Journal
BIOTECHNIQUES
Volume 55, Issue 3, Pages 133-+Publisher
BIOTECHNIQUES OFFICE
DOI: 10.2144/000114077
Keywords
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Funding
- NIH [P01 AG017242]
- Ministry of Education and Science of Russian Federation [14.B37.21.1966]
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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, costeffective, 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 (similar to 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.
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