Abstract
Quantification of deletions in mtDNA is a long-standing problem in mutational analysis. We describe here an approach that combines the power of single-molecule PCR of the entire mitochondrial genome with the enrichment of the deletions by restriction digestion. This approach is indispensable if information about wide range of deletion types in a sample is critical, such as in studies concerning distribution of deletion breakpoints (as opposed to approaches where fraction of a single deletion or a limited set of deletions is used as a proxy for total deletion load). Because deletions in a sample are quantified almost exhaustively, the other important application of this approach involves studies where only small amounts of tissue, such as biopsies, are available.
The original version of this chapter was revised: The spelling of the fifth author’s name was corrected. The erratum to this chapter is available at: DOI10.1007/978-1-4939-3040-1_18
An erratum to this chapter can be found at http://dx.doi.org/10.1007/978-1-4939-3040-1_18
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Kudin AP, Zsurka G, Elger CE, Kunz WS (2009) Mitochondrial involvement in temporal lobe epilepsy. Exp Neurol 218:326–332
Brooks NE, Cadena SM, Vannier E, Cloutier G, Carambula S, Myburgh KH, Roubenoff R, Castaneda-Sceppa C (2010) Effects of resistance exercise combined with essential amino acid supplementation and energy deficit on markers of skeletal muscle atrophy and regeneration during bed rest and active recovery. Muscle Nerve 42:927–935
Cloutier G, Kraytsberg Y, Khrapko K, Brooks N, Safd A, Roubenoff R, Castaneda-Sceppa C (2014) Bedrest increases burden of mitochondrial DNA deletions in human muscle (956.1). FASEB J 28:956.1
Payne BA, Wilson IJ, Hateley CA, Horvath R, Santibanez-Koref M, Samuels DC, Price DA, Chinnery PF (2011) Mitochondrial aging is accelerated by anti-retroviral therapy through the clonal expansion of mtDNA mutations. Nat Genet 43:806–810
Khrapko K (2011) The timing of mitochondrial DNA mutations in aging. Nat Genet 43:726–727
Kraytsberg Y, Simon DK, Turnbull DM, Khrapko K (2009) Do mtDNA deletions drive premature aging in mtDNA mutator mice? Aging Cell 8:502–506
Mita S, Rizzuto R, Moraes CT, Shanske S, Arnaudo E, Fabrizi GM, Koga Y, DiMauro S, Schon EA (1990) Recombination via flanking direct repeats is a major cause of large-scale deletions of human mitochondrial DNA. Nucleic Acids Res 18:561–567
Samuels DC, Schon EA, Chinnery PF (2004) Two direct repeats cause most human mtDNA deletions. Trends Genet 20:393–398
Guo X, Popadin K, Markuzon N, Orlov YL, Kraytsberg Y, Krishnan KJ, Zsurka G, Turnbull DM, Kunz WS, Khrapko K (2010) Repeats, longevity and the sources of mtDNA deletions: evidence from “deletional spectra”. Trends Genet 26:340–343
Damas J, Carneiro J, Gonçalves J, Stewart JB, Samuels DC, Amorim A, Pereira F (2012) Mitochondrial DNA deletions are associated with non-B DNA conformations. Nucleic Acids Res 40:7606–7621
Kraytsberg Y, Kudryavtseva E, McKee AC, Geula C, Kowall NW, Khrapko K (2006) Mitochondrial DNA deletions are abundant and cause functional impairment in aged human substantia nigra neurons. Nat Genet 38:518–520
Bender A, Krishnan KJ, Morris CM, Taylor GA, Reeve AK, Perry RH, Jaros E, Hersheson JS, Betts J, Klopstock T et al (2006) High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease. Nat Genet 38:515–517
Kajander OA, Poulton J, Spelbrink JN, Rovio A, Karhunen PJ, Jacobs HT (1999) The dangers of extended PCR in the clinic [letter]. Nat Med 5:965–966
Soong NW, Hinton DR, Cortopassi G, Arnheim N (1992) Mosaicism for a specific somatic mitochondrial DNA mutation in adult human brain. Nat Genet 2:318–323
Corral-Debrinski M, Horton T, Lott MT, Shoffner JM, Beal MF, Wallace DC (1992) Mitochondrial DNA deletions in human brain: regional variability and increase with advanced age. Nat Genet 2:324–329
Meissner C, Bruse P, Mohamed SA, Schulz A, Warnk H, Storm T, Oehmichen M (2008) The 4977 bp deletion of mitochondrial DNA in human skeletal muscle, heart and different areas of the brain: a useful biomarker or more? Exp Gerontol 43:645–652
Kraytsberg Y, Nekhaeva E, Chang C, Ebralidse K, Khrapko K (2004) Analysis of somatic mutations via long-distance single molecule PCR. In: Demidov VV, Broude NE (eds) DNA amplification: current technologies and applications. Horizon, Norfolk, UK, pp 97–110
Kraytsberg Y, Bodyak N, Myerow S, Nicholas A, Ebralidze K, Khrapko K (2009) Quantitative analysis of somatic mitochondrial DNA mutations by single-cell single-molecule PCR. Methods Mol Biol 554:329–369
Vermulst M, Wanagat J, Kujoth GC, Bielas JH, Rabinovitch PS, Prolla TA, Loeb LA (2008) DNA deletions and clonal mutations drive premature aging in mitochondrial mutator mice. Nat Genet 40:392–394
Taylor SD, Ericson NG, Burton JN, Prolla TA, Silber JR, Shendure J, Bielas JH (2014) Targeted enrichment and high-resolution digital profiling of mitochondrial DNA deletions in human brain. Aging Cell 13:29–38
Bielas JH, Loeb LA (2005) Quantification of random genomic mutations. Nat Methods 2:285–290
Khrapko K, Turnbull D (2014) Mitochondrial DNA mutations in aging. Prog Mol Biol Transl Sci 127:29–62
Nicholas A, Kraytsberg Y, Guo X, Khrapko K (2009) On the timing and the extent of clonal expansion of mtDNA deletions: evidence from single-molecule PCR. Exp Neurol 218:316–319
Popadin K, Safdar A, Kraytsberg Y, Khrapko K (2014) When man got his mtDNA deletions? Aging Cell 13:579–582
Acknowledgements
This research was supported in part by the Ellison Medical Foundation (to K.K.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Kraytsberg, Y. et al. (2016). Quantitation of Mitochondrial DNA Deletions Via Restriction Digestion/Long-Range Single-Molecule PCR. In: McKenzie, M. (eds) Mitochondrial DNA. Methods in Molecular Biology, vol 1351. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3040-1_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3040-1_4
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3039-5
Online ISBN: 978-1-4939-3040-1
eBook Packages: Springer Protocols