Date of Award

8-1-2011

Document Type

Masters Thesis

Degree Name

M.S.

Organizational Unit

College of Natual Science and Mathematics

First Advisor

Phillip B. Danielson, Ph.D.

Second Advisor

James Fogleman

Third Advisor

Nancy Lorenzon

Fourth Advisor

Davor Balzar

Keywords

Denaturing High-Performance Liquid Chromatography (DHPLC), Forensic validation, Human mitochondrial DNA

Abstract

The objective of this research was the evaluation and forensic validation of Denaturing High-Performance Liquid Chromatography (DHPLC) as a sequencingindependent 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.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Rights Holder

Sarah E. Lewis

Provenance

Received from ProQuest

File Format

application/pdf

Language

en

File Size

102 p.

Discipline

Biology

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