Comet assay

The single cell gel electrophoresis assay SCGEalso known as comet assay is an uncomplicated and sensitive technique for comet assay detection of DNA damage at the level of the individual eukaryotic cell. The term "comet" refers to the pattern of DNA migration through the electrophoresis gel, which often resembles a comet, comet assay.

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. We present a procedure for the comet assay, a gel electrophoresis—based method that can be used to measure DNA damage in individual eukaryotic cells. It is versatile, relatively simple to perform and sensitive. Although most investigations make use of its ability to measure DNA single-strand breaks, modifications to the method allow detection of DNA double-strand breaks, cross-links, base damage and apoptotic nuclei.

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.

Giovannelli, L. Ophthalmic Res.

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.

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.

Comet assay

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. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage.

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ISSN 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. Gowher, H. Article PubMed Google Scholar. Overnight lysis improves the efficiency of detection of DNA damage in the alkaline comet assay. SpheroidChip: patterned agarose microwell compartments harboring HepG2 spheroids are compatible with genotoxicity testing. PubMed Abstract Google Scholar. The types of damage detected encompass DNA strand breaks and alkali-labile sites e. Cytogenetic status and oxidative stress parameters in patients with thyroid diseases. Seasonal variations as predictive factors of the comet assay parameters: a retrospective study. In an in vitro study the cells would be exposed to a test agent — typically UV light , ionising radiation , or a genotoxic chemical — to induce DNA damage in the encapsulated cells. Structural basis for inhibition of DNA replication by aphidicolin. Google Scholar Collins, A. Anyone you share the following link with will be able to read this content:.

The comet assay single cell gel electrophoresis is the most common method for measuring DNA damage in eukaryotic cells or disaggregated tissues. The assay depends on the relaxation of supercoiled DNA in agarose-embedded nucleoids the residual bodies remaining after lysis of cells with detergent and high salt , which allows the DNA to be drawn out towards the anode under electrophoresis, forming comet-like images as seen under fluorescence microscopy.

Technical recommendations to perform the alkaline standard and enzyme-modified comet assay in human biomonitoring studies. Article Google Scholar Gowher, H. Genetic susceptibility of newborn daughters to oxidative stress. ISSN Hum Reprod. Radiation-induced heat-labile sites that convert into DNA double-strand breaks. Cell Res. Issue Date : March The pH of the lysis solution can be adjusted usually between neutral and alkaline pH depending upon the type of damage the researcher is investigating. Yeast 28 , 55—61 Technical updates to the comet assay in vivo for assessing DNA damage in zebrafish embryos from fresh and frozen cell suspensions.