Comet assay

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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.

Comet assay

The single cell gel electrophoresis assay SCGE , also 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 term "comet" refers to the pattern of DNA migration through the electrophoresis gel, which often resembles a comet. 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. This is followed by visual analysis with staining of DNA and calculating fluorescence to determine the extent of DNA damage. This can be performed by manual scoring or automatically by imaging software. This mono-suspension is cast on a microscope slide. A glass cover slip is held at an angle and the mono-suspension is applied to the point of contact between the coverslip and the slide. As the coverslip is lowered onto the slide the molten agarose spreads to form a thin layer.

Planarians customize their stem cell responses following genotoxic stress as a function of exposure time and regenerative state. Agarwal, A. The comet assay comet assay used to follow the accumulation of DNA breaks repair intermediates with time of incubation.

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 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.

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.

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International Programme on Chemical Safety. Measuring oxidative damage to DNA and its repair with the comet assay. Hininger, I. DNA damage and repair measured in different genomic regions using the comet assay with fluorescent in situ hybridization. Evans, M. Heterogeneity in DNA damage using the comet assay. Ethics declarations Competing interests The authors declare no competing interests. Cytogenetic status of healthy children assessed with the alkaline comet assay and the cytokinesis-block micronucleus cytome assay. NTP Carcinogenicity Database. In parallel with the development of the comet assay for DNA damage measurement, assays for DNA repair—an essential element in the genotoxic cellular response—have been developed. Cotelle S, F. Azqueta drafted the paper and revised the manuscript; all other co-authors contributed to the Materials and Procedure sections; A. Assessment of cytogenetic damage and oxidative stress in personnel occupationally exposed to the pulsed microwave radiation of marine radar equipment. Potassium bromate as positive assay control for the Fpg-modified comet assay. Measurement of DNA breaks and oxidative damage in polymorphonuclear and mononuclear white blood cells: a novel approach using the comet assay.

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.

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, CAS Google Scholar. Kohn, K. In vitro analysis of early genotoxic and cytotoxic effects of okadaic acid in different cell types of the mussel Mytilus galloprovincialis. IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. Diagnostic Res. DNA Repair Amst. Frenzilli, G. Correspondence to Amaya Azqueta. Search Search articles by subject, keyword or author. Fry, R. Vesterdal, L. Guecheva, T. Monitoring air pollution effects on children for supporting public health policy: the protocol of the prospective cohort MAPEC study. Ge, J.