Forensic utility of mitochondrial DNA analysis based on denaturing high-performance liquid chromatography.

Aim: To determine the forensic utility for pairwise DNA comparisons and DNA mixture resolution with denaturing high-performance liquid chromatography (DHPLC) of human mitochondrial DNA (mtDNA).

Methods: MtDNA hypervariable regions (HV) 1 and 2 from the mtDNA D-loop were amplified by the polymerase chain reaction and mixed between known and unknown sample sources. The DNA mixtures were denatured and reannealed, and the resultant homo- and heteroduplices were evaluated by temperature-modulated heteroduplex analysis by the DHPLC method.

Results: All 144 pairwise comparisons of HV1 and HV2 mtDNA fragments were successfully resolved by the DHPLC method. Forensic proficiency test standards were successfully resolved and DHPLC match/non-match results agreed with sequencing results provided by the test providers. The DHPLC method successfully identified one questioned sample that was prepared by the test provider as a body fluid mixture. MtDNA amplicon mixtures could be separated into their constitutive components by DHPLC and fraction collection approaches.

Conclusions: DHPLC methods provide the forensic scientist with a powerful tool to rapidly screen mtDNA and may result in standardized methods to resolve mtDNA mixtures. These advances will allow mtDNA analysis in cases not previously examined by current sequencing-based approaches and could allow more forensic case samples to be entered into the proposed mtDNA Combined DNA Index System (CODIS trade mark ) databank as a result of mtDNA mixture resolution.