Application of Oxford Nanopore Technology in Detecting Single and Multiple mtDNA Deletions

Mitochondrial DNA (mtDNA) plays a central role in oxidative phosphorylation, and its structural integrity is essential for normal cellular function. Due to its proximity to the respiratory chain and lack of protective histones, mtDNA is highly susceptible to variation; among these, single and multiple deletions are major contributors to mitochondrial disease. Conventional methods (Southern blotting, long-range PCR, short-read NGS) have been widely used to study such deletions; however, their effectiveness is limited by amplification bias and indirect inference. Oxford Nanopore Technology (ONT) offers a promising alternative for detecting large-scale mtDNA deletions by enabling amplification-free, long-read sequencing and real-time data acquisition. ONT also supports integrated workflows for basecalling, alignment, and quality control (e.g., on GridION), and provides read-level views that reveal deletion breakpoints and the co-presence of undeleted molecules. This thesis applies ONT to detect single and multiple mtDNA deletions in muscle-derived Mus musculus DNA, using long-range PCR only as a comparator. LR-PCR gels showed sub-genome-length bands consistent with assay bias, whereas ONT resolved deletions directly at the molecule level and distinguished single dominant events from coexisting deletions within the same sample.