Abstract
Mitochondrial DNA (mtDNA) deletions underpin mitochondrial dysfunction in human tissues in aging and disease. The multicopy nature of the mitochondrial genome means these mtDNA deletions can occur in varying mutation loads. At low levels, these deletions have no impact, but once the proportion of molecules harbouring a deletion exceeds a threshold level, then dysfunction occurs. The location of the breakpoints and the size of the deletion impact upon the mutation threshold required to cause deficiency of an oxidative phosphorylation complex, and this varies for each of the different complexes. Furthermore, mutation load and deletion species can vary between adjacent cells in a tissue, with a mosaic pattern of mitochondrial dysfunction observed. As such, it is often important for understanding human aging and disease to be able to characterise the mutation load, breakpoints and size of deletion(s) from a single human cell. Here, we detail protocols for laser micro-dissection and single cell lysis from tissues, and the subsequent analysis of deletion size, breakpoints and mutation load using long-range PCR, mtDNA sequencing and real-time PCR, respectively.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Damas J, Carneiro J, Amorim A, Pereira F (2014) MitoBreak: the mitochondrial DNA breakpoints database. Nucleic Acids Res 42(Database issue):D1261–D1268. https://doi.org/10.1093/nar/gkt982
Rocha MC, Rosa HS, Grady JP, Blakely EL, He L, Romain N et al (2018) Pathological mechanisms underlying single large-scale mitochondrial DNA deletions. Ann Neurol 83(1):115–130. https://doi.org/10.1002/ana.25127
Pitceathly RD, Rahman S, Hanna MG (2012) Single deletions in mitochondrial DNA--molecular mechanisms and disease phenotypes in clinical practice. Neuromuscul Disord: NMD 22(7):577–586. https://doi.org/10.1016/j.nmd.2012.03.009
Nicholls TJ, Zsurka G, Peeva V, Schöler S, Szczesny RJ, Cysewski D et al (2014) Linear mtDNA fragments and unusual mtDNA rearrangements associated with pathological deficiency of MGME1 exonuclease. Hum Mol Genet 23(23):6147–6162. https://doi.org/10.1093/hmg/ddu336
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. https://doi.org/10.1007/978-1-59745-521-3_21
Lehmann D, Tuppen HAL, Campbell GE, Alston CL, Lawless C, Rosa HS et al (2019) Understanding mitochondrial DNA maintenance disorders at the single muscle fibre level. Nucleic Acids Res 47(14):7430–7443. https://doi.org/10.1093/nar/gkz472
Rygiel KA, Tuppen HA, Grady JP, Vincent A, Blakely EL, Reeve AK et al (2016) Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis. Nucleic Acids Res 44(11):5313–5329. https://doi.org/10.1093/nar/gkw382
Campbell GR, Ziabreva I, Reeve AK, Krishnan KJ, Reynolds R, Howell O et al (2011) Mitochondrial DNA deletions and neurodegeneration in multiple sclerosis. Ann Neurol 69(3):481–492. https://doi.org/10.1002/ana.22109
Rygiel KA, Grady JP, Taylor RW, Tuppen HAL, Turnbull DM (2015) Triplex real-time PCR--an improved method to detect a wide spectrum of mitochondrial DNA deletions in single cells. Sci Rep 5:9906. https://doi.org/10.1038/srep09906
Basu S, Xie X, Uhler JP, Hedberg-Oldfors C, Milenkovic D, Baris OR, Kimoloi S, Matic S, Stewart JB, Larsson N-G, Wiesner RJ, Oldfors A, Gustafsson CM, Falkenberg M, Larsson E (2020) Accurate mapping of mitochondrial DNA deletions and duplications using deep sequencing. PLoS Genet 16(12):e1009242
Murphy JL, Ratnaike TE, Shang E, Falkous G, Blakely EL, Alston CL et al (2012) Cytochrome c oxidase-intermediate fibres: importance in understanding the pathogenesis and treatment of mitochondrial myopathy. Neuromuscul Disord: NMD 22(8):690–698. https://doi.org/10.1016/j.nmd.2012.04.003
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Tuppen, H.A.L., Reeve, A.K., Vincent, A.E. (2023). Single Cell Analysis of Mitochondrial DNA Deletions. In: Nicholls, T.J., Uhler, J.P., Falkenberg, M. (eds) Mitochondrial DNA. Methods in Molecular Biology, vol 2615. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2922-2_29
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2922-2_29
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2921-5
Online ISBN: 978-1-0716-2922-2
eBook Packages: Springer Protocols