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 sequence results. Here, we describe a reliable method for the preparation of mtDNA-enriched samples from eukaryotic cells ready for direct sequencing. This protocol utilizes a conventional miniprep kit, in conjunction with a paramagnetic bead-based purification step.
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References
Brown WM, George M Jr, Wilson AC (1979) Rapid evolution of animal mitochondrial DNA. Proc Natl Acad Sci U S A 76(4):1967–1971
Baklouti-Gargouri S, Ghorbel M, Ben MA, Mkaouar-Rebai E, Cherif M, Chakroun N, Sellami A, Fakhfakh F, Ammar-Keskes L (2013) Mitochondrial DNA mutations and polymorphisms in asthenospermic infertile men. Mol Biol Rep 40(8):4705–4712
Greaves LC, Reeve AK, Taylor RW, Turnbull DM (2012) Mitochondrial DNA and disease. J Pathol 226(2):274–286
Larsson NG, Clayton DA (1995) Molecular genetic aspects of human mitochondrial disorders. Annu Rev Genet 29:151–178
Mezghani N, Mnif M, Mkaouar-Rebai E, Kallel N, Charfi N, Abid M, Fakhfakh F (2013) A maternally inherited diabetes and deafness patient with the 12S rRNA m.1555A > G and the ND1 m.3308 T > C mutations associated with multiple mitochondrial deletions. Biochem Biophys Res Commun 431(4):670–674
Van Den Ouweland JM, Lemkes HH, Trembath RC, Ross R, Velho G, Cohen D, Froguel P, Maassen JA (1994) Maternally inherited diabetes and deafness is a distinct subtype of diabetes and associates with a single point mutation in the mitochondrial tRNA(Leu(UUR)) gene. Diabetes 43(6):746–751
Bindoff LA, Engelsen BA (2012) Mitochondrial diseases and epilepsy. Epilepsia 53 Suppl 4:92–97
Khrapko K, Vijg J (2009) Mitochondrial DNA mutations and aging: devils in the details? Trends Genet 25(2):91–98
Ameur A, Stewart JB, Freyer C, Hagstrom E, Ingman M, Larsson NG, Gyllensten U (2011) Ultra-deep sequencing of mouse mitochondrial DNA: mutational patterns and their origins. PLoS Genet 7(3):e1002028
Mondal R, Ghosh SK (2013) Accumulation of mutations over the complete mitochondrial genome in tobacco-related oral cancer from northeast India. Mitochondrial DNA 24(4):432–439
Defontaine A, Lecocq FM, Hallet JN (1991) A rapid miniprep method for the preparation of yeast mitochondrial DNA. Nucleic Acids Res 19(1):185
Peloquin JJ, Bird DM, Platzer EG (1993) Rapid miniprep isolation of mitochondrial DNA from metacestodes, and free-living and parasitic nematodes. J Parasitol 79(6):964–967
Quispe-Tintaya W, White RR, Popov VN, Vijg J, Maslov AY (2013) Fast mitochondrial DNA isolation from mammalian cells for next-generation sequencing. Biotechniques 55(3):133–136
Acknowledgment
This research was supported by NIH grant P01 AG017242 (JV) and by the Ministry of Education and Science of Russian Federation grant 14.B37.21.1966 (VNP and AYM).
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Quispe-Tintaya, W., White, R.R., Popov, V.N., Vijg, J., Maslov, A.Y. (2015). Rapid Mitochondrial DNA Isolation Method for Direct Sequencing. In: Weissig, V., Edeas, M. (eds) Mitochondrial Medicine. Methods in Molecular Biology, vol 1264. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2257-4_9
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DOI: https://doi.org/10.1007/978-1-4939-2257-4_9
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