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

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).

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.

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.

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.