Date of Award
The objective of this research was the evaluation and forensic validation of Denaturing High-Performance Liquid Chromatography (DHPLC) as a sequencing-independent means of detecting the presence of sequence differences in pair-wise mixtures of non-concordant amplicons of human mitochondrial DNA (mtDNA). The reproducibility and efficacy of DHPLC results, including amplification reproducibility, injection reproducibility, and column-to-column reproducibility were measured, showing negligible assay-to-assay variability. In addition, cross-contamination on the DHPLC columns demonstrated very low level DNA carryover between a high-abundance sample and subsequent zero-volume injections.
The accuracy with which DHPLC technology can be used to screen both evidence and control samples in the context of a forensic laboratory was evaluated. This was demonstrated by a number of pair-wise comparisons of each of the forensically relevant amplicons from 95 unrelated individuals in the study, and was in 100% agreement with sequencing data. 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.
Lewis, Sarah Elizabeth, "Sequence Detection And Comparative Analysis Of The Hv1 And Hv2 Control Regions Of Human Mitochondrial Dna By Denaturing High-Performance Liquid Chromatography" (2011). Electronic Theses and Dissertations. 365.
Recieved from ProQuest
Sarah Elizabeth Lewis