Browsing by Subject "copy number"
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Item Discovering the Optimal Hair Sections for Mitochondrial DNA Quantification via a Multiplex Real-Time PCR Assay(2015-05-01) Nakhla, Meriam I.; Warren, Joseph E.; Planz, John V.; Hodge, Lisa M.Hair is among the frequently encountered evidence found in crime scenes. The average person loses approximately 100 hairs a day. Because these hairs are telogen strands, or at the end of their life-phase, there is very little tissue present to obtain nuclear DNA. Hair shafts, however, contain mitochondrial DNA that can be used for identification purposes. There are two areas of concern involving mtDNA analysis of hair shafts: 1) will there be enough mtDNA present to obtain a full profile, and 2) and has the integrity of mtDNA been compromised due to oxidative properties, and/or the keratinization of the hair. The purpose of this project is to elucidate whether the amount of mitochondrial DNA changes from the proximal to the distal end of the hair shaft. Five hair samples were obtained from five subjects and the hairs were dissected at every fourth centimeter. DNA was extracted from each hair section, and subjected to mitochondrial DNA quantification (via the control region of the genome), as well as assessed for any deletions seen within the coding region as a sign of damage that may have occurred, using an assay validated by the University of North Texas- Health Science Center (UNTHSC, Fort Worth, Texas). It was found that there was generally a gradual decrease in mitochondria copy number throughout the hair strands from the proximal to the distal end. Also, it was found that mitochondrial DNA is more susceptible to damage towards the distal end. Mitochondrial DNA sequencing was performed on specific samples to observe any relationship between the concentration of mitochondria and the stability of the sequence.Item Optimization and Evaluation of qPCR Duplex Assay for mtDNA Copy Number Quantification(2020-05) Johnson, Gretchen A.; Planz, John V.; Phillips, Nicole R.; Zascavage, Roxanne R.Purpose: The mitochondrial genome (mtDNA) encodes thirteen essential proteins in oxidative phosphorylation, the cell's primary energy-generating process. Depending on the cell type and stage of development, each cell contains an average of 103 to 104 copies of mtDNA. Current methods of quantification of mtDNA copy number can be imprecise due to low efficiencies of assays and inherent imbalance of mtDNA copy number with nuclear DNA (nDNA) copy number. Accurate quantification of both mtDNA and nDNA is important when calculating the ratio of mtDNA to nDNA. The goal of this project is to optimize a duplex assay that will give precise and accurate estimates in human samples. Methods: Here we employ synthetic oligomer standards for an absolute real-time qPCR assay. The significance of using absolute qPCR is that the standard curve allows for the direct comparison of unknowns to obtain a copy number. The mitochondrial target is a site in the minor arc (MinArc), and the nuclear target is a single copy locus ([beta]2M). The accuracy of this assay was evaluated using a standard reference material (SRM2372a) and the precision was evaluated via replications. Results: This design resulted in high R2 values for the standards as well as sufficiently high efficiencies. The precision of the assay was analyzed over 6 replicated runs and was deemed effectively reproducible. The accuracy was assessed with the use of a standard reference material (SRM 2372a) and was found to be problematic [Romsos et al., 2018]. This could be from a possible dilution bias of the SRM, effectively changing the copy number ratios in a difficult to predict way [Malik et al., 2011]. An attempt to mathematically correct the data was made but did not provide any solution. Conclusion: The optimization of this assay is ongoing due to the error in accuracy. The assay has proven to be precise and reproducible with sufficient efficiency. Possible future directions include sonication of samples and SRMs to examine if dilution bias could be the cause of inaccurate SRM quantification. Other methods of possibly reducing dilution bias mentioned in Malik et al. [2011] include manual shearing and the use of DNA carriers such as tRNA. Another avenue of future research could include a different method of mathematically correcting the data post run to improve accuracy. This assay has the potential to provide data which can be used to indicate overall mitochondrial health and can be utilized in various research areas such as aging, cancer, forensics and neurodevelopment.