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Assessment of Human Mitochondrial DNA Open Access Deposited

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Human mitochondrial DNA (mtDNA) quantification and sequence analysis is important in many scientific disciplines, including biomedical research and practice, reproductive genetics, environmental genetics, genetic genealogy, ancient DNA studies, and forensic DNA analysis. Furthermore, in advance of mtDNA sequence analysis, whether via traditional Sanger sequencing or with advanced massively parallel sequencing (MPS) technology, mtDNA must be successfully amplified to generate sufficient template. Therefore the objective of this project was to assess mtDNA samples, both quantitatively and qualitatively. The goals were to 1) develop an accurate and precise method for quantifying mtDNA in extracts of various human tissue, 2) ascertain its purity with respect to the presence or absence of amplification inhibitors, and 3) describe and interpret the degree of mtDNA degradation or fragmentation to facilitate mtDNA copy number and sequence determinations. Two methodologies were developed, a duplex quantitative PCR (qPCR) assay, which addressed the first two goals, and a triplex qPCR assay, which addressed all three goals. The triplex assay includes the same short mtDNA amplification target as the duplex, plus a novel internal positive control (IPC) system, which replaces the duplex IPC and was specifically developed and tested for use in the triplex assay. The triplex assay also includes a longer mtDNA target to enable assessment of degradation via a degradation index. The triplex is a hybrid qPCR assay that combines absolute quantification of the short target, which requires a standard curve, and relative quantification, which compares amplification of the long target relative to the short, using the ΔΔCt method. In support of assessing mtDNA degradation, fragmentation was simulated in silico and a model of fragmentation was developed. Equations describing copy number for any length of mtDNA target, even if not directly amplified, termed virtual copy number (VCN), were also developed based on the short and long mtDNA targets amplified in the triplex. The model and VCN values were evaluated with controlled fragmentation experiments and tested with extracts of human sera, calcified tissue, and hair. Finally, guidelines for interpreting mtDNA copy number and degradation state were established to ensure successful mtDNA analysis, particularly for ancient DNA and forensic DNA studies.

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