The QPCR assay for analysis of mitochondrial DNA damage, repair, and relative copy number.
The quantitative polymerase chain reaction (QPCR) assay allows measurement of DNA damage in the mitochondrial and nuclear genomes without isolation of mitochondria. It also permits measurement of relative mitochondrial genome copy number. Finally, it can be used for measurement of DNA repair in vivo when employed appropriately. In this manuscript we briefly review the methodology of the QPCR assay, discuss its strengths and limitations, address considerations for measurement of mitochondrial DNA repair, and describe methodological changes implemented in recent years. We present QPCR assay primers and reaction conditions for five species not previously described in a methods article: Caenorhabditis elegans, Fundulus heteroclitus, Danio rerio, Drosophila melanogaster, and adenovirus. Finally, we illustrate the use of the assay by measuring repair of ultraviolet C radiation-induced DNA damage in the nuclear but not mitochondrial genomes of a zebrafish cell culture.
Hunter
Senyene E
SE
Nicholas School of the Environment, Duke University, Durham, NC 27708-0328, USA.
Jung
Dawoon
D
Di Giulio
Richard T
RT
Meyer
Joel N
JN
D000818:Animals; D001483:Base Sequence; D004249:DNA Damage; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair; D004272:DNA, Mitochondrial / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D018628:Gene Dosage; D054629:Genome, Mitochondrial; D006801:Humans; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D004272:DNA, Mitochondrial