The Use of Bacterial Repair Endonucleases in the Comet Assay
The comet assay is a sensitive electrophoretic method for measuring DNA breaks at the level of single cells, used widely in genotoxicity experiments, in 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 oxidized purines, oxidized 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.
Key wordsComet assay DNA damage Altered bases Electrophoresis Repair endonucleases
I thank Katja Lange and Anna Tirado for excellent technical support.
- 2.Dusinska M, Collins A (1996) Detection of oxidised purines and UV-induced photoproducts in DNA of single cells, by inclusion of lesion-specific enzymes in the comet assay. Altern Lab Anim 24:405–411Google Scholar