Attempts to quantify mitochondrial DNA deletions in Single Drosophila melanogaster flies

Huzzard, Lewis and Clancy, David (2020) Attempts to quantify mitochondrial DNA deletions in Single Drosophila melanogaster flies. Masters thesis, Lancaster University.

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Abstract

Research to date has not clearly described the role mitochondrial DNA (mtDNA) deletions may have in normal ageing. Therefore, a high throughput method of mtDNA deletion detection and quantification is required. The goal of this project was to develop such an assay using individual Drosophila melanogaster, which allowed for rapid generation of aged animals and manipulation of conditions which could affect deletion generation. An assay composed of DNA extraction and Quantitative Polymerase Chain Reaction (QPCR) was designed for deletion amplification. Primers were designed to amplify deletions within the cytochrome oxidase (COX) region with primers in nadh ubiquinone oxidoreductase 1 (ND1) used as a control to quantify total mtDNA. Optimising QPCR methods for specific primer pairs improved target amplification and replicability. Redesign of an existing DNA extraction kit improved overall mtDNA yield for assay development but still lacked consistency. QPCR inhibitors present in commercial extraction kits were present in minor concentrations in extracts and likely impacted amplification efficiency. The lack of sufficient mtDNA extraction from single Drosophila for consistent deletion amplification lead to mispriming and nonspecific amplification of nuclear DNA (nDNA). Repeated amplification of one deletion across multiple extracts suggests QPCR preferentially amplifies the shortest available target sequence, corresponding to the largest deletion. Redesign of the DNA extraction method to yield higher mtDNA concentration whilst reducing inhibitors would assist in reducing nonspecific amplification. mtDNA enrichment may be required to remove nDNA if nonspecific amplification still occurs. Differential amplification of deletions depending on size renders comparison and quantification difficult. Targeted amplification of a specific common deletion may eliminate this issue at the cost of quantifying just one deletion. The likelihood of single Drosophila harbouring sufficient numbers of a specific deletion should be determined to assess if quantification of mtDNA deletion levels in single Drosophila is viable.