QPCR: a tool for analysis of mitochondrial and nuclear DNA damage in ecotoxicology.
The quantitative PCR (QPCR) assay for DNA damage and repair has been used extensively in laboratory species. More recently, it has been adapted to ecological settings. The purpose of this article is to provide a detailed methodological guide that will facilitate its adaptation to additional species, highlight its potential for ecotoxicological and biomonitoring work, and critically review the strengths and limitations of this assay. Major strengths of the assay include very low (nanogram to picogram) amounts of input DNA; direct comparison of damage and repair in the nuclear and mitochondrial genomes, and different parts of the nuclear genome; detection of a wide range of types of DNA damage; very good reproducibility and quantification; applicability to properly preserved frozen samples; simultaneous monitoring of relative mitochondrial genome copy number; and easy adaptation to most species. Potential limitations include the limit of detection (approximately 1 lesion per 10(5) bases); the inability to distinguish different types of DNA damage; and the need to base quantification of damage on a control or reference sample. I suggest that the QPCR assay is particularly powerful for some ecotoxicological studies.
Meyer
Joel N
JN
Nicholas School of the Environment, Duke University, Durham, Box 90328, NC 27708-0328, USA. joel.meyer@duke.edu
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000378:metabolism*; D054750:Ecotoxicology / Q000379:methods*; D004784:Environmental Monitoring; D005819:Genetic Markers; D006801:Humans; D009152:Mutagenicity Tests; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D018570:Risk Assessment; D013045:Species Specificity
D004272:DNA, Mitochondrial; D005819:Genetic Markers