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Comparative Analysis of the HV1 and HV2 Regions of Human Mitochondrial DNA by Denaturing High‐Performance Liquid Chromatography
Author(s) -
Kristinsson Richard,
Lewis Sarah E.,
Danielson Phillip B.
Publication year - 2009
Publication title -
journal of forensic sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.715
H-Index - 96
eISSN - 1556-4029
pISSN - 0022-1198
DOI - 10.1111/j.1556-4029.2008.00940.x
Subject(s) - amplicon , denaturing high performance liquid chromatography , mitochondrial dna , concordance , heteroplasmy , biology , genetics , sequence (biology) , dna sequencing , sequence analysis , microbiology and biotechnology , dna , polymerase chain reaction , gene
Denaturing high‐performance liquid chromatography (DHPLC) was evaluated as a sequencing‐independent means of detecting the presence of sequence differences in pair‐wise mixtures of nonconcordant amplicons of human mitochondrial DNA (mtDNA). A total of 920 pair‐wise combinations of HV1 and HV2 mtDNA amplicons from 95 individuals were assayed by DHPLC for sequence concordance/nonconcordance. For the 72 combinations of amplicons from different individuals who shared identical DNA sequences, DHPLC assays consistently indicated sequence concordance between the samples. This was in 100% agreement with sequencing data. For the 849 combinations of amplicons which differed in sequence, DHPLC detected the presence of sequence nonconcordance in all but 13 assays to yield 98.5% concordance with sequencing. Thus, DHPLC can be used to detect a diversity of sequence differences (transitions, transversions, insertions, and deletions) in the mtDNA D‐loop. Accordingly, DHPLC may have utility as a presumptive indicator of mtDNA sequence concordance samples, as a screen for heteroplasmy/situational mixtures, and as a means for the physical fractionation of the individual contributors to an mtDNA mixture prior to sequencing.