QPCR: a tool for analysis of mitochondrial and nuclear DNA damage in ecotoxicology

Abstract

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.

Fig. 1

Schematic rendering of the basis of the QPCR assay as it functions in the mitochondrial genome. The circular mitochondrial genome is represented as a white circle, the long amplicon (10–15 kb) is represented as a grey crescent that amplifies the majority of this genome, and the small amplicon (~200 bases) is shaded black. Primers are represented as filled arrows. Lesions are represented as stars that would inhibit or block the progression of the DNA polymerase used in the PCR reaction, thus reducing the amplification of the long product under quantitative conditions. Amplification of the short product is not inhibited except by very high levels of damage: since the target is so small, in a large population of mitochondrial genomes, very few will have damage in the region amplified by the small product primers

Fig. 2

Schematic outline of how a QPCR experiment is carried out (adapted with permission from Ayala-Torres et al. 2000). The genotoxin-exposed biological sample of interest (e.g., cells in a petri dish or a fish from a polluted site) is sampled, and total genomic DNA is extracted, quantified, and QPCR-amplified using the same amount of template genomic DNA input. Relative amplification of all samples is compared to amplification of control/reference samples to calculate DNA damage (lesion frequency)