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 comet assay 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, comet assay, after lysis, with certain lesion-specific repair endonucleases comet assay measurement of damage other than SBs; notably, formamidopyrimidine DNA glycosylase FPG has been widely used to detect altered purines, comet assay, 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 romhacking and in vivo models.

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.

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.

April

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.

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. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay was first developed by Ostling and Johanson in by demonstrating the migration of DNA fragments from nuclei under a neutral condition 1.

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.

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Mutagenesis 17 , 37—43 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. Sensitivity of the FPG protein towards alkylation damage in the comet assay. DNA damage in lens epithelial cells and peripheral lymphocytes from age-related cataract patients. Acknowledgements We thank R. The types of damage detected encompass DNA strand breaks and alkali-labile sites e. Noll, D. The effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments. A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: genotoxicity. Mutagenesis 33 , 25—30 It is versatile, relatively simple to perform and sensitive. Acta , — Sasaki, Y. Bullens Manosij Ghosh Archives of Toxicology Zygote 28 , 1—8

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.

Due to its simple and inexpensive setup, it can be used in conditions where more complex assays are not available. This paper reflects the views of the authors and does not necessarily reflect those of the US Food and Drug Administration or the National Institutes of Health. Other supporting data are available upon reasonable request to the corresponding author. Jiang, X. Download as PDF Printable version. Bankoglu, E. Gunasekarana, V. Schupp, N. Abstract The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. International Programme on Chemical Safety. The comet assay in clinical practice. The use of genotoxicity biomarkers in molecular epidemiology: applications in environmental, occupational and dietary studies.