Development of Multiple Assays using 46 SNPs for Comprehensive mtDNA Haplogrouping and Application to Highly Degraded...

Six multiplex PCR systems using single-base extension reactions to analyze 46 mitochondrial DNA (mtDNA)-coding region single nucleotide polymorphisms (SNPs) that define 42 haplogroups, that is, 24 major mtDNA haplogroups and 18 subclades, were devised. To improve the usefulness of the established systems for the analysis of degraded DNA samples, novel primers to render amplicons with sizes <150 bp were designed. By applying these systems to 214 Japanese individuals, 24 different haplogroups (power of discrimination = 93.4%) were found. To assess the effectiveness of our systems in grouping degraded DNA, an ancient bone sample of a Jomon skeleton was analyzed and then classified as haplogroup N9b. We conclude that the present systems are powerful screening tools for major haplogroups of mtDNA in addition to the prevalent subhaplogroups in the Japanese population and that these systems are capable of analyzing highly degraded DNA samples in forensic studies.