The Comet Assay: A Sensitive and Quantitative Method for Analysis of DNA Damage

The comet assay (single-cell gel electrophoresis) is a simple method for measuring DNA strand breaks in cells that are embedded in agarose and lysed to remove membranes and soluble cell constituents (including most histones). The assay depends on the ability of breaks to relax supercoiling, which allows DNA still attached to a nuclear matrix to move toward the anode under electrophoresis, forming a ‘comet tail’ when viewed by fluorescence microscopy. The percentage of DNA in the tail indicates the frequency of DNA breaks. An additional step — digestion with lesion-specific endonucleases — is introduced after lysis in order to detect different kinds of DNA damage. The assay can be calibrated to give quantitative measures of DNA damage. It has been widely used in genotoxicity testing and human biomonitoring, and also in ecogenotoxicology, as well as in basic research. We here discuss the development of the method, its principles, applications, and limitations, and attempt to dispel some fallacies that trouble the assay. We provide a detailed protocol, including a recent modification that increases the assay's throughput.

[1]  P. Olive,et al.  Heterogeneity in Radiation-Induced DNA Damage and Repair in Tumor and Normal Cells Measured Using the “Comet” Assay , 1990 .

[2]  Amaya Azqueta,et al.  Towards a more reliable comet assay: optimising agarose concentration, unwinding time and electrophoresis conditions. , 2011, Mutation research.

[3]  Amaya Azqueta,et al.  The influence of scoring method on variability in results obtained with the comet assay. , 2011, Mutagenesis.

[4]  Andrew R Collins,et al.  The use of bacterial repair endonucleases in the comet assay. , 2011, Methods in molecular biology.

[5]  G. Brunborg,et al.  Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation. , 2010, Toxicology letters.

[6]  David H Phillips,et al.  Variation in the measurement of DNA damage by comet assay measured by the ECVAG inter-laboratory validation trial. , 2010, Mutagenesis.

[7]  Steffen Loft,et al.  An ECVAG† trial on assessment of oxidative damage to DNA measured by the comet assay , 2009, Mutagenesis.

[8]  Alexander Rapp,et al.  Introducing a true internal standard for the Comet assay to minimize intra- and inter-experiment variability in measures of DNA damage and repair , 2009, Nucleic acids research.

[9]  G. Brunborg,et al.  Single‐cell gel electrophoresis (the comet assay): Loops or fragments? , 2008, Electrophoresis.

[10]  Awadhesh N Jha,et al.  Ecotoxicological applications and significance of the comet assay. , 2008, Mutagenesis.

[11]  A. Collins,et al.  The comet assay in human biomonitoring: gene-environment interactions. , 2008, Mutagenesis.

[12]  Gunnar Brunborg,et al.  The comet assay: topical issues. , 2008, Mutagenesis.

[13]  Catherine C. Smith,et al.  Recommendations for design of the rat comet assay. , 2008, Mutagenesis.

[14]  E. Cemeli,et al.  The comet assay in male reproductive toxicology , 2009, Cell Biology and Toxicology.

[15]  A. Collins,et al.  Comet assay-based methods for measuring DNA repair in vitro; estimates of inter- and intra-individual variation , 2009, Cell Biology and Toxicology.

[16]  V. Arlt,et al.  The 32P-postlabeling assay for DNA adducts , 2007, Nature Protocols.

[17]  M. Fenech,et al.  An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans. , 2006, Carcinogenesis.

[18]  Andrew Collins,et al.  Detection of Alu sequences and mtDNA in comets using padlock probes. , 2006, Mutagenesis.

[19]  Andrew Collins,et al.  Establishing the background level of base oxidation in human lymphocyte DNA: results of an interlaboratory validation study , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[20]  A. Collins,et al.  The comet assay for DNA damage and repair , 2004, Molecular biotechnology.

[21]  R R Tice,et al.  Recommendations for conducting the in vivo alkaline Comet assay. 4th International Comet Assay Workshop. , 2003, Mutagenesis.

[22]  Escodd.  Comparative analysis of baseline 8-oxo-7,8-dihydroguanine in mammalian cell DNA, by different methods in different laboratories: an approach to consensus. , 2002, Carcinogenesis.

[23]  A. Collins,et al.  Oxidative stress in humans: validation of biomarkers of DNA damage. , 2002, Carcinogenesis.

[24]  R. Tyrrell,et al.  Inter-laboratory Validation of Procedures for Measuring 8-oxo-7,8-dihydroguanine/8-oxo-7,8-dihydro-2′-deoxyguanosine in DNA , 2002, Free radical research.

[25]  B. Riis.  Comparison of Results from Different Laboratories in Measuring 8-oxo-2′-deoxyguanosine in Synthetic Oligonucleotides , 2002, Free radical research.

[26]  A. Collins,et al.  Detection of alkylation damage in human lymphocyte DNA with the comet assay. , 2001, Acta biochimica Polonica.

[27]  J. Mclean,et al.  Comet assay: rapid processing of multiple samples. , 2000, Mutation research.

[28]  A. Collins.  Comparison of different methods of measuring 8-oxoguanine as a marker of oxidative DNA damage , 2000 .

[29]  A. Collins,et al.  Single cell gel electrophoresis: Detection of DNA damage at different levels of sensitivity , 1999, Electrophoresis.

[30]  J. Férard,et al.  Comet assay in genetic ecotoxicology: A review , 1999, Environmental and molecular mutagenesis.

[31]  E. Seeberg,et al.  Release of normal bases from intact DNA by a native DNA repair enzyme , 1998, The EMBO journal.

[32]  J. Cadet,et al.  Problems in the measurement of 8-oxoguanine in human DNA. Report of a workshop, DNA oxidation, held in Aberdeen, UK, 19-21 January, 1997. , 1997, Carcinogenesis.

[33]  A. Collins,et al.  The comet assay: what can it really tell us? , 1997, Mutation research.

[34]  A. Collins,et al.  Oxidative DNA damage in human cells: the influence of antioxidants and DNA repair. , 1997, Biochemical Society transactions.

[35]  D. Mitchell,et al.  UV‐sensitive rodent mutant cell lines of complementation groups 6 and 8 differ phenotypically from their human counterparts , 1997, Environmental and molecular mutagenesis.

[36]  J. Ravanat,et al.  Determination of 8-oxoguanine in DNA by gas chromatography--mass spectrometry and HPLC--electrochemical detection: overestimation of the background level of the oxidized base by the gas chromatography--mass spectrometry assay. , 1995, Chemical research in toxicology.

[37]  A. Collins,et al.  Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. , 1993, Carcinogenesis.

[38]  P. Olive,et al.  DNA double-strand breaks measured in individual cells subjected to gel electrophoresis. , 1991, Cancer research.

[39]  M. Dizdaroglu.  Chemical determination of free radical-induced damage to DNA. , 1991, Free radical biology & medicine.

[40]  R. Tice,et al.  A simple technique for quantitation of low levels of DNA damage in individual cells. , 1988, Experimental cell research.

[41]  K. Johanson,et al.  Bleomycin, in contrast to gamma irradiation, induces extreme variation of DNA strand breakage from cell to cell. , 1987, International journal of radiation biology and related studies in physics, chemistry, and medicine.

[42]  A. Ootsuyama,et al.  Formation of 8-hydroxyguanine moiety in cellular DNA by agents producing oxygen radicals and evidence for its repair. , 1986, Carcinogenesis.

[43]  K J Johanson,et al.  Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. , 1984, Biochemical and biophysical research communications.

[44]  B. Rydberg,et al.  ESTIMATION OF DNA STRAND BREAKS IN SINGLE MAMMALIAN CELLS , 1978 .

[45]  T. Lindahl,et al.  Rate of depurination of native deoxyribonucleic acid. , 1972, Biochemistry.