NCJ Number
226638
Date Published
July 2005
Length
106 pages
Annotation
This report describes the methodology and presents the results of research that involved the developmental validation of denaturing high-performance liquid chromatography (DHPLC) for the rapid and accurate resolution of mitochondrial DNA (mtDNA) mixtures.
Abstract
The research shows that DHPLC is an accurate method for rapid sequence-specific fractionation of heteroplasmic or multicontributor DNA mixtures prior to sequencing. Fractionation allows the mtDNA sequence of individual contributors to be unambiguously determined without secondary amplification or excessive manipulation. The demonstrated reproducibility of electrophoretic sequence profiles and correlation with changes in DNA quantity ratios makes it possible to determine the linkage phase of individual amplicons from even partially resolved mtDNA mixtures. In addition, DHPLC can be used to streamline the entire mtDNA analysis process flow by enabling analysts to assess sample complexity, accurately quantify PCR product yield without gels, purify target amplicons to facilitate improved dye terminator labeling, and confirm negative controls without sequencing. This approach has also been validated by using casework-type samples. Detailed descriptions of methods and materials address mtDNA extraction and sequencing and DHPLC analysis. 31 figures, 8 tables, 37 references, and appended draft standard operating procedures for DHPLC analysis of mtDNA