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Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells.

Publication ,  Journal Article
Santos, JH; Meyer, JN; Mandavilli, BS; Van Houten, B
Published in: Methods in molecular biology (Clifton, N.J.)
January 2006

In this chapter, we describe a gene-specific quantitative polymerase chain reaction (QPCR)-based assay for the measurement of DNA damage, using amplification of long DNA targets. This assay has been extensively used to measure the integrity of both nuclear and mitochondrial genomes exposed to different genotoxins, and has proved particularly valuable in identifying reactive oxygen species-mediated mitochondrial DNA (mtDNA) damage. QPCR can be used to quantify the formation of DNA damage, as well as the kinetics of damage removal. One of the main strengths of the assay is that it permits monitoring the integrity of mtDNA directly from total cellular DNA without the need for isolating mitochondria, or a separate step of mtDNA purification. Here we discuss advantages and limitations of using QPCR to assay DNA damage in mammalian cells. In addition, we give a detailed protocol for the QPCR assay that helps facilitate its successful deployment in any molecular biology laboratory.

Duke Scholars

Published In

Methods in molecular biology (Clifton, N.J.)

DOI

EISSN

1940-6029

ISSN

1064-3745

Publication Date

January 2006

Volume

314

Start / End Page

183 / 199

Related Subject Headings

  • Templates, Genetic
  • Rats
  • Polymerase Chain Reaction
  • Mitochondria
  • Mice
  • Humans
  • Developmental Biology
  • DNA, Mitochondrial
  • DNA Repair
  • DNA Damage
 

Citation

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Santos, J. H., Meyer, J. N., Mandavilli, B. S., & Van Houten, B. (2006). Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells. Methods in Molecular Biology (Clifton, N.J.), 314, 183–199. https://doi.org/10.1385/1-59259-973-7:183
Santos, Janine H., Joel N. Meyer, Bhaskar S. Mandavilli, and Bennett Van Houten. “Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells.Methods in Molecular Biology (Clifton, N.J.) 314 (January 2006): 183–99. https://doi.org/10.1385/1-59259-973-7:183.
Santos JH, Meyer JN, Mandavilli BS, Van Houten B. Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells. Methods in molecular biology (Clifton, NJ). 2006 Jan;314:183–99.
Santos, Janine H., et al. “Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells.Methods in Molecular Biology (Clifton, N.J.), vol. 314, Jan. 2006, pp. 183–99. Epmc, doi:10.1385/1-59259-973-7:183.
Santos JH, Meyer JN, Mandavilli BS, Van Houten B. Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells. Methods in molecular biology (Clifton, NJ). 2006 Jan;314:183–199.

Published In

Methods in molecular biology (Clifton, N.J.)

DOI

EISSN

1940-6029

ISSN

1064-3745

Publication Date

January 2006

Volume

314

Start / End Page

183 / 199

Related Subject Headings

  • Templates, Genetic
  • Rats
  • Polymerase Chain Reaction
  • Mitochondria
  • Mice
  • Humans
  • Developmental Biology
  • DNA, Mitochondrial
  • DNA Repair
  • DNA Damage