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 comet assay extent of damage that can be inflicted experimentally without killing 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.

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. Olive, P. Neri, M. Worldwide interest in the comet assay: a bibliometric study.

Pfuhler, S. Sul, D.

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.

Federal government websites often end in. The site is secure. 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.

Comet assay

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. The technique was later developed by Singh et al. Both assays allow visualization of fragmented DNA, and provide a straightforward way to quantitatively evaluate DNA damage.

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The slides are then immersed in a solution that cause the cells to lyse. A further application of the comet assay is as a valuable experimental tool for human biomonitoring as well as in clinical studies. NanoGenotoxicology: present and the future. 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. Earthworm comet assay for assessing the risk of weathered petroleum hydrocarbon contaminated soils: need to look further than target contaminants. Studying the kinetics of repair of induced damage will help in our understanding of cellular responses to genotoxic chemicals. Cytogenetic status and oxidative stress parameters in patients with thyroid diseases. Role of inducible nitrogen oxide synthase in benzene-induced oxidative DNA damage in the bone marrow of mice. Souto, E. Neri, M. Figure 1: Typical dose response relationships for human cells exposed to ionizing radiation using the two methods described in this protocol.

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.

Jiang, X. Norishadkam, M. Validation of the 3D reconstructed human skin comet assay, an animal-free alternative for following-up positive results from standard in vitro genotoxicity assays. Reiter, M. Sign up for Nature Briefing. Krol, K. 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. Cancer Res. You are using a browser version with limited support for CSS. Hininger, I. The comet assay: past, present, and future. A comparative performance test of standard, medium- and high-throughput comet assays. DNA strand breakage and DNA structure influence staining with propidium iodide using the alkaline comet assay.