Molecular Genetics
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/29934
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Browsing Molecular Genetics by Author "Phillips, Nicole"
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Item Optimization and Evaluation of qPCR Duplex Assay for mtDNA Copy Number Quantification(2020) Phillips, Nicole; Planz, John; Johnson, Gretchen A.Purpose: The mitochondrial genome (mtDNA) encodes thirteen essential proteins for oxidative phosphorylation. Depending on the cell type and stage of development, each cell contains an average of 10^3 to 10^4 copies of mtDNA. Current methods of mtDNA copy number quantification 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 components 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 ratio estimates. Methods: 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 (β2M). The accuracy of this assay was evaluated using a standard reference material (SRM2372a) and the precision was evaluated via replications. Results: This assay was optimized so both targets amplify with similar efficiency to give precise and accurate ratio estimates. The precision was demonstrated over repeated runs and shown to be accurate based on SRM quantification. Conclusion: This protocol demonstrates reproducible quantification in both mitochondrial and nuclear targets while reducing time and resources. The data generated can be used to indicate overall mitochondrial health and can be utilized in research areas such as aging, cancer, forensics and neurodevelopment.Item The genomic architecture of the latent variable δ homolog (dEQ) in Mexican Americans and non-Hispanic whites(2020) Phillips, Nicole; Silzer, Talisa; Barber, Robert; Royall, Don; Palmer, Raymond; Colmenarez, MicaelaThe latent variable δ homolog (dEQ) has been established as a reliable indicator of cognitive performance and a predictor of future dementia. Here, we sought to identify genetic variants underlying dEQ in both non-Hispanic White (NWH) and Mexican American (MA) populations, hypothesizing that novel genomic risk loci for dementia will be implicated in the MA cohort. Genotyping was performed on the TARCC cohort using the Illumina® MEGA Array, which includes ~1.7 million SNPs. dEQ was generated by Donald Royall and Ray Palmer (UT Health Science Center @ San Antonio) for the entire genotyped cohort (nNHW= 1572; nMA=1030). Association testing was conducted using PLINK (Purcell et al., 2007) according to standard procedures (Anderson et al., 2010). The primary genetic association with dEQ in NHW is the TOMM40/APOE locus (rs4420638, p=9.477x10-32); the association for this locus in the MA cohort was much less significant (p=3.886x10-5). Among MAs, three interesting and unique loci emerged as suggestive: XIRP2 (chr2, rs7595556 p=8.165x10-6), KIF13A (chr6, rs7766167, p= 3.404x10-6), and LINC00907 (chr18, rs237972, p=7.88x10-7). The loci discovered in the MA cohort have not previously been implicated in dementia/Alzheimer's disease; however, variants in these genes have been associated with metabolic phenotypes that are particularly relevant to the pathophysiology of cognitive decline in MAs: diabetic neuropathy, epigenetic aging, and childhood obesity. Further investigation of these genomic regions may illuminate mechanisms by which metabolic syndromes may confer risk for earlier onset of age-related cognitive decline in MAs.