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 Comet assay 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, 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

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. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. 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. This is a preview of subscription content, access via your institution. The majority of the data shown here as examples or anticipated results are available in the original papers. Other supporting data are available upon reasonable request to the corresponding author.

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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 comet assay is a versatile method for measuring DNA strand breaks in individual cells. It can also be applied to cells isolated from treated animals. In this review, we highlight advantages and limitations of this in vivo comet assay in a regulatory context. Modified versions of the standard protocol detect oxidized DNA bases and may be used to reveal sites of DNA base loss, DNA interstrand crosslinks, and the extent of DNA damage induced indirectly by reactive oxygen species elicited by chemical-induced oxidative stress. The assay is, however, at best semi-quantitative, and we discuss possible approaches to improving DNA damage quantitation and highlight the necessity of optimizing protocol standardization to enhance the comparability of results between laboratories. As a genotoxicity test in vivo , the in vivo comet assay has the advantage over the better established micronucleus erythrocyte test that it can be applied to any organ, including those that are specific targets of chemical carcinogens or those that are the first sites of contact of ingested or inhaled mutagens. We illustrate this by examples of its use in risk assessment for the food contaminants ochratoxin and furan. We suggest that improved quantitation is required to reveal the full potential of the comet assay and enhance its role in the battery of in vivo approaches to characterize the mechanisms of toxicity and carcinogenicity of chemicals and to aid the determination of safe human exposure limits. Keywords: DNA damage; comet assay; risk assessment.

Comet assay

Federal government websites often end in. The site is secure. Maintenance of DNA integrity is of pivotal importance for cells to circumvent detrimental processes that can ultimately lead to the development of various diseases. In the face of a plethora of endogenous and exogenous DNA-damaging agents, cells have evolved a variety of DNA repair mechanisms that are responsible for safeguarding genetic integrity.

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DNA damage in leukocytes and buccal and nasal epithelial cells of individuals exposed to air pollution in Mexico City. Influence of mus and mus mutations of Drosophila melanogaster on the genotoxicity of model chemicals in somatic cells in vivo measured with the comet assay. The impact of single- and double-strand DNA breaks in human spermatozoa on assisted reproduction. Free Radic. Sperm DNA damage output parameters measured by the alkaline comet assay and their importance. Article Google Scholar Smith, C. The alkaline comet assay single cell gel electrophoresis is the most widely used method for measuring DNA damage in eukaryotic cells Neri et al. 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. A sensitive new method for rapid detection of abnormal methylation patterns in global DNA and within CpG islands. Pogribny, I. Advanced search. Earthworm comet assay for assessing the risk of weathered petroleum hydrocarbon contaminated soils: need to look further than target contaminants.

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.

Kundi, M. Speit, G. Part A 83A , — Radiation-induced heat-labile sites that convert into DNA double-strand breaks. Biomarkers of intermediate endpoints in environmental and occupational health. The comet assay as a rapid test in biomonitoring occupational exposure to DNA-damaging agents and effect of confounding factors. The comet assay: a sensitive genotoxicity test for the detection of DNA damage. The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with chemicals selected from the IARC monographs and U. Correspondence to Peggy L Olive. Oliveira, R. Standardisation of the in vitro comet assay: influence of lysis time and lysis solution composition on the detection of DNA damage induced by X-rays. Environmental Science and Pollution Research The bromodeoxyuridine comet assay: detection of maturation of recently replicated DNA in individual cells. Lanier, C.