Abstract
Compromised mitochondrial DNA structural integrity can have functional consequences in mitochondrial gene expression and replication leading to metabolic and degenerative diseases, aging and cancer. Gel electrophoresis coupled with Southern blot and probe hybridization and long PCR are established methods for detecting mtDNA damage. But each has its respective shortcomings: gel electrophoresis is at best semi-quantitative and long PCR does not offer information on the structure. To overcome these limitations, we developed a new method with real-time PCR to accurately quantify the mtDNA structural damage/repair and copy number change. We previously showed that the different mtDNA structures (supercoiled, relaxed circular, and linear) have profound influences on the outcome of the real-time PCR amplification. The supercoiled structure is inhibitory to the PCR amplification, while relaxed structures are readily amplified. We will illustrate the use of this new method by quantifying the kinetics of mtDNA damage and repair in LNCaP prostate cancer cells induced by exogenous H2O2 treatments. The use of this new method on clinical samples for spontaneous mtDNA damage level will also be highlighted.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wallace, D.C. (2005) A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: A dawn for evolutionary medicine. Annu. Rev. Genet. 39, 359–407.
Turrens, J.F. and Boveris, A. (1980) Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem. J. 191, 421–427.
Richter, C. (1995) Oxidative damage to mitochondrial DNA and its relationship to ageing. Int. J. Biochem. Cell Biol. 27, 647–653.
Toyokuni S., Okamoto K., Yodoi J. and Hiai H. (1995) Persistent oxidative stress in cancer. FEBS Lett. 358, 1–3.
Herrmann, P.C., Gillespie, J.W., Charboneau L., Bichsel, V.E., Paweletz, C.P., Calvert, V.S., Kohn, E.C., Emmert-Buck, M.R., Liotta, L.A., et al. (2003) Mitochondrial proteome: Altered cytochrome c oxidase subunit levels in prostate cancer. Proteomics. 3, 1801–1810.
Fliss, M.S., Usadel H., Caballero, O.L., Wu L., Buta, M.R., Eleff, S.M., Jen J. and Sidransky D. (2000) Facile detection of mitochondrial DNA mutations in tumors and bodily fluids. Science. 287, 2017–2019.
Parrella, P., Xiao, Y., Fliss, M., Sanchez-Cespedes, M., Mazzarelli, P., Rinaldi, M., Nicol, T., Gabrielson, E., Cuomo, C., et al. (2001) Detection of mitochondrial DNA mutations in primary breast cancer and fine-needle aspirates. Cancer Res. 61, 7623–7626.
Chen, J.Z., Gokden, N., Greene, G.F., Mukunyadzi, P. and Kadlubar, F.F. (2002) Extensive somatic mitochondrial mutations in primary prostate cancer using laser capture microdissection. Cancer Res. 62, 6470–6474.
Radloff, R., Bauer, W. and Vinograd, J. (1967) A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: The closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. USA. 57, 1514–1521.
Robberson, D.L. and Clayton, D.A. (1972) Replication of mitochondrial DNA in mouse L cells and their thymidine kinase-derivatives: Displacement replication on a covalently-closed circular template. Proc. Natl. Acad. Sci USA. 69, 3810–3814.
Bogenhagen, D. and Clayton, D.A. (1978) Mechanism of mitochondrial DNA replication in mouse L-cells: Introduction of superhelical turns into newly replicated molecules. J. Mol. Biol. 119, 69–81.
Clayton, D.A. (1982) Replication of animal mitochondrial DNA. Cell. 28, 693–705.
Chen, J., Kadlubar, F.F. and Chen, J.Z. (2007) DNA supercoiling suppresses real-time PCR: A new approach to the quantification of mitochondrial DNA damage and repair. Nucleic Acids Res. 35(4), 1377–1388.
Ayala-Torres, S., Chen, Y., Svoboda, T., Rosenblatt, J. and Van Houten, B. (2000) Analysis of gene-specific DNA damage and repair using quantitative polymerase chain reaction. Methods. 22, 135–147.
Pfaffl M.W. (2001) A new mathematical model for relative quantification for real-time RT-PCR. Nucleic Acids Res. 29(9), e45.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Chan, S.W., Chen, J.Z. (2009). Measuring mtDNA Damage Using a Supercoiling-Sensitive qPCR Approach. In: Stuart, J.A. (eds) Mitochondrial DNA. Methods in Molecular Biology™, vol 554. Humana Press. https://doi.org/10.1007/978-1-59745-521-3_12
Download citation
DOI: https://doi.org/10.1007/978-1-59745-521-3_12
Publisher Name: Humana Press
Print ISBN: 978-1-934115-60-2
Online ISBN: 978-1-59745-521-3
eBook Packages: Springer Protocols