Rapid screening for Native American mitochondrial and Y-chromosome haplogroups detection in routine DNA analysis.

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.