Abstract
The comet assay or single-cell gel electrophoresis assay is a relatively simple and sensitive technique for quantitatively measuring DNA damage and repair at the single-cell level in all types of tissue where a single-cell suspension can be obtained. Isolated cells are mixed with agarose, positioned on a glass slide, and then lysed in a high-salt solution which removes all cell contents except the nuclear matrix and DNA, which is finally subjected to electrophoresis. Damaged DNA is electrophoresed from the nuclear matrix into the agarose gel, resembling the appearance of a comet, while undamaged DNA remains largely within the proximity of the nuclear matrix. By choosing different pH conditions for electrophoresis, different damage types and levels of sensitivity are produced: a neutral (pH 8–9) electrophoresis mainly detects DNA double-strand breaks, while alkaline (pH ≥ 13) conditions detect double- and single-strand breaks as well as alkali-labile sites. This protocol describes a standard comet assay study for the analysis of DNA damage and outlines important variations of this protocol.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ostling O, Johanson KJ (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123:291–298
Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191
Olive PL, Banath JP, Durand RE (1990) Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the “comet” assay. Radiat Res 122:86–94
Klaude M, Eriksson S, Nygren J, Ahnstrom G (1996) The comet assay: mechanisms and technical considerations. Mutat Res 363:89–96
Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221
Fairbairn DW, Olive PL, O’Neill KL (1995) The comet assay: a comprehensive review. Mutat Res 339:37–59
Collins AR (2004) The comet assay for DNA damage and repair: principles, applications, and limitations. Mol Biotechnol 26:249–261
Anderson D, Schmid TE, Baumgartner A, Cemeli-Carratala E, Brinkworth MH, Wood JM (2003) Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the Comet assay). Mutat Res 544:173–178
Sipinen V, Laubenthal J, Baumgartner A, Cemeli E, Linschooten JO, Godschalk RW, Van Schooten FJ, Anderson D, Brunborg G (2010) In vitro evaluation of baseline and induced DNA damage in human sperm exposed to benzo[a]pyrene or its metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide, using the comet assay. Mutagenesis 25:417–425
Cemeli E, Baumgartner A, Anderson D (2009) Antioxidants and the Comet assay. Mutat Res 681:51–67
Collins AR, Azqueta A (2011) DNA repair as a biomarker in human biomonitoring studies; further applications of the comet assay. Mutat Res 736(1–2):122–129
Baumgartner A, Cemeli E, Anderson D (2009) The comet assay in male reproductive toxicology. Cell Biol Toxicol 25:81–98
Albertini RJ, Anderson D, Douglas GR, Hagmar L, Hemminki K, Merlo F, Natarajan AT, Norppa H, Shuker DE, Tice R et al (2000) IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. International Programme on Chemical Safety. Mutat Res 463:111–172
Kumaravel TS, Vilhar B, Faux SP, Jha AN (2009) Comet assay measurements: a perspective. Cell Biol Toxicol 25:53–64
Collins AR, Oscoz AA, Brunborg G, Gaivao I, Giovannelli L, Kruszewski M, Smith CC, Stetina R (2008) The comet assay: topical issues. Mutagenesis 23:143–151
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Anderson, D., Laubenthal, J. (2013). Analysis of DNA Damage via Single-Cell Electrophoresis. In: Makovets, S. (eds) DNA Electrophoresis. Methods in Molecular Biology, vol 1054. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-565-1_14
Download citation
DOI: https://doi.org/10.1007/978-1-62703-565-1_14
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-564-4
Online ISBN: 978-1-62703-565-1
eBook Packages: Springer Protocols