Abstract： Standard methods for mitochondrial DNA (mtDNA) extraction do not provide the level of enrichment for mtDNA sufficient for direct sequencing and must be followed by long-range-PCR amplification, which can bias the sequencing results. Here, we describe a fast, cost-effective, and reliable method for preparation of mtDNA enriched samples from eukaryotic cells ready for direct sequencing. Our protocol utilizes a conventional miniprep kit, paramagnetic bead-based purification, and an optional, limited PCR amplification of mtDNA. The first two steps alone provide more than 2000-fold enrichment for mtDNA when compared with total cellular DNA (~200-fold in comparison with current commercially available kits) as demonstrated by real-time PCR. The percentage of sequencing reads aligned to mtDNA was about 22% for non-amplified samples and greater than 99% for samples subjected to 10 cycles of long-range-PCR with mtDNA specific primers.
Keywords: next-generation sequencing, mitochondrial DNA
In mammalian cells, mitochondria are often present in thousands of copies, depending on the cell type. Mitochondrial genomes lack histone protection and reside in close proximity to reactive oxygen species. These factors, as well as limited fidelity of mtDNA replication and repair machineries, ensure a much higher mutation rate in the mitochondrial genome than in the nuclear genome (1,2), leading to heterogeneity within the mtDNA population (3,4). However, any deleterious effects of random mutations in mtDNA are compensated by the presence of multiple mitochondria in each cell. This decreased selection pressure allows mutated mtDNA to accumulate over time, making mtDNA a powerful indicator of detrimental effects of endogenous and environmental damaging agents, as well as overall somatic deterioration. It is also known that inherited mutations in the mtDNA can cause human diseases or mitochondrial disorders such as maternally inherited diabetes and deafness (5,6), mitochondrial myopathy (7), and even accelerated aging (8).