The Use of Bacterial Repair Endonucleases in the Comet Assay

Abstract

The comet assay is a sensitive electrophoretic method for measuring DNA breaks at the level of single cells, used widely in genotoxicity experiments, biomonitoring, and in fundamental research. Its sensitivity and range of application are increased by the incorporation of an extra step, after lysis of agarose-embedded cells, in which the DNA is digested with lesion-specific endonucleases (DNA repair enzymes of bacterial or phage origin). Enzymes with specificity for oxidised purines, oxidised pyrimidines, alkylated bases, UV-induced cyclobutane pyrimidine dimers, and misincorporated uracil have been employed. The additional enzyme-sensitive sites, over and above the strand breaks detected in the standard comet assay, give a quantitative estimate of the number of specific lesions present in the cells.