Abstractto identify an effective method of evaluating the radiosensitivity of human tumor cell lines in vitro. Single cell gel electrophoresis scge or the comet assay is a versatile, sensitive yet simple and economical technique. Investigating oxidative dna damage and its repair using. Comet assay or single cell gel electrophoresis assay detects single or double strand breaks measured at the individual cell level. The neutral comet assay allows the detection of doublestrand breaks by subjecting lysed cell nuclei to an electrophoretic field at neutral ph. The most widely used method for assessment of dna damage is the alkaline comet assay.
The comet assay is a damage genetic biomarker based on the migration of negatively charged dna fragments produced by mutagenic agents in individual cells. Leucocytes isolated from simply frozen whole blood can be. The comet assay which can measure residual microdna damage is appropriate for the study of dna repair. Regardless of the nature or location of the dsbs, chinese hamster v79 cells with sphase dna content showed about 23 times less damage by all agents than cells with g1 or g2mphase dna content. The comet assay is an extremely sensitive dna damage assay. Evaluating in vitro dna damage using comet assay protocol. Comet assay is a noninvasive technique that can serve as a powerful tool in the detection of these genetic level dna damages. The comet assay, also called single cell gel electrophoresis scge, is a sensitive and rapid technique for quantifying and analyzing dna damage in individual cells. Sc, the university of british columbia, 1990 a thesis submitted in partial fulfillment of the requirements for the degree of master of science in the faculty of graduate studies department of. The comet assay which can measure residual micro dna damage is appropriate for the study of dna repair. Dna damage was measured as the percentage of comet tail dna. The comet assay for dna damage and repair the comet assay for dna damage and repair collins, andrew 20040606 00. Trevigens comet analysis software can rapidly evaluate large numbers of cells, related.
Essentially, anything that can cause dna damage or denaturation except the factors being researched is to be avoided. 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. In the comet assay, when a damaged dnacontaining single cell suspension embedded in low melting agarose is. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.
The comet assay in toxicology 2nd edition pdf is written by diana anderson, diana anderson, alok dhawan, alok dhawan, andrew collins, michael plewa, tomas gichner, narendra singh, declan mckenna, ashutosh kumar, s piperakis, maria dusinska, emilio rojas, siegfried knasmueller, giada frenzilli, sheena lewis, andreas hartmann, mark browne, d lovell and you can download for free. The comet assay is a quantitative method for measuring dna damage primarily strand breaks. The last decade witnessed an increase in the use of comet assay for dna damage monitoring in cancer patients and controls. 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. In the comet assay, when a damaged dnacontaining single cell suspension embedded. May 23, 2019 comet assays are used to determine the extent of dna damage in a cell. These cryopreserved cell preparations contain different levels of dna damage to be used as controls when performing the comet assay. This unit describes alkaline comet assay procedures for assessing dna strand breaks and oxidative base alterations. Nested pcr for mtdna4977bp deletion and comet assay for. Dna damage risk assessment in comet assay by the use of buccal mucosa cells has great advantages in comparison. Dna repair can be monitored by incubating cells after treatment with damaging. The assay depends on the relaxation of supercoiled dna in agaroseembedded 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. We have used video image analysis to define appropriate features of the comet as a measure of dna damage, and have quantified damage and repair by ionizing radiation. Comet assay iv tm is the latest in a highly successful series of comet scoring systems originally developed by perceptive instruments, now a part of instem.
Assessment of dna damage by comet assay in buccal epithelial. In addition fpgmodified comet assay prove to be more sensitive for detection of gamma irradiation induced dna damage than the standard assay. The comet assay for dna damage and repair applications in. Detection of dna damage using comet assay image analysis. Detection of dna damage at the level of an individual eukaryotic cell warrants high significance in the fields of toxicology, pharmaceuticals, genotoxicity testing, environmental human biomonitoring, diagnosis of genetic disorders. Oct 15, 2015 the most widely used method for assessment of dna damage is the alkaline comet assay. To evaluate the dna damage in the buccal cells of patients with oral submucous fibrosis using comet assay. The comet assay is a singlecell gel electrophoresis method that can measure a variety of types of dna damage, and repair of damage, in individual cells. But as the comet assay can provide a measure of both dna content and dna damage, it is possible to analyze damage in any phase of the cell cycle. Validation of the 3d skin comet assay using full thickness. So the comet assay is a wellestablished, simple, versatile, visual, rapid, and sensitive method used extensively to assess dna damage quantitatively and qualitatively at single cell level. Evaluation of dna damage using singlecell gel electrophoresis comet assay.
The comet assay, or single cell gel electrophoresis scge, is well established as a direct measure of dna damage in genotoxicity studies. The comet single cell gel electrophoresis assay is a method for obtaining the amount of dna fragmentation of single cells. Due to their oxidative damage etiology, varicocele patients could be a group likely to be successfully treated with antioxidants. Heterogeneity in radiationinduced dna damage and repair. The comet assay methodology is able to detect both chromosomal damage. The comet assay single cell gel electrophoresis is the most common method for measuring dna damage in eukaryotic cells or disaggregated tissues. Interpretation of comet results is complicated by the fact that there is no simple relationship between the amount of dna damage caused by a specific chemical and the biological impact of that damage. We present a procedure for the comet assay, a gel electrophoresisbased method that can be used to measure dna damage in individual eukaryotic cells. Dna damage is commonly measured at the level of individual cells using. The comet assay can be used to detect dna damage caused by double strand breaks, single.
Usage of the standard and modified comet assay in assessment. Besides potential differences in baseline dna damage, smokers and nonsmokers were compared with regard to their mutagen sensitivity and dna repair capacity. Comet assay, the alkaline version in particular, has become a very popular method for the analysis of dna damage caused. Comet assay or single cell gel electrophoresis assay detects single or double strand breaks. The comet assay is a sensitive and efficient technique for analyzing dna damage in cells, caused by strand breaks, dna lesions, alkali labile sites and dna crosslinking with protein 5,6. The assay depends on the relaxation of supercoiled dna in agaroseembedded 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. Evaluation of storage conditions for assessing dna damage using the comet assay dante villavicencio submitted to the facultyof the universitygraduate schoo l in partial fulfillment of the requirements for the degree master of science in the department of pharmacologyand toxicology, indiana university october 2006. It is a valuable tool in population monitoring, for example in assessing the role of oxidative stress in human disease, and in monitoring the effects of dietary antioxidants.
If all factors except oxidative stress causing dna damage are kept constant, the amount of dna damage is a good indirect parameter of oxidative stress. For detailed explanation please see calculation of results section in product manual. Electrophoresis at high ph results in structures resembling comets, observed by. This is a sensitive and rapid method for detecting dna strand breakdown in individual cells, and.
Comet assays are used to determine the extent of dna damage in a cell. It is widely used to estimate dna damage caused by chemicals or radiation, for example, to test individual sensitivities to ionizing radiation in cancer patients. The comet assay or single cell gel electrophoresis scge assay is a rapid, sensitive and relatively simple method for detecting dna damage at the level of individual cells singh et al. Evaluation of dna damage in the buccal cells of patients. A sensitive method for detecting dna damage in individual cells. Alkaline and neutral comet assay profiles of sperm dna damage. Preservation of human blood cells for dna damage analysis with the comet assay conventionally involves the isolation of mononuclear cells by centrifugation, suspension in freezing medium and slow freezing to. The comet assay for dna damage appeals to researchers because of its simplicity. Epifluorescence microscopy visualization of dna damage using the oxiselect comet assay kit. Pdf comet assay for dna damage and repair researchgate. Comet assay interest group is a free forum for exchange of information on comet assay worldwidejoining this group you will have free access to the latest developments in this field as well as access to archived discussions at nih listserv. Unlike the other assays described above, the comet assay measures transient genetic damage that is not a fixed change to the dna.
The comet assay is a dna damage quantification method that is efficient and easy to perform, and has low timebudget demands and high reproducibility. The comet assay or singlecell gel electrophoresis 1 is widely used to assess dna damage. Single cell gel electrophoresis as a tool to assess genetic damage in. As the frequency of dna breaks increases, so does the fraction of the dna extending towards the anode, forming the comet tail. The mechanism of formation of comets under neutral or alkaline conditions is best. Alkaline comet assay for assessing dna damage in individual. Dna damage is commonly measured at the level of individual cells using the socalled comet assay singlecell gel electrophoresis. Ionizing radiation ir can result in serious genomic instability and genotoxicity by causing dna damage. Typically, neutral comet is used to detect dna doublestranded breaks while alkali comet detects a wider breadth of dna lesions including dna singlestranded breaks and abasic sites.
Comet assay as an indirect measure of systemic oxidative. Choose from the standard 3well comet assay kit or the higherthroughput 96well comet assay kit. The comet assay is a versatile and sensitive method for measuring single and doublestrand breaks in dna. Download the comet assay in toxicology 2nd edition pdf free. Detection of dna doublestrand breaks through the cell. Essentially, anything that can cause dna damage or denaturation. As such, this is one of the techniques used in the area of cancer research for the evaluation of genotoxicity and effectiveness of chemoprevention. Opencomet is a plugin for the image processing program imagej. May 18, 20 the comet assay single cell gel electrophoresis is the most common method for measuring dna damage in eukaryotic cells or disaggregated tissues. It has been widely used in genotoxicity testing and. Download the comet assay in toxicology 2nd edition pdf. The comet assay for dna damage and repair, molecular. Nowadays the comet assay is a widely accepted method for the evaluation of dna damage and its repair in eukaryotic cells, other than in humans, and can be done both in vitro and in vivo in yeast.
Alkaline and neutral comet assay profiles of sperm dna. Investigating oxidative dna damage and its repair using the. Dna damage is a precursor of cancer, but the link is a weak one, as almost all damage is repaired. Analysis of the shape and length of the comet tail, just like the dna content in the tail, gives an assessment of dna damage. Evaluation of dna damage in the buccal cells of patients with. Comet assay to detect doublestranded breaks in dna a comet assay is a technique used to determine the amount of doublestrand breaks in dna dna damage in cells. This sensitivity needs to be handled carefully as it is also vulnerable to physical changes which can affect the reproducibility of results. The comet assay is a wellpublished method for measuring cellular dna damage. Dna damage measured by the comet assay in eight agronomic. The software applies state of the image processing methods to automatically locate and score comets to characterize and quantify the degree of dna damage revealed by the comet assay. Jun 27, 2006 we present a procedure for the comet assay, a gel electrophoresisbased method that can be used to measure dna damage in individual eukaryotic cells. The study was undertaken to evaluate gamma radiationinduced dna damage in aedes aegypti. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and doublestrand dna breaks in vitro. Residual protein content measured after lysis of sphase cells embedded in agarose did not differ.
Electrophoresis at high ph results in structures resembling comets. The alkaline comet assay single cell gel electrophoresis is the most widely used method for measuring dna damage in eukaryotic cells neri. Select the appropriate set of control cells when performing either the alkaline or neutral comet assay. Follow the instructions to install and launch opencomet. Factors influencing the sensitivity of the alkaline comet. Opencomet can analyze a batch of comet assay images, identify the comets and measure their parameters. The assay was optimized for lysing solution, lysing time, electrophoresis time, and propidium iodide concentration using chinese hamster v79 cells. Comet assay measures dna breaks, induced by different factors. An electric current is applied to the gel that causes dna to move electrophoresis, and. It is compatible with 4x images which allows for rapid screening of up to 300 comets in seconds when analyzing cometchip. Comet assay iv is the professional choice for those seeking accuracy, reproducibility and glp compliance. The nucleus of an individual cell is placed on a microscope slide coated with an agarose gel.
A comet assay for dna damage and repair after exposure to. Our comet assays provide a fast, convenient way to screen for general dna damage, regardless of the source. Automated comet assay imaging and dualmask analysis to. Few genotoxic agents break dna directly, but many lesions can be converted to breaks, and dna repair pathways involve breaks as intermediates. The comet assay was employed to demonstrate the extent of dna damage produced in adult male a. The assay can be calibrated to give quantitative measures of dna damage. The theoretical value was then compared to responses to external xray or. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing. Pdf the comet assay, also called single cell gel electrophoresis scge, is a sensitive and rapid.
The comet assay for dna damage and repair springerlink. The comet assay, also known as single cell gel electrophoresis scre, helps scientists to determine whether there has been deoxyribonucleic acid dna damage to a. Factors influencing the sensitivity of the alkaline comet assay for detection of dna damage after low doses of radiation by christopher david fjell b. Jun 05, 2019 the comet assay, also known as single cell gel electrophoresis scre, helps scientists to determine whether there has been deoxyribonucleic acid dna damage to a single cell from apoptosis cell. The comet assay singlecell gel electrophoresis is a simple method for measuring deoxyribonucleic acid dna strand breaks in eukaryotic cells. Apart from casecontrol studies, reports described the determination of dna damage prior to baseline value and after chemoradiotherapy, the treatment resulted in significantly elevated dna damage. Comet assay and foci dna damage quantification metasystems.
These results reinforce the fact that there is a relation between oxidative dna fragmentation assessed by the alkaline comet assay and enzymatic dna damage assessed by the neutral comet assay. The neutral comet assay allows the detection of doublestrand breaks by subjecting lysed cell nuclei to an electrophoretic field at neutral ph sakkas et al. This information can provide a great deal of useful information regarding the diagnosis and progression of diseases, the. Comet assay software for measuring dna damage and repair. The comet assay is not the only way to measure oxidative dna damage, but it is one of the most sensitive and accurate, being relatively free of artefacts. Dna damage and repair measured by comet assay in cancer. Detection of deoxyribonucleic acid dna damage at the level of an individual eukaryotic cell warrants high significance in the fields of toxicology, pharmaceuticals, genotoxicity testing, environmental human biomonitoring, diagnosis of genetic disorders etc. The data obtained demonstrate the feasibility of using the comet assay for detecting induced dna damage in crops. Evaluation of gamma radiationinduced dna damage in aedes. Comet assay single cell gel electrophoresis sigmaaldrich. Ap biology 2017 frq 6 student samples college board. It is now in widespread use in genetic toxicology and oncology.
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