Mitochondrial DNA screening by melting curve analysis using peptide nucleic acid probes

Analysis of single nucleotide polymorphisms (SNPs) in mitochondrial (mt)DNA hypervariable regions (HV) 1/2 is valuable in forensic investigations. We developed a method for mtDNA screening of the HV1 and HV2 regions by melting curve analysis, using peptide nucleic acid (PNA) probes. This method focuses on melting peak patterns obtained by thermal dissociation of PNA/DNA duplexes in amplified mtDNA products. Five PNA probe sets were designed to detect 25 SNPs in the two HV regions. We also detected non-target SNPs based on unexpected melting temperature (T_m) shifts. In fact, 62 SNPs (42 SNPs in HV1 and 20 in HV2) were identified, including the 25 target SNPs. Using this method, 46 melting peak patterns, including 8 pattern groups, were obtained in 60 unrelated individuals. The peak patterns were compared to 55 haplotypes identified by Sanger sequencing. The results obtained from analysis of target mtDNA SNPs were entirely consistent with those obtained by Sanger sequencing. Screening the HV1 and HV2 regions of mtDNA by this method may help minimize unnecessary recourse to full sequence analysis, allows to rapidly exclude samples that do not match evidence and reference samples, and may reduce turnaround times and analysis costs. Overall, this method may be effective and helpful in forensic investigations.