Comet assay

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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.

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. The agarose forms a matrix of carbohydrate fibres that encapsulate the cells, anchoring them in place. The agarose is considered to be osmotic -neutral, therefore solutions can penetrate the gel and affect the cells without cells shifting position.

Radiation Research. Sensitivity of the FPG protein towards alkylation damage in comet assay comet assay. Biomarkers 2064—70

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 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.

Comet assay

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.

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Part C. Contribution of apoptosis to observed DNA damage in mussel cells. Cell Biol. Mutagenesis 18 , 45—51 In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Yeast 28 , 55—61 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. Mutagenesis 18 , 45—51 New revival of an old biomarker: characterisation of buccal cells and determination of genetic damage in the isolated fraction of viable leucocytes. Skip to main content Thank you for visiting nature. 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 comet assay single cell gel electrophoresis is the most common method for measuring DNA damage in eukaryotic cells or disaggregated tissues.

McAllister et al. Contribution of apoptosis to observed DNA damage in mussel cells. Gunasekarana, V. The bromodeoxyuridine comet assay: detection of maturation of recently replicated DNA in individual cells. Collins, A. Nat Protoc 18 , — Fourth International Workgroup on Genotoxicity testing: results of the in vivo comet assay workgroup. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The comet has been modified for use with sperm cells as a tool for male infertility diagnosis [19] [20] [21]. So—what can we hope for in the next 30 years? Merk, O. Published : 27 June