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.
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References
Collins, A.R., Duthie, S.J., and Dobson, V.L. (1993) Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis 14, 1733–1735.
Dusinska, M., and 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. Alternatives to Laboratory Animals 24, 405–411.
Collins, A.R., Dusinska, M., and Horska, A. (2001) Detection of alkylation damage in human lymphocyte DNA with the comet assay. Acta Biochimica Polonica 48, 611–614.
Collins, A.R., Mitchell, D.L., Zunino, A., de Wit, J., and Busch, D. (1997) UV-sensitive rodent mutant cell lines of complementation groups 6 and 8 differ phenotypically from their human counterparts. Environmental and Molecular Mutagenesis 29, 152–160.
Duthie, S.J., and McMillan, P. (1997) Uracil misincorporation in human DNA detected using single cell gel electrophoresis. Carcinogenesis 18, 1709–1714.
Collins, A.R. (2004) The comet assay for DNA damage and repair. Molecular Biotechnology 26, 249–261.
Collins, A.R., and Horvathova, E. (2001) Oxidative DNA damage, antioxidants and DNA repair; applications of the comet assay. Biochemical Society Transactions 29, 337–341.
Collins, A.R., Azqueta Oscoz, A., Brunborg, G. et al. (2008) The comet assay: topical issues. Mutagenesis 23, 143–151.
Collins, A.R., Cadet, J., Moller, L., Poulsen, H.E., and Vina, J. (2004) Are we sure we know how to measure 8-oxo-7,8-dihydroguanine in DNA from human cells? Archives of Biochemistry and Biophysics 423, 57–65.
Speit, G., Schütz, P., Bonzheim, I., Trenz, K., and Hoffmann, H. (2004) Sensitivity of the FPG protein towards alkylation damage in the comet assay. Toxicology Letters 146, 151–158.
Berdal, K.G., Johansen, R.F., and Seeberg, E. (1998) Release of normal bases from intact DNA by a native DNA repair enzyme. EMBO Journal 17, 363–367.
Smith, C.C., O’Donovan, M.R., and Martin, E.A. (2006) hOGG1 recognizes oxidative damage using the comet assay with greater specificity than FPG or ENDOIII. Mutagenesis 21(3), 185–190.
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I thank Katja Lange and Anna Tirado for excellent technical support.
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Collins, A.R. (2011). The Use of Bacterial Repair Endonucleases in the Comet Assay. In: Gautier, JC. (eds) Drug Safety Evaluation. Methods in Molecular Biology, vol 691. Humana Press. https://doi.org/10.1007/978-1-60761-849-2_8
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DOI: https://doi.org/10.1007/978-1-60761-849-2_8
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