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

Wilber Quispe-Tintaya, Ryan R. White, Vasily N. Popov, Jan Vijg, Alexander Y. Maslov

Research output: Contribution to journalArticle

28 Scopus citations

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. Te 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. Te 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.

Original languageEnglish (US)
Pages (from-to)133-136
Number of pages4
JournalBioTechniques
Volume55
Issue number3
DOIs
StatePublished - Sep 1 2013

Keywords

  • Mitochondrial DNA
  • Next-generation sequencing

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

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