The comet assay: A method to measure DNA damage in individual cells

Nature Protocols

Volumn 1, Issue 1, 2006, Pages 23-29

  Olive, Peggy L ^a   Banáth, Judit P ^a  

^a BRITISH COLUMBIA CANCER RESEARCH CENTRE   (Canada)

Author keywords

[No Author keywords available]

Indexed keywords

APOPTOSIS; ARTICLE; CELL NUCLEUS; COMET ASSAY; DNA DAMAGE; DNA REPAIR; DOUBLE STRANDED DNA BREAK; EUKARYOTIC CELL; GENETICS; METHODOLOGY;

APOPTOSIS; CELL NUCLEUS; COMET ASSAY; DNA BREAKS, DOUBLE-STRANDED; DNA DAMAGE; DNA REPAIR; EUKARYOTIC CELLS;

EID: 34250654965     PISSN: 17542189     EISSN: 17502799     Source Type: Journal    
DOI: 10.1038/nprot.2006.5     Document Type: Article

References (44)

1. Ostling, O. & Johanson, K.J. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123, 291-298 (1984).
2. Singh, N.P., McCoy, M.T., Tice, R.R. & Schneider, E.L. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184-191 (1988).
3. Rydberg, B. The rate of strand separation in alkali of DNA of irradiated mammalian cells. Radiat. Res. 61, 274-287 (1975).
4. Kohn, K.W. & Grimek-Ewig, R.A. Alkaline elution analysis, a new approach to the study of DNA single-strand interruptions in cells. Cancer Res. 33, 1849-1853 (1973).
5. Olive, P.L. Cell proliferation as a requirement for development of the contact effect in Chinese hamster V79 spheroids. Radiat. Res. 117, 79-92 (1989).
6. Olive, P.L., Banáth, J.P. & Durand, R.E. Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay. Radiat. Res. 122, 86-94 (1990).
7. Fairbairn, D.W., Olive, P.L. & O'Neill, K.L. The comet assay: A comprehensive review. Mutat. Res. 339, 37-59 (1995).
8. Salagovic, J., Gilles, J., Verschaeve, L. & Kalina, I. The comet assay for the detection of genotoxic damage in the earthworms: A promising tool for assessing the biological hazards of polluted sites. Folia Biol. (Praha.) 42, 17-21 (1996).
9. Moller, P. Genotoxicity of environmental agents assessed by the alkaline comet assay. Basic Clin. Pharmacol. Toxicol. 96 (Suppl. 1), 1-42 (2005).
10. Kiskinis, E., Suter, W. & Hartmann, A. High-throughput Comet assay using 96-well plates. Mutagenesis 17, 37-43 (2002).
11. Frieauff, W., Hartmann, A. & Suter, W. Automatic analysis of slides processed in the Comet assay. Mutagenesis 16, 133-137 (2001).
12. Tice, R.R. et al. Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35, 206-221 (2000).
13. Bauch, T., Bocker, W., Mallek, U., Muller, W.U. & Streffer, C. Optimization and standardization of the "comet assay" for analyzing the repair of DNA damage in cells. Strahlenther. Onkol. 175, 333-340 (1999).
14. Banath, J.P., Kim, A. & Olive, P.L. Overnight lysis improves the efficiency of detection of DNA damage in the alkaline comet assay. Radiat. Res. 155, 564-571 (2001).
15. Olive, P.L., Wlodek, D. & Banáth, J.P. DNA double-strand breaks measured in individual cells subjected to gel electrophoresis. Cancer Res. 51, 4671-4676 (1991).
16. Collins, A.R., Duthie, S.J. & Dobson, V.L. Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogen. 14, 1733-1735 (1993).
17. Merk, O. & Speit, G. Detection of crosslinks with the comet assay in relationship to genotoxicity and cytotoxicity. Environ. Mol. Mutagen. 33, 167-172 (1999).
18. Olive, P.L., Frazer, G. & Banáth, J.P. Radiation-induced apoptosis measured in TK6 human B lymphoblast cells using the comet assay. Radiat. Res. 136, 130-136 (1993).
19. Ostling, O. & Johanson, K.J. Bleomycin, in contrast to gamma irradiation, induces extreme variation of DNA strand breakage from cell to cell. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 52 683-691 (1987).
20. Olive, P.L., Johnston, P.J., Banáth, J.P. & Durand, R.E. The comet assay: A new method to examine heterogeneity associated with solid tumors. Nat. Med. 4, 103-105 (1998).
21. Olive, P.L., Durand, R.E., LeRiche, J.C., Olivotto, I.A. & Jackson, S.M. Gel electrophoresis of individual cells to quantify hypoxic fraction in human breast cancers. Cancer Res. 53, 733-736 (1993).
22. Olive, P.L. & Banáth, J.P. Multicell spheroid response to drugs predicted with the comet assay. Cancer Res. 57, 5528-5533 (1997).
23. Almeida, G.M., Duarte, T.L., Steward, W.P. & Jones, G.D. Detection of oxaliplatin-induced DNA crosslinks in vitro and in cancer patients using the alkaline comet assay. DNA Repair (Amst.) 5, 219-225 (2006).
24. Olive, P.L. et al. The comet assay in clinical practice. Acta Oncol. 38, 839-844 (1999).
25. Dorie, M.J. et al. DNA damage measured by the comet assay in head and neck cancer patients treated with tirapazamine. Neoplasia. 1, 461-467 (1999).
26. Speit, G. & Hartmann, A. The comet assay: A sensitive genotoxicity test for the detection of DNA damage. Methods Mol. Biol. 291, 85-95 (2005).
27. Strauss, G.H., Peters, W.P. & Everson, R.B. Measuring DNA damage in individual cells of heterogeneous mixtures: A novel application of an immunological typing technique. Mutat. Res. 304, 211-216 (1994).
28. Santos, S.J., Singh, N.P. & Natarajan, A.T. Fluorescence in situ hybridization with comets. Exp. Cell Res. 232, 407-411 (1997).
29. Rapp, A., Hausmann, M. & Greulich, K.O. The comet-FISH technique: A tool for detection of specific DNA damage and repair. Methods Mol. Biol. 291, 107-119 (2005).
30. Speit, G. & Hartmann, A. The comet assay: A sensitive genotoxicity test for the detection of DNA damage. Methods Mol. Biol. 291, 85-95 (2005).
31. Hartmann, A. et al. Recommendations for conducting the in vivo alkaline Comet assay. 4th International Comet Assay Workshop. Mutagenesis 18, 45-51 (2003).
32. Albertini, R.J. et al. IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. International Programme on Chemical Safety. Mutat. Res. 463, 111-172 (2000).
33. Duez, P., Dehon, G., Kumps, A. & Dubois, J. Statistics of the Comet assay: A key to discriminate between genotoxic effects. Mutagenesis 18, 159-166 (2003).
34. Verde, P.E. et al. Application of public-domain statistical analysis software for evaluation and comparison of comet assay data. Mutat. Res. 604, 71-82 (2006).
35. Olive, P.L. & Durand, R.E. Heterogeneity in DNA damage using the comet assay. Cytometry A 66, 1-8 (2005).
36. Olive, P.L. & Johnston, P.J. DNA damage from oxidants: Influence of lesion complexity and chromatin organization. Oncol. Res. 9, 287-294 (1997).
37. Sasaki, Y.F. et al. The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with 208 chemicals selected from the IARC monographs and U.S. NTP Carcinogenicity Database. Crit. Rev. Toxicol. 30, 629-799 (2000).
38. Cedervall, B. et al. Methods for the quantification of DNA double-strand breaks determined from the distribution of DNA fragment sizes measured by pulsed-field gel electrophoresis. Radiat. Res. 143, 8-16 (1995).
39. Olive, P.L. DNA damage and repair in individual cells: Applications of the comet assay in radiobiology. Int. J. Radiat. Biol. 75, 395-405 (1999).
40. Speit, G. & Hartmann, A. The contribution of excision repair to the DNA effects seen in the alkaline single cell gel test (comet assay). Mutagenesis 10, 555-559 (1995).
41. Alapetite, C., Wachter, T., Sage, E. & Moustacchi, E. Use of the alkaline comet assay to detect DNA repair deficiencies in human fibroblasts exposed to UVC, UVB, UVA and γ rays. Int. J. Radiat. Biol. 69, 359-369 (1996).
42. Rydberg, B. Radiation-induced heat-labile sites that convert into DNA double-strand breaks. Radiat. Res. 153, 805-812 (2000).
43. Collins, A.R. The comet assay for DNA damage and repair: Principles, applications, and limitations. Mol. Biotechnol. 26, 249-261 (2004).
44. Olive, P.L., Banáth, J.P. & Fjell, C.D. DNA strand breakage and DNA structure influence staining with propidium iodide using the alkaline comet assay. Cytometry 16, 305-312 (1994).