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Attempts to Quantify Mitochondrial DNA Deletions in Single Drosophila Melanogaster Flies

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Attempts to Quantify Mitochondrial DNA Deletions in Single Drosophila Melanogaster Flies Attempts to Quantify Mitochondrial DNA Deletions in Single Drosophila melanogaster Flies Masters by Research Thesis Lewis Huzzard BSc (Hons) Lancaster University July 2020 I, Lewis Huzzard, confirm that the work presented in this thesis is my own and has not been submitted in substantially the same form for the award of a higher degree elsewhere. Where information has been derived from other sources, I confirm this has been indicated in the thesis. Submitted in fulfilment of the requirements for the degree of Research Masters 1 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. 2 Table of Contents Abstract ............................................................................................................................. 2 Acknowledgements .......................................................................................................... 5 Literature Review .............................................................................................................. 6 Introduction .............................................................................................................................. 6 Concepts of Ageing .................................................................................................................... 7 Evolutionary Theory of Ageing .............................................................................................. 7 Mechanistic Theory of Ageing ............................................................................................... 8 Oxidative Theory of Ageing ..................................................................................................... 10 Free Radical Theory of Ageing ............................................................................................ 10 Redox Homeostasis ............................................................................................................. 11 The Role of Reactive Oxygen Species in Ageing .................................................................. 13 Mitochondrial Theory of Ageing ............................................................................................. 14 The Mitochondrial Theory of Ageing ................................................................................... 15 Mitochondrial DNA ............................................................................................................. 19 Mitochondrial DNA Repair .................................................................................................. 20 Mitochondrial DNA Replication ........................................................................................... 22 Causes of Mitochondrial DNA Damage ............................................................................... 24 Accumulation of Mitochondrial DNA Damage .................................................................... 25 Mitochondrial Heteroplasmy .............................................................................................. 27 The Role of Mitochondrial DNA Damage in Ageing ............................................................ 30 Mitochondrial DNA Deletion ................................................................................................... 31 Accumulation of Mitochondrial DNA Deletions .................................................................. 31 Impacts of Mitochondrial DNA Deletion ............................................................................. 34 The Common Deletion ......................................................................................................... 35 The Role of Mitochondrial DNA Deletions in Ageing ........................................................... 37 Detection & Quantification of Mitochondrial DNA Deletions ............................................. 39 Experimental Aims .................................................................................................................. 42 Age-Related Functional Decline .......................................................................................... 42 Non-genetic Manipulation of Lifespan ................................................................................ 43 cytochrome c oxidase .......................................................................................................... 44 NADH ubiquinone oxidoreductase 1 ................................................................................... 44 Experimental Aims .............................................................................................................. 45 Methods .......................................................................................................................... 46 Drosophila Egg Hatching & Food ............................................................................................. 46 Fly Ageing ................................................................................................................................ 47 DNA Extraction ........................................................................................................................ 48 Zymo Quick-DNA Tissue/Insect Microprep Kit. .................................................................... 48 Invitrogen Purelink Genomic DNA Mini Kit ......................................................................... 49 Qiagen QIAamp DNA Micro Kit. .......................................................................................... 50 Optimised Zymo Quick-DNA Tissue/Insect Microprep Kit. .................................................. 51 Primers .................................................................................................................................... 52 QPCR ........................................................................................................................................ 53 Agarose Gel Separation & Imaging .......................................................................................... 54 DNA Purification from Agarose Gel ......................................................................................... 54 Sequencing .............................................................................................................................. 55 Results ............................................................................................................................. 55 Drosophila Lifespan Data ........................................................................................................ 55 3 DNA Extraction ........................................................................................................................ 56 Comparing DNA Extraction Kits .......................................................................................... 56 Qiagen Extraction Kit Contamination ................................................................................. 58 Optimisation of the Zymo Extraction Kit ............................................................................. 60 ND1 Primer Pair ....................................................................................................................... 61 Optimisation of ND1 Amplification ..................................................................................... 62 Yui Primer Pair ......................................................................................................................... 66 Optimisation of Yui
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