title: Fast mitochondrial DNA isolation from mammalian cells for next-generation sequencing.
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.
authors.affiliation: Albert Einstein College of Medicine, Department of Genetics, New York, NY, USA.
authors.forename: Wilber
authors.initials: W
authors.lastname: Quispe-Tintaya
authors.forename: Ryan R
authors.initials: RR
authors.lastname: White
authors.forename: Vasily N
authors.initials: VN
authors.lastname: Popov
authors.forename: Jan
authors.initials: J
authors.lastname: Vijg
authors.forename: Alexander Y
authors.initials: AY
authors.lastname: Maslov
mesh_terms: D000818:Animals; D001709:Biotechnology / Q000379:methods; D002460:Cell Line; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D051379:Mice; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004272:DNA, Mitochondrial
keywords: mitochondrial DNA; next-generation sequencing