Comet assay

The single cell gel electrophoresis comet assay SCGEalso known as comet assay is an uncomplicated and sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell.

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.

Comet assay

The alkaline comet assay single cell gel electrophoresis is the most widely used method for measuring DNA damage in eukaryotic cells Neri et al. It detects strand breaks SBs and alkali-labile sites at frequencies from a few hundred to several thousand breaks per cell—a biologically useful range, extending from low endogenous damage levels to the extent of damage that can be inflicted experimentally without killing cells. Digestion of the nucleoids, after lysis, with certain lesion-specific repair endonucleases allows measurement of damage other than SBs; notably, formamidopyrimidine DNA glycosylase FPG has been widely used to detect altered purines, which are converted to breaks by the enzyme. Since the first report by Ostling and Johanson the comet assay has been widely used in genotoxicity testing of chemicals, in both in vitro and in vivo models. An advantage with the latter is that cells from various tissues can be studied, in a wide variety of eukaryotic organisms. This approach is very useful since Drosophila melanogaster is a valuable model for all kinds of processes related to human health, including DNA damage responses. The use of plants as well as a wide range of terrestrial and aquatic species in the comet assay has dramatically increased in the last decade Costa et al. A recent validation study has indicated that the in vitro comet assay combined with FPG may be an effective complementary line-of-evidence in ERA even in particularly challenging natural scenarios such as estuarine environments Costa et al. During the past decade the production and use of nano-sized materials has significantly increased, and as a consequence so has human exposure to these types of materials. Identifying and understanding the hazards of nanomaterials NMs in relation to human health is not a simple matter. Not only is the chemical composition of NMs responsible for their genotoxicity, but also shape, specific surface area, size, size distribution, and zeta potential determine the effects of these materials on the genome. Although there is still a debate about the suitability of standard genotoxicity assays for studying the effects of NMs, so far the most used method in nanogenotoxicology, thanks to its robustness, versatility, and reliability, has been the comet assay Azqueta and Dusinska, In addition to investigating the genotoxicity of radiation and various chemicals, the plant comet assay has recently also been used to study the genotoxic impact of NPs Santos et al. A further application of the comet assay is as a valuable experimental tool for human biomonitoring as well as in clinical studies.

Knudsen, L.

Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites e. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups.

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.

Comet assay

Federal government websites often end in. The site is secure. DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA.

Mountain standard time to gmt

On the search for an intelligible comet assay descriptor. Kohn, K. Neri, M. A comparative performance test of standard, medium- and high-throughput comet assays. Pereira, V. Mutagenesis 21 , — Gelaleti, R. DNA damage in lens epithelium of cataract patients in vivo and ex vivo. DNA damage in lens epithelial cells and peripheral lymphocytes from age-related cataract patients. Dusinska, M. Augustyniak, M.

The comet assay single cell gel electrophoresis is the most common method for measuring DNA damage in eukaryotic cells or disaggregated tissues.

Sperm DNA damage output parameters measured by the alkaline comet assay and their importance. Article Google Scholar Feretti, D. Sorry, a shareable link is not currently available for this article. A novel contact assay for testing genotoxicity of chemicals and whole sediments in zebrafish embryos. Google Scholar Wong, C. Genetic susceptibility of newborn daughters to oxidative stress. Retrieved Sensitivity and variability of visual scoring in the comet assay. Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily. S2CID Risom, L. This is followed by visual analysis with staining of DNA and calculating fluorescence to determine the extent of DNA damage.