Analysis of DNA damage and repair in nuclear and mitochondrial DNA of animal cells using quantitative PCR.

This chapter was written as a guide to using the long-amplicon quantitative PCR (QPCR) assay for the measurement of DNA damage in mammalian as well as nonmammalian species such as Caenorhabditis elegans (nematodes), Drosophila melanogaster (fruit flies), and two species of fish (Fundulus heteroclitus and Danio rerio). Since its development in the early 1990s (Kalinowski et al., Nucleic Acids Res 20:3485-3494, 1992; Salazar and Van Houten, Mutat Res 385:139-149, 1997; Yakes and Van Houten, Proc Natl Acad Sci USA 94:514-519, 1997), the QPCR assay has been widely used to measure DNA damage and repair kinetics in nuclear and mitochondrial genomes after genotoxin exposure (Yakes and Van Houten, Proc Natl Acad Sci USA 94:514-519, 1997; Santos et al., J Biol Chem 278:1728-1734, 2003; Mandavilli et al., Mol Brain Res 133:215-223, 2005). One of the main strengths of the assay is that the labor-intensive and artifact-generating step of mitochondrial isolation is not needed for the accurate measurement of mitochondrial DNA copy number and damage. Below we present the advantages and limitations of using QPCR to assay DNA damage in animal cells and provide a detailed protocol of the QPCR assay that integrates its usage in newly developed animal systems.