Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells.
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.
Santos
Janine H
JH
Laboratory of Molecular Genetics, National Institute of Environmental Health Science, NIH, Research Triangle Park, NC, USA.
Meyer
Joel N
JN
Mandavilli
Bhaskar S
BS
Van Houten
Bennett
B