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The Comet Assay

Principles, Applications, and Limitations

  • Protocol
In Situ Detection of DNA Damage

Part of the book series: Methods in Molecular Biology ((MIMB,volume 203))

Abstract

Over the past decade, the comet assay, or single cell gel electrophoresis (SCGE) has become one of the standard methods for assessing DNA damage, with applications in genototoxicity testing, human biomonitoring and molecular epidemiology, ecotoxicology, as well as fundamental research in DNA damage and repair. The assay attracts adherents by its simplicity, sensitivity, versatility, speed and economy, and the number of publications it spawns rises each year. It is sometimes used without too much thought as to how it works or what sort of information it provides; the fact that it is so successful at demonstrating DNA damage is enough to justify its use. This is a shame, as it is capable of subtle manipulation to tell us not just how much damage is present in cells, but what form it takes. Although it is essentially a method for measuring DNA breaks, the introduction of lesion-specific endonucleases allows detection of, for example, UV-induced pyrimidine dimers, oxidized bases and alkylation damage.

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References

  1. Cook P. R., Brazell I. A. and Jost E. (1976) Characterization of nuclear structures containing superhelical DNA. J. Cell Sci. 22, 303–324.

    PubMed  CAS  Google Scholar 

  2. Östling O. and Johanson K. J. (1984) Microelectrophoretic study of radiationinduced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123, 291–298.

    Article  PubMed  Google Scholar 

  3. Singh N. P., McCoy M. T., Tice R. R. and Schneider E. L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exper. Cell Res. 175, 184–191.

    Article  CAS  Google Scholar 

  4. Olive P. L., Banáth J. P. and Durand R. E. (1990) Heterogeneity in radiationinduced DNA damage and repair in tumor and normal cells measured using the ‘comet’ assay. Rad. Res. 122, 86–94.

    Article  CAS  Google Scholar 

  5. Collins A. R., Dobson V. L., Dušinská M., Kennedy G. and Štětina R. (1997) The comet assay: what can it really tell us? Mut. Res. 375, 183–193.

    Article  CAS  Google Scholar 

  6. Angelis K. J., Dušinská M. and Collins A. R. (1999) Single cell gel electrophoresis; detection of DNA damage at different levels of sensitivity. Electrophoresis 20, 1923–1933.

    Article  Google Scholar 

  7. Olive P. L., Wlodek D. and Banáth J. P. (1991) DNA double-strand breaks measured in individual cells subjected to gel electrophoresis. Cancer Res. 51, 4671–4676.

    PubMed  CAS  Google Scholar 

  8. 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.

    Article  PubMed  CAS  Google Scholar 

  9. Dušinská 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.

    Google Scholar 

  10. 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. Env. Mol. Mutagenesis 29, 152–160.

    Article  CAS  Google Scholar 

  11. Collins A. R., Dušinská M., and Horská A. (2001) Detection of alkylation damage in human lymphocyte DNA with the comet assay. Acta Biochemica Polonica 48, 611–614.

    CAS  Google Scholar 

  12. McGlynn A. P., Wasson G., O’Connor J., McKelvey-Martin V. J. and Downes C. S. (1999) The bromodeoxyuridine comet assay: Detection of maturation of recently replicated DNA in individual cells. Cancer Res. 59, 5912–5916.

    PubMed  CAS  Google Scholar 

  13. Gedik C. M., Ewen S. W. B. and Collins A. R. (1992) Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells. Intl. J. Rad. Biol. 62, 313–320.

    Article  CAS  Google Scholar 

  14. Green M. H. L., Waugh A. P. W., Lowe J. E., Harcourt S. A., Cole J. and Arlett C. F. (1994) Effect of deoxyribonucleosides on the hypersensitivity of human peripheral blood lymphocytes to UV-B and UV-C irradiation. Mut. Res. 315, 25–32.

    CAS  Google Scholar 

  15. Santos S. J., Singh N. P. and Natarajan A. T. (1997) Fluorescence in situ hybridization with comets. Exper. Cell Res. 232, 407–411.

    Article  CAS  Google Scholar 

  16. McKelvey-Martin V. J., Ho E. T. S., McKeown S. R., McCarthy P. J., Rajab N. F. and Downes C. S. (1998) Emerging applications of the single cell gel electrophoresis (Comet) assay. I. Management of invasive transitional cell human bladder carcinoma. II. Fluorescent in situ hybridization Comets for the identification of damaged and repaired DNA sequences in individual cells. Mutagenesis 13, 1–8.

    Article  PubMed  CAS  Google Scholar 

  17. Rapp A., Bock C., Dittmar H. and Greulich K. O. (1999) Comet-fish used to detect UV-A sensitive regions in the whole human genome and on chromosome 8. Neoplasma 46, 99–101.

    Google Scholar 

  18. Singh N. P. (2000) A simple method for accurate estimation of apoptotic cells. Exper. Cell Res. 256, 328–337.

    Article  CAS  Google Scholar 

  19. Olive P. L. and Banáth J. P. (1993) Induction and rejoining of radiation-induced DNA single-strand breaks—tail moment as a function of position in the cell-cycle. Mut. Res. 294, 275–283.

    CAS  Google Scholar 

  20. Collins A., Dušinská M., Franklin M., Somorovská M., Petrovská H., Duthie S., Fillion L., Panayiotidis M., Rašlová K. and Vaughan N. (1997) Comet assay in human biomonitoring studies: reliability, validation, and applications. Env. Mol. Mutagenesis 30, 139–146.

    Article  CAS  Google Scholar 

  21. Somorovská M., Szabová E., Vodićka P., Tulinská J., Barancoková M., Fábry R., Líšková A., Riegerová Z., Petrovská H., Kubová J., Rausová K., Dušinská M. and Collins A. (1999) Biomonitoring of genotoxic risk in workers in a rubber factory: comparison of the Comet assay with cytogenetic methods and immunology. Mut. Res. 445, 181–192.

    Google Scholar 

  22. Collins A. R., Rašlová K., Somorovská M., Petrovská H., Ondrušová A., Vohnout B., Fábry R. and Duáinská M. (1998) DNA damage in diabetes: correlation with a clinical marker. Free Radical Biol. Med. 25, 373–377.

    Article  CAS  Google Scholar 

  23. Betti C., Davini T., Giannessi L., Loprieno N. and Barale R. (1994) Microgel electrophoresis assay (comet test) and SCE analysis in human lymphocytes from 100 normal subjects. Mut. Res. 307, 323–333.

    Article  CAS  Google Scholar 

  24. Jenkinson A. M., Collins A. R., Duthie S. J., Wahle K. W. J. and Duthie G. G. (1999) The effect of increased intakes of polyunsaturated fatty acids and vitamin E on DNA damage in human lymphocytes. FASEB J. 13, 2138–2142.

    PubMed  CAS  Google Scholar 

  25. Pool-Zobel B. L., Bub A., Muller H., Wollowski I. and Rechkemmer G. (1997) Consumption of vegetables reduces genetic damage in humans: first results of a human intervention trial with carotenoid-rich foods. Carcinogenesis 18, 1847–1850.

    Article  PubMed  CAS  Google Scholar 

  26. Duthie S. J., Ma A., Ross M. A. and Collins A. R. (1996) Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes. Cancer Res. 56, 1291–1295.

    PubMed  CAS  Google Scholar 

  27. Novotná B. (2000) Increased DNA fragmentation detected by comet assay in peripheral blood of heterozygotes for a frameshift mutation in the DNA repair protein nibrin. DNA Repair Workshop, Smolenice, Slovakia, October 2000, Abstracts p.33.

    Google Scholar 

  28. Collins A., Cadet J., Epe B. and Gedik C. (1997) Problems in the measurement of 8-oxoguanine in human DNA. Report of a workshop, DNA Oxidation, held in Aberdeen, UK, 19-21 January, 1997. Carcinogenesis 18, 1833–1836.

    Article  PubMed  CAS  Google Scholar 

  29. Collins A. R., Dušinská M., Horváthová E., Munro E., Savio M. and Štětina R. (2001) Inter-individual differences in repair of base oxidation, measured in vitro with the comet assay. Mutagenesis 16, 297–301.

    Article  PubMed  CAS  Google Scholar 

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© 2002 Humana Press Inc.

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Collins, A.R. (2002). The Comet Assay. In: Didenko, V.V. (eds) In Situ Detection of DNA Damage. Methods in Molecular Biology, vol 203. Humana Press. https://doi.org/10.1385/1-59259-179-5:163

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  • DOI: https://doi.org/10.1385/1-59259-179-5:163

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-952-0

  • Online ISBN: 978-1-59259-179-4

  • eBook Packages: Springer Protocols

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