Typing of mitochondrial DNA coding region SNPs of forensic and anthropological interest using SNaPshot minisequencing.
The development of new methodologies for high-throughput SNP analysis is one of the most stimulating areas in genetic research. Here, we describe a rapid and robust assay to simultaneously genotype 17 mitochondrial DNA (mtDNA) coding region SNPs by minisequencing using SNaPshot. SNaPshot is a methodology based on a single base extension of an unlabeled oligonucleotide with labeled dideoxy terminators. The set of SNPs implemented in this multiplexed SNaPshot reaction allow us to allocate common mitochondrial West Eurasian haplotypes into their corresponding branch in the mtDNA skeleton, with special focus on those haplogroups lacking unambiguous diagnostic positions in the first and second hypervariable regions (HVS-I/II; by far, the most common segments analyzed by sequencing). Particularly interesting is the set of SNPs that subdivide haplogroup H; the most frequent haplogroup in Europe (40-50%) and one of the most poorly characterized phylogenetically in the HVS-I/II region. In addition, the polymorphic positions selected for this multiplex reaction increase considerably the discrimination power of current mitochondrial analysis in the forensic field and can also be used as a rapid screening tool prior to full sequencing analysis. The method has been validated in a sample of 266 individuals and shows high accuracy and robustness avoiding both the use of alternative time-consuming classical strategies (i.e. RFLP typing) and the need for high quantities of DNA template.
Quintáns
B
B
Unidad de Genética, Instituto de Medicina Legal, Universidad de Santiago de Compostela, San Francisco s/n, Santiago de Compostela, Galicia 15782, Spain.
Alvarez-Iglesias
V
V
Salas
A
A
Phillips
C
C
Lareu
M V
MV
Carracedo
A
A
D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000032:analysis*; D005060:Europe; D018732:Forensic Anthropology / Q000379:methods*; D005554:Forensic Medicine / Q000379:methods*; D014644:Genetic Variation; D006239:Haplotypes; D006801:Humans; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide
D017931:DNA Primers; D004272:DNA, Mitochondrial