Molecular Biotechnology

, 26:249 | Cite as

The comet assay for DNA damage and repair

Principles, applications, and limitations
  • Andrew R. CollinsEmail author


The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage.

Index Entries

Comet assay single-cell gel electrophoresis DNA damage DNA repair genotoxicity testing molecular epidemiology 


  1. 1.
    Cook, P. R., Brazell, I. A., and Jost, E. (1976) Characterization of nuclear structures containing superhelical DNA. J. Cell Sci. 22, 303–324.PubMedGoogle Scholar
  2. 2.
    Östling, O. and Johanson, K. J. (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123, 291–298.PubMedCrossRefGoogle Scholar
  3. 3.
    Koppen, G. and Angelis, K. J. (1998) Repair of X-ray induced DNA damage measured by the comet assay in roots of Vicia faba. Env. Mol. Mutagenesis 32, 281–285.CrossRefGoogle Scholar
  4. 4.
    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. Exp. Cell Res. 175, 184–191.PubMedCrossRefGoogle Scholar
  5. 5.
    Olive, P. L., Banáth, J. P., and Durand, R. E. (1990) Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the “comet” assay. Radiat. Res. 122, 86–94.PubMedCrossRefGoogle Scholar
  6. 6.
    Collins, A. R., Dobson, V. L., Dušinská, M., Kennedy, G., and Štětina, R. (1997) The comet assay: what can it really tell us? Mutat. Res. 375, 183–193.PubMedGoogle Scholar
  7. 7.
    Collins, A. R. and Dušinská, M. (2002) Oxidation of cellular DNA measured with the comet assay. In Methods in Molecular Biology, vol. 186, Oxidative Stress Biomarkers and Antioxidant Protocols (Armstrong, D., ed.). Humana Press, Totowa, NJ, pp. 147–159.Google Scholar
  8. 8.
    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.CrossRefGoogle Scholar
  9. 9.
    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.PubMedGoogle Scholar
  10. 10.
    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.PubMedCrossRefGoogle Scholar
  11. 11.
    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. Altern. Lab. Anim. 24, 405–411.Google Scholar
  12. 12.
    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. Environ. Mol. Mutagen. 29, 152–160.PubMedCrossRefGoogle Scholar
  13. 13.
    Collins, A. R., Dušinská, M., and Horská, A. (2001) Detection of alkylation damage in human lymphocyte DNA with the comet assay. Acta Biochim. Pol. 48, 611–614.PubMedGoogle Scholar
  14. 14.
    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.PubMedGoogle Scholar
  15. 15.
    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. Int. J. Radiat. Biol. 62, 313–320.PubMedGoogle Scholar
  16. 16.
    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. Mutat. Res. 315, 25–32.PubMedGoogle Scholar
  17. 17.
    Collins, A. R., Ma, A-G., and Duthie, S. J. (1995) The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells. Mutat. Res. 336, 69–77.PubMedGoogle Scholar
  18. 18.
    Santos, S. J., Singh, N. P., and Natarajan, A. T. (1997) Fluorescence in situ hybridization with comets. Exp. Cell Res. 232, 407–411.PubMedCrossRefGoogle Scholar
  19. 19.
    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.PubMedCrossRefGoogle Scholar
  20. 20.
    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
  21. 21.
    Collins, A. R., Horváthová, E., Dušinská, M., and Shaposhnikov, S. (submitted) Gene-specific DNA damage and repair measured by the comet assay with fluorescent in situ hybridization.Google Scholar
  22. 22.
    Singh, N. P. (2000) A simple method for accurate estimation of apoptotic cells. Exp. Cell Res. 256, 328–337.PubMedCrossRefGoogle Scholar
  23. 23.
    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. Mutat. Res. 294, 275–283.PubMedGoogle Scholar
  24. 24.
    Collins, A. R., Dušinská, M., Gedik, C. M., and Štětina, R. (1996) Oxidative damage to DNA: Do we have a reliable biomarker? Environ. Health Perspect. 104(Suppl 3), 465–469.PubMedCrossRefGoogle Scholar
  25. 25.
    Hartmann, A., Agurell, E., Beevers, C., et al. (2003) Recommendations for conducting the in vivo alkaline comet assay. Mutagenesis 18, 45–51.PubMedCrossRefGoogle Scholar
  26. 26.
    Tice, R. R., Agurell, E., Anderson, D., et al. (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35, 206–221.PubMedCrossRefGoogle Scholar
  27. 27.
    Duthie, S. J. and Dobson, V. L. (1999) Dietary flavonoids protect human colonocyte DNA from oxidative attack in vitro. Eur. J. Nutr. 38, 28–34.PubMedCrossRefGoogle Scholar
  28. 28.
    Dixon, D. R., Pruski, A. M., Dixon, L. R. J., and Jha, A. N. (2002) Marine invertebrate eco-genotoxicology: a methodological overview. Mutagenesis 17, 495–507.PubMedCrossRefGoogle Scholar
  29. 29.
    Verschaeve, L. and Gilles, J. (1995) Single-cell gel-electrophoresis assay in the earthworm for the detection of genotoxic compounds in soils. Bull. Env. Contam. Toxicol. 54, 112–119.CrossRefGoogle Scholar
  30. 30.
    Somorovská, M., Szabová, E., Vodička, P., et al. (1999) Biomonitoring of genotoxic risk in workers in a rubber factory: comparison of the Comet assay with cytogenetic methods and immunology. Mutat. Res. 445, 181–192.PubMedGoogle Scholar
  31. 31.
    Collins, A. R., Rašlová, K., Somorovská, M., et al. (1998) DNA damage in diabetes: correlation with a clinical marker. Free Radic. Biol. Med. 25, 373–377.PubMedCrossRefGoogle Scholar
  32. 32.
    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. Mutat. Res. 307, 323–333.PubMedGoogle Scholar
  33. 33.
    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.PubMedGoogle Scholar
  34. 34.
    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.PubMedCrossRefGoogle Scholar
  35. 35.
    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.PubMedGoogle Scholar
  36. 36.
    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, Abstract p. 33.Google Scholar
  37. 37.
    Green, M. H. L., Lowe, J. E., Harcourt, S. A., et al. (1992) UV-C sensitivity of unstimulated and stimulated human lymphocytes from normal and xeroderma pigmentosum donors in the comet assay: A potential diagnostic technique. Mutat. Res. 273, 137–144.PubMedGoogle Scholar
  38. 38.
    ESCODD (2003) Measurement of DNA oxidation in human cells by chromatographic and enzymic methods. Free Radic. Biol. Med. 34, 1089–1099.CrossRefGoogle Scholar
  39. 39.
    ESCODD, Gedik, C. M., and Collins, A. (submitted) Base oxidation in human lymphocyte DNA: results of an inter-laboratory validation study.Google Scholar
  40. 40.
    Frankenberg-Schwager, M. (1989) Review of repair kinetics for DNA damage induced in eukaryotic cells in vitro by ionizing radiation. Radiother Oncol. 14, 307–320.PubMedCrossRefGoogle Scholar
  41. 41.
    Torbergsen, A. C. and Collins, A. R. (2000) Recovery of human lymphocytes from oxidative DNA damage: the apparent enhancement of DNA repair by carotenoids is probably simply an antioxidant effect. Eur. J. Nutr. 39, 80–85.PubMedCrossRefGoogle Scholar
  42. 42.
    Collins, A. R. and Horváthová, E. (2001) Oxidative DNA damage, antioxidants and DNA repair: applications of the comet assay. Biochem. Soc. Trans. 29(Pt 2), 337–341.PubMedCrossRefGoogle Scholar
  43. 43.
    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.PubMedCrossRefGoogle Scholar
  44. 44.
    Collins, A. R., Harrington, V., Drew, J., and Melvin, R. (2003) Nutritional modulation of DNA repair in a human intervention study. Carcinogenesis 24, 511–515.PubMedCrossRefGoogle Scholar
  45. 45.
    Collins, A., Dušinská, M., Franklin, M., et al. (1997) Comet assay in human biomonitoring studies: reliability, validation, and applications. Env. Mol. Mutagen. 30, 139–146.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2004

Authors and Affiliations

  1. 1.Department of NutritionUniversity of OsloOsloNorway

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