This study evaluated denaturing high-performance liquid chromatography (DNPLC) as a sequencing-independent means of detecting the presence of sequence differences in pair-wise mixtures of nonconcordant amplicons of human mitochondrial DNA (mtDNA).
The study demonstrates that DHPLC analysis of pair-wise combinations of identical mtDNA amplicons accurately and reliably produces a single chromatographic peak consistent with sequence concordance. These results were in complete agreement with DNA sequence data. Conversely, in pair-wise combinations of nonidentical amplicons, DHPLC successfully detected a diversity of sequence differences throughout the HV1 and HV2 regions. Based on this evaluation, DHPLC may have significant forensic usefulness in several areas. These include as a presumptive test of mtDNA concordance between known and questioned samples, as a screen for mixed samples prior to direct sequencing, and as a preparatory tool for the physical fractionation of the individual contributors to an mtDNA mixture prior to sequencing. Although DHPLC cannot be considered a replacement for direct sequencing of mtDNA, it has advantages as a potential screening tool. First, the assay is relatively simple and fast. It uses raw PCR products, which avoids the time and expense of amplicons cleanup. Compared to alternate mtDNA screening strategies based on oligonucleotide probes or linear arrays, DHPLC consumes less DNA and is not limited by the need to design probes for the detection of known mutations at predetermined polymorphic sites. The study also shows DHPLC can be used to resolve mixtures of nonidentical mtDNA amplicons into a series of homo- and heteroduplex peaks. Since these can be differentially eluted in time, it should be possible to physically recover and concentrate the DNA contained in any given fraction of eluent. A total of 920 pair-wise combinations of HV1 and V2 mtDNA amplicons from 95 individuals were assayed by DHPLC for sequence concordance/nonconcordance. 3 tables, 4 figures, and 33 references