Measurement of DNA base and nucleotide excision repair activities in mammalian cells and tissues using the comet assay – A methodological overview

• In vitro estimation of base and nucleotide excision repair using the comet assay.
• Protocols for measuring DNA repair in cultured cells.
• Protocols for measuring DNA repair in blood mononuclear cells.
• Protocols for measuring DNA repair in human and animal tissues.

There is an increasing demand for phenotyping assays in the field of human functional genetics. DNA repair activity is representative of this functional approach, being seen as a valuable biomarker related to cancer risk. Repair activity is evaluated by incubating a cell extract with a DNA substrate containing lesions specific for the DNA repair pathway of interest. Enzymic incision at the lesion sites can be measured by means of the comet assay (single cell gel electrophoresis). The assay is particularly applicable for evaluation of base and nucleotide excision repair pathways (BER and NER). Substrate DNA containing oxidised purines gives a measure of BER, while UV-induced photolesions are the substrate for NER. While applications of comet-based DNA repair assays continue to increase, there are no commonly accepted standard protocols, which complicates inter-laboratory comparisons of results.Here we provide a comprehensive summary of protocols for the comet-based BER- and NER-specific in vitro DNA repair assays that can be applied to a wide spectrum of biological material – cultured cell lines, blood cells, animal tissue samples and human biopsies. Our intention is to provide a detailed and user-friendly account of the assays, including practical tips and recommendations to help in setting them up. By proposing standard protocols, we hope to facilitate comparison of results obtained in different laboratories.