the power of discrimination between individuals (SNPs positions 16,519 and 16,362). It was done using a modified version of a previously published multiplex SNaPshot minisequencing reaction established to resolve European haplogroups, adding SNPs targeting Africans (L0, L1, L2, L3, and L*) and Asians (A, B, C, and D) haplogroups based on SNPs described at PhyloTree.org build 2. PCR primers were designed using PerlPrimer software and checked with the Autodimer program. Thirty-three primer-pairs were used to amplify 42 SNPs. Using this panel, we were able to successfully classify 160 individuals into their correct haplogroups. Complete SNP profiles were obtained from 10. pg of total DNA. We conclude that it is possible to build and genotype more than 40 mtDNA SNPs in a single multiplex PCR and SNaPshot reaction, with sensitivity and reliability, resolving haplogroup classification in admixture populations. © 2011.