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, 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. 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.

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. Abstract The comet assay single-cell gel electrophoresis is a simple method for measuring deoxyribonucleic acid DNA strand breaks in eukaryotic cells. Publication types Review.

The role of comet assay DNA fragmentation testing in predicting intra-uterine insemination outcome: a systematic review and meta-analysis. Acta Biochim.

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.

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.

Comet assay

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. The assay has been modified to detect various base alterations, by including digestion of nucleoids with a lesion-specific endonuclease. We describe here recent technical developments, theoretical aspects, limitations as well as advantages of the assay, and modifications to measure cellular antioxidant status and different types of DNA repair. We briefly describe the applications of this method in genotoxicity testing, human biomonitoring, and ecogenotoxicology. Abstract The comet assay single cell gel electrophoresis is the most common method for measuring DNA damage in eukaryotic cells or disaggregated tissues. Publication types Review. Substances Genetic Markers.

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Cotelle S, F. Read Edit View history. DNA damage in leukocytes and buccal and nasal epithelial cells of individuals exposed to air pollution in Mexico City. Mandon, M. Fairbairn, D. 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. Detection of oxidised purines and UV-induced photoproducts in DNA of single cells, by inclusion of lesion-specific enzymes in the comet assay. 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. Reprints and permissions. Vestergaard, S. Zebrafish embryos as an alternative to animal experiments—a commentary on the definition of the onset of protected life stages in animal welfare regulations. Hartmann, A. Mrowicka, M.

The comet assay single-cell gel electrophoresis is a simple method for measuring deoxyribonucleic acid DNA strand breaks in eukaryotic cells.

Kooter, I. Validation of freezing tissues and cells for analysis of DNA strand break levels by comet assay. The comet assay as a rapid test in biomonitoring occupational exposure to DNA-damaging agents and effect of confounding factors. Comet assay in neural cells as a tool to monitor DNA damage induced by chemical or physical factors relevant to environmental and occupational exposure. 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. Suitability of human and mammalian cells of different origin for the assessment of genotoxicity of metal and polymeric engineered nanoparticles. A comparative performance test of standard, medium- and high-throughput comet assays. Carcinogenesis 14 , — The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility. Article Google Scholar Brennan, L. 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. This is a preview of subscription content, access via your institution. A simple technique for quantitation of low levels of DNA damage in individual cells.