Quantitation of Mitochondrial DNA Deletions Via Restriction Digestion/Long-Range Single-Molecule PCR

  • Yevgenya Kraytsberg
  • Xinhong Guo
  • Saisai Tao
  • Alexandra Kuznetsov
  • Catherine MacLean
  • Daniel Ehrlich
  • Evan Feldman
  • Igor Dombrovsky
  • Deye Yang
  • Gregory J. Cloutier
  • Carmen Castaneda-Sceppa
  • Konstantin KhrapkoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1351)


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.

Key words

PCR Mitochondrial DNA Mutation Deletions Restriction digestion Exercise 



This research was supported in part by the Ellison Medical Foundation (to K.K.).


  1. 1.
    Kudin AP, Zsurka G, Elger CE, Kunz WS (2009) Mitochondrial involvement in temporal lobe epilepsy. Exp Neurol 218:326–332CrossRefPubMedGoogle Scholar
  2. 2.
    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 42:927–935CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    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 28:956.1Google Scholar
  4. 4.
    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 43:806–810CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Khrapko K (2011) The timing of mitochondrial DNA mutations in aging. Nat Genet 43:726–727CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Kraytsberg Y, Simon DK, Turnbull DM, Khrapko K (2009) Do mtDNA deletions drive premature aging in mtDNA mutator mice? Aging Cell 8:502–506CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    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 18:561–567CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Samuels DC, Schon EA, Chinnery PF (2004) Two direct repeats cause most human mtDNA deletions. Trends Genet 20:393–398CrossRefPubMedGoogle Scholar
  9. 9.
    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 26:340–343CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    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 40:7606–7621CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    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 38:518–520CrossRefPubMedGoogle Scholar
  12. 12.
    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 38:515–517CrossRefPubMedGoogle Scholar
  13. 13.
    Kajander OA, Poulton J, Spelbrink JN, Rovio A, Karhunen PJ, Jacobs HT (1999) The dangers of extended PCR in the clinic [letter]. Nat Med 5:965–966CrossRefPubMedGoogle Scholar
  14. 14.
    Soong NW, Hinton DR, Cortopassi G, Arnheim N (1992) Mosaicism for a specific somatic mitochondrial DNA mutation in adult human brain. Nat Genet 2:318–323CrossRefPubMedGoogle Scholar
  15. 15.
    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 2:324–329CrossRefPubMedGoogle Scholar
  16. 16.
    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 43:645–652CrossRefPubMedGoogle Scholar
  17. 17.
    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 97–110Google Scholar
  18. 18.
    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 554:329–369CrossRefPubMedGoogle Scholar
  19. 19.
    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 40:392–394CrossRefPubMedGoogle Scholar
  20. 20.
    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 13:29–38CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Bielas JH, Loeb LA (2005) Quantification of random genomic mutations. Nat Methods 2:285–290CrossRefPubMedGoogle Scholar
  22. 22.
    Khrapko K, Turnbull D (2014) Mitochondrial DNA mutations in aging. Prog Mol Biol Transl Sci 127:29–62CrossRefPubMedGoogle Scholar
  23. 23.
    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 218:316–319CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Popadin K, Safdar A, Kraytsberg Y, Khrapko K (2014) When man got his mtDNA deletions? Aging Cell 13:579–582CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Yevgenya Kraytsberg
    • 1
  • Xinhong Guo
    • 2
  • Saisai Tao
    • 1
  • Alexandra Kuznetsov
    • 1
  • Catherine MacLean
    • 1
  • Daniel Ehrlich
    • 1
  • Evan Feldman
    • 1
  • Igor Dombrovsky
    • 1
  • Deye Yang
    • 3
  • Gregory J. Cloutier
    • 4
  • Carmen Castaneda-Sceppa
    • 4
  • Konstantin Khrapko
    • 5
    Email author
  1. 1.Beth Israel Deaconess Medical CenterBostonUSA
  2. 2.College of BiologyHunan UniversityChangshaPeople’s Republic of China
  3. 3.Heart Centre, The Affiliated HospitalHangzhou Normal UniversityHangzhouPeople’s Republic of China
  4. 4.Bouve College of Health SciencesNortheastern UniversityBostonUSA
  5. 5.Department of BiologyNortheastern UniversityBostonUSA

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