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Biotechniques. 2013 Sep;55(3):133-6. doi: 10.2144/000114077.

Fast mitochondrial DNA isolation from mammalian cells for next-generation sequencing.

Author information

1
Albert Einstein College of Medicine, Department of Genetics, New York, NY, USA.

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:

mitochondrial DNA; next-generation sequencing

PMID:
24003945
PMCID:
PMC4353588
DOI:
10.2144/000114077
[Indexed for MEDLINE]
Free PMC Article

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