title: Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells.
abstract: In this chapter, we describe a gene-specific quantitative PCR (QPCR)-based assay for the measurement of DNA damage, using amplification of long DNA targets. This assay has been used extensively to measure the integrity of both nuclear and mitochondrial genomes exposed to different genotoxins and has proven to be particularly valuable in identifying reactive oxygen species-mediated mitochondrial DNA damage. QPCR can be used to quantify both 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 mitochondrial DNA purification. Here we discuss advantages and limitations of using QPCR to assay DNA damage in mammalian cells. In addition, we give a detailed protocol of the QPCR assay that helps facilitate its successful deployment in any molecular biology laboratory.
authors.affiliation: Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh, 5117 Centre Avenue, Pittsburgh, PA, 15213, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Furda
authors.forename: Janine H
authors.initials: JH
authors.lastname: Santos
authors.forename: Joel N
authors.initials: JN
authors.lastname: Meyer
authors.forename: Bennett
authors.initials: B
authors.lastname: Van Houten
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics; D056915:DNA Copy Number Variations; D004249:DNA Damage; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial