COMET ASSAY INTEREST GROUP

The Comet Assay or single cell gel electrophoresis (SCGE) assay is a rapid, sensitive and relatively simple method for detecting DNA damage at the level of individual cells (Singh et al., 1988). It combines the simplicity of biochemical techniques for detecting DNA single strand breaks (strand breaks and incomplete excision repair sites), alkali-labile sites, and cross-linking, with the single cell approach typical of cytogenetic assays. 

This was first introduced by Ostling and Johanson in 1984. This was a neutral assay in which the lysis and electrophoresis were done under neutral conditions. Staining was done with acridine orange. The image obtained looked like a “comet” with a distinct head, comprising of intact DNA and a tail, consisting of damaged or broken pieces of DNA hence the name “Comet” Assay. The approach of Ostling and Johanson was based on previous work published by P Cook et al., 1976, who developed a method for investigating nuclear structure based on the high salt lysis of cells in the presence of non-ionic detergents. 

The more versatile alkaline method of the comet assay was developed by N.P. Singh and co workers in 1988. This method was developed to measure low levels of strand breaks with high sensitivity. Several reviews have been published in recent years to highlight the procedures, advantages and limitations of this assay in genotoxicological, ecotoxicological and biomonitoring studies (Collins, 2004; Dixon et al., 2002; Fairbairn et al. 1995; Lee and Steinert, 2003). The assay has also been successfully implemented in plant cells under laboratory conditions (Gichner et al., 2004, Gichner et al., 2006). 

The main advantages of the Comet Assay include: (a) the collection of data at the level of the individual cell, allowing more robust statistical analyses (b) the need for a small number of cells per sample (<10,000) (c) sensitivity for detecting DNA damage and (d) use of any eukaryote single cell population both in vitro and in vivo, including cells obtained from exposed human populations and aquatic organisms for eco-genotoxicological studies and environmental monitoring (Collins et al., 1997; Dixon et al., 2002; Lee and Steinert, 2003; Jha, 2004). 

The importance of this assay has also being realised in regulatory genotoxicological studies (Tice et al., 2000, Hartmann et al., 2003, Burlinson et al., 2007) and there is a move to replace some traditional assays (e.g. liver UDS assay) in regulatory genotoxicological studies with in vivo Comet assay. In combination with certain bacterial enzymes (e.g. formamidopyrimidine glycosylase, endonuclease III, uracil-DNA glycosylases etc.), which recognise oxidised purines and pyrimidine bases, this assay has been used to determine oxidative DNA damage which has been implicated in several health conditions (Collins et al., 1993; Collins et al., 1997a, Collins et al., 2001, Kruman et al., 2002). This assay has also been used to show protective effects of different dietary factors in chemo-preventive studies (Bichler et al., 2007; Collins et al., 2001). 

In combination with the fluorescence in situ hybridisation (FISH) technique (Comet-FISH), the application of this assay has also been extended to determine sequence or gene specific damage and repair (Santos et al., 1997; McKenna et al., 2003) as well as of possible diagnostic use (Kumaravel and Bristow, 2005). In addition, the assay is being used in translational research to assess whether tumour radio-sensitivity (Fisher et al., 2007) and chemo-sensitivity (Smith et al., 2007) can be determined. This would allow clinicians to individualize patient management, allocating cancer therapy to those for whom it will be of most benefit and reducing the likelihood of patients receiving toxic (and as such ineffective) therapy. 

In view of its wide applications and uses, based on PubMed/Web of science, in the last 10 years, more than 5000 papers have been published in peer-reviewed scientific journals, which reflect its popularity. 

The Comet Assay is based on the ability of negatively charged loops/fragments of DNA to be drawn through an agarose gel in response to an electric field. The extent of DNA migration depends directly on the DNA damage present in the cells. It should be noted that DNA lesions consisting of strand breaks after treatment with alkali either alone or in combination with certain enzymes (e.g. endonucleases) increases DNA migration, whereas DNA-DNA and DNA-protein cross-links result in retarded DNA migration compared to those in concurrent controls (Tice et al., 2000). In this assay, a suspension of cells is mixed with low melting point agarose and spread onto a microscope glass slide. Following lysis of cells with detergent at high salt concentration, DNA unwinding and electrophoresis is carried out at a specific pH. Unwinding of the DNA and electrophoresis at neutral pH (7-8) predominantly facilitates the detection of double strand breaks and cross links; unwinding and electrophoresis at pH 12.1-12.4 facilitates the detection of single and double strand breaks, incomplete excision repair sites and cross links; while unwinding and electrophoresis at a pH greater than 12.6 expresses alkali labile sites (ALS) in addition to all types of lesions listed above (Miyamae et al., 1997). When subjected to an electric field, the DNA migrates out of the cell, in the direction of the anode, appearing like a 'comet'. The size and shape of the comet and the distribution of DNA within the comet correlate with the extent of DNA damage (Fairbairn et al., 1995). Principles of image analysis are described by B Vilhar. This website serves as a single key resource for all up to date information on the comet assay. This website also provides a NIH Listserv© for any discussions about the comet assay. There are also links to various Contract Research Laboratories (CROs), who offer comet assay on a commercial basis.

REFERENCES

Bichler J, Cavin C, Simic T, Chakraborty A, Ferk F, Hoelzl C, Schulte-Hermann R, Kundi M, Haidinger G, Angelis K, Knasmüller S. Coffee consumption protects human lymphocytes against oxidative and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trp-P-2) induced DNA-damage: Results of an experimental study with human volunteers. Food Chem. Toxicol. 2007 Epub ahead of print.

Burlinson B, Tice RR, Speit G, Agurell E, Brendler-Schwaab SY, Collins AR, Escobar P, Honma M, Kumaravel TS, Nakajima M, Sasaki YF, Thybaud V, Uno Y, Vasquez M, Hartmann A. In Vivo Comet Assay Workgroup, part of the Fourth International Workgroup on Genotoxicity Testing: results of the in vivo Comet Assay workgroup. Mutat. Res. 2007; 627: 31-5.

Collins AR, Duthie, SJ, Dobson VL. Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis. 1993; 14: 1733–735.Collins A, Dusinska M, Franklin M, Somorovska M, Petrovska H, Duthie S, Fillion L, Panayiotidis M, Raslova K, Vaughan N. Comet Assay in human biomonitoring studies: reliability, validation, and applications. Environ. Mol. Mutagen. 1997; 30: 139–46.

Collins AR, Dusinská M, Horská A. Detection of alkylation damage in human lymphocyte DNA with the comet assay. Acta Biochim. Pol. 2001; 48: 611–14.

Collins AR. Comet Assay for DNA damage and repair: principles, applications and limitations. Mol. Biotechnol. 2004; 26: 249-61.

Dixon DR, Pruski AM, Dixon LRJ, Jha AN. Marine invertebrate eco-genotoxicology: a methodological overview. Mutagenesis. 2002; 17: 495-507.

Fairbairn DW, Olive PL, O’Neill KL. The Comet Assay: A comprehensive review. Mutat. Res. 1995; 339: 37-59.

Fisher AE, Burke D, Routledge MN. Can irradiation of rectal tumour cells from patient biopsy predict outcome of radiotherapy? Proceedings of the Genome Stability network/United Kingdom Environmental Mutagen Society Joint Congress, University of Cardiff, 1 – 4 July 2007.

Gichner T, Mukherjee A, Veleminsky J. DNA staining with the fluorochromes EtBr, DAPI and YOYO-1 in the comet assay with tobacco plants after treatment with ethyl methanesulphonate, hyperthermia and DNase-I. Mutat Res. 2006; 605: 17-21.

Hartmann A, Agurell E, Beevers C, Brendler-Schwaab S, Burlinson B, Clay P, Collins A, Smith A, Speit G, Thybaud V, Tice RR; 4th International Comet Assay Workshop. Recommendations for conducting the in vivo alkaline Comet Assay. Mutagenesis. 2003; 18: 45-51.

Jha AN, Genotoxicological studies in aquatic organisms: an overview. Mutat. Res. 2004; 552: 1-17.

Kruman II, Kumaravel TS, Lohani A, Pedersen WA, Cutler RG, Kruman Y, Haughey N, Lee J, Evans M, Mattson MP. Folic acid deficiency and homocysteine impair DNA repair in hippocampal neurons and sensitize them to amyloid toxicity in experimental models of Alzheimer's disease. J. Neurosci. 2002; 22:1752-62.

Kumaravel TS, Bristow RG. Detection of genetic instability at HER-2/neu and p53 loci in breast cancer cells using Comet-FISH. Breast Cancer Res. Treat. 2005; 91: 89-93

Ostling O, Johanson KJ. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 1984; 123: 291-98.

Santos SJ, Singh NP, Natarajan AT. Fluorescence in situ hybridization with comets. Exp. Cell. Res. 1997; 232: 407-11.

Smith AJO, Almeida GM, Thomas AL, Jones GD. Comet assay measures of irinotecan-induced DNA damage in vitro and in vivo. Proceedings of the Genome Stability network/United Kingdom Environmental Mutagen Society Joint Congress University of Cardiff, 1 – 4 July 2007.

Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF. Single cell gel/Comet Assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 2000; 35: 206-21.

Vilhar B. Help! There is a comet in my computer! A dummy’s guide to image analysis used in the comet assay. University of Ljubljana, http://www.botanika.biologija.org/exp/comet/Comet-principles.pdf (accessed 07, 2007)