Evaluation of Molecular Techniques Using a Synthetic Mitochondrial Genome

The mitochondrion is responsible for the bulk of cellular energy production through the process of oxidative phosphorylation. The mitochondrial genome (mtGenome) is subject to a high mutation rate due to its proximity to reactive oxygen species produced in energy production. Over 250 pathogenic mutations have been characterized, and studies have demonstrated mtDNA variations at the cellular, tissue, and individual level. Some of the characterization techniques include long range PCR and sequencing. Sanger sequencing has been the gold standard, but next-generation sequencing technologies are now available. These methods may be evaluated using synthetic DNA of known base composition. This project utilizes the first synthetic mtGenome to optimize a LR PCR protocol and evaluate sequence quality using Sanger, MiSeq System, and Ion Personal Genome Machine System sequencing platforms.