Protocol

DNA Repair Protocols

Volume 314 of the series Methods in Molecular Biology™ pp 183-199

Quantitative PCR-Based Measurement of Nuclear and Mitochondrial DNA Damage and Repair in Mammalian Cells

  • Janine H. SantosAffiliated withLaboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH
  • , Joel N. MeyerAffiliated withLaboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH
  • , Bhaskar S. MandavilliAffiliated withLaboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH
  • , Bennett Van HoutenAffiliated withLaboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH

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Abstract

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.

Key Words

Mammalian cells DNA quantitative damage repair QPCR