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. Comet assays are used to determine the extent of dna damage in a cell. Detection of dna doublestrand breaks through the cell. Our comet assays provide a fast, convenient way to screen for general dna damage, regardless of the source. The resulting baseless sugars are alkalilabile, and under the conditions of the. 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. 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. The comet assay, or single cell gel electrophoresis assay scge, is a common technique for measurement of dna damage in individual cells. Pdf the comet assay, also called single cell gel electrophoresis scge, is a sensitive and rapid technique for quantifying and analyzing. Comet assay different types of dna damage in rbcs of c. The levels of dna damage measured were expressed as % tail dna and olive tail moment otm. The last decade witnessed an increase in the use of comet assay for dna damage monitoring in cancer patients and controls. Hedgehog comets can be produced by treating cells with 0. Dna repair measured by the comet assay 617 and alkaline comet assays do, however, differ in one important respect.
Dna repair can be monitored by incubating cells after treatment with damaging. These two papers studied the kinetics of repair by performing the comet assay on dna damaged cells at different times after incubation, in what has been called the cellular repair assay or the challenge assay au et al. Choose from the standard 3well comet assay kit or the higherthroughput 96well comet assay kit. Measurement of dna damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Comet assay iv is the professional choice for those seeking accuracy, reproducibility and glp compliance. Dna damage is a precursor of cancer, but the link is a weak one, as almost all damage is repaired. The standard comet assay is used to monitor the capacity of cells to rejoin breaks.
Students were asked to identify one property of dna and to provide reasoning to support how this property. Investigating oxidative dna damage and its repair using the. The comet assay was first developed by ostling and johanson in 1984 by. 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 in arabidopsis thaliana as measured by. Dna damage in mouse organs and in human sperm cells by. After the exposure period was complete the possible dna damage on the rats brain, liver, kidney, skin, and testicular tissues was detected through the single cell gel electrophoresis assay comet method. Parameters of the standard comet assay showed significantly higher values in samples exposed to 4 gy than in samples exposed to radiation dose of 0. 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. Pdf variation in the measurement of dna damage by comet. The focus of this metaanalysis is on studies that investigate potential effects of cigarette smoking on dna using the comet assay. Pdf dna damage and repair detected by the comet assay in. Few genotoxic agents break dna directly, but many lesions can be converted to breaks, and dna repair pathways involve breaks as intermediates.
This can be done in a few minutes either by yourself or your it department. Evaluating in vitro dna damage using comet assay ncbi nih. All the comet assay results files produced by the comet assay iv system can be used with the excelbased spreadsheet generator, which is supplied free with every comet assay iv system. Select the appropriate set of control cells when performing either the alkaline or neutral comet assay. The comet assay for dna damage and repair applications in. The comet assay can be used to detect dna damage caused by double strand breaks, single strand breaks, alkali labile sites, oxidative base damage, and dna crosslinking with dna or protein. As such, this is one of the techniques used in the area of cancer research for the evaluation of genotoxicity and effectiveness of chemoprevention. Dna damage induced by high fluoride and low iodine in rat brains. Residual protein content measured after lysis of s.
Comet assay as an indirect measure of systemic oxidative. Additionally, severe oxidative injury resulted from msg exposure in two doses that could lead to dna damage, the comet assay was used to evaluate dna damage and strand breaks 24, this is greatly. 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 for dna damage appeals to researchers because of its. The comet assay, also known as singlecell gel electrophoresis scge, has become an attractive assay in the assessment of dna damage. The comet assay is a versatile technique for detecting damage and with adjustments to the protocol can be used to quantify the presence of a wide variety of dna altering lesions damage. Comet assay kit 3well slides ab238544 is a fast and sensitive kit for the measurement of cellular dna damage. Requirement for human mps1ttk in oxidative dna damage repair and cell survival through mdm2 phosphorylation. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress. Under alkaline conditions ph, the comet assay can detect single and double stranded breaks, resulting, for example, from direct interactions with dna, alkali labile sites or as a consequence of transient dna strand breaks resulting from dna excision repair. This is a sensitive and rapid method for detecting dna strand breakdown in individual cells, and. 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. The comet assay is a wellpublished method for measuring cellular dna damage.
The in vivo comet assay has recently been implemented into regulatory genotoxicity testing of pharmaceuticals with inclusion into the ich s2r1 guidance. 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. As the frequency of dna breaks increases, so does the fraction of the dna extending towards the anode, forming the comet tail. Gebhart1 1institute of human genetics, university of erlangennuremberg, schwabachanlage 10, d91054 erlangen. While the comet assay has widespread use, a common issue in all its applications is the process of analyzing the microscope images. Cometassay in combination with pnafish detects mutagen. It is a convenient way to screen for general dna damage, regardless of the source or nature of the damage. Opencomet can analyze a batch of comet assay images, identify the comets and measure their parameters. Comet assay results, measurements and data processing.
Comet assay in combination with pnafish detects mutageninduced dna damage and specific repeat sequences in the damaged dna of transformed cells g. The comet assay singlecell gel electrophoresis is a simple method for measuring deoxyribonucleic acid dna strand breaks in eukaryotic cells. Under an electrophoretic field, damaged cellular dna containing fragments and strand breaks is separated from intact dna, yielding a classic comet. Unlike the other assays described above, the comet assay measures transient genetic damage that is not a fixed change to the dna. Combining a novel comet assay procedure with automated. In repair experiments residual dna damage could be observed after postirradiation incubation for 60 minutes. The comet assay for dna damage and repair springerlink. O 2uv dna damage normal cell dna repair damaged ageing cell ber. This question focused on the analysis of dna using a comet assay. These cryopreserved cell preparations contain different levels of dna damage to be used as controls when performing the comet assay. Minimum criteria for the acceptance of in vivo alkaline comet. Detection of alkylation damage in human lymphocyte dna. Variation in the measurement of dna damage by comet assay.
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. Opencomet is a plugin for the image processing program imagej. Its simplicity, sensitivity, speed and economy make it a prime choice in genotoxicity testing, ecogenotoxicity, human epidemiology studies as well as mechanistic studies of dna damage and repair. The amount of dna damage was measured as% tail dna value.
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. The alkaline comet assay single cell gel electrophoresis is the most widely used method for measuring dna damage in eukaryotic cells neri. Inhibition of h2o2induced dna damage in single cell gel. The recommendations from the international workshops held for the comet assay have resulted in establishment of guidelines, and the oecd has adopted a guideline for the in vivo comet assay as a test for assessing dna damage in animals. Cometassay electrophoresis starter kits archives trevigen. Students were presented with a diagram demonstrating the results of a comet assay in a cell with dna damage and a written description of the assay. Getting started with comet assay iv is as simple as installing the software and connecting the camera. Cells should be lysed at 04 c to minimize dna damage by endonucle ases. Extent of dna damage is usually visually estimated by comet tail measurement. The comet assay is well established for quantifying dna damage in mammalian cells and is compatible with detection of a wide array of dna damaging agents. A comet assay for dna damage and repair after exposure to.
Comet assay single cell gel electrophoresis sigmaaldrich. Comet assay or single cell gel electrophoresis assay detects single or double strand breaks measured at the individual cell level. Chapter 1 the comet assay in toxicology rsc publishing. The comet assay is a relatively simple, sensitive, and quantitative method to study dna damage including oxidative damage and repair at the level of the individual cell. 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. The single cell gel electrophoresis or comet assay is widely used to detect dna damage following exposure to genotoxic compounds with cells isolated from tissue or circulatory fluids from various organisms. Essentially, anything that can cause dna damage or denaturation except the factors being researched is to be avoided. The comet assay is used to study processes involving dna damage in many fields such as environmental toxicology, biomonitoring, radiation biology, nutritional studies and cancer studies.
Nov 30, 2012 the in vivo alkaline comet assay detects primary dna damage in various organs and tissues of exposed animals and can be used to assess the genotoxicity of a great variety of chemical compounds, which include food additives, flavourings, food contact materials, foodborne by. In vivo alkaline comet assay and enzymemodified alkaline. Investigating oxidative dna damage and its repair using. Therefore, to further evaluate evidence for smokingrelated dna effects in the comet assay, we now used a metaanalysis approach based on a literature search. The more breaks are present, the more loops are relaxed, and the more intense is the fluorescence of the comet tail relative to the nucleoid core when the nucleoids are stained. If the treated cells are incubated for half an hour, hedgehog comets are no longer seen the dna damage has been repaired by the cell. The oxiselect comet assay is a fast and sensitive kit for the measurement of cellular dna damage. Fluoride not only penetrates the bloodbrain barrier, but it can also induce dna damage in brain cells. Each kit provides sufficient reagents to perform up to 75 assays. We present a procedure for the comet assay, a gel electrophoresisbased method that can be used to measure dna damage in individual eukaryotic cells. Alok dhawan, mahima bajpayee and devendra parmar new chemicals are being added each year to the existing burden of toxic substances in the environment.
Oxiselect 96well comet assay kit cell biolabs, inc. Assessing dna damage in hemocytes of the freshwater mussel elliptio complanata with the comet assay. 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. Ionizing radiation ir can result in serious genomic instability and genotoxicity by causing dna damage. This sensitivity needs to be handled carefully as it is also vulnerable to physical changes which can affect the reproducibility of results.
The nucleus of an individual cell is placed on a microscope slide coated with an agarose gel. Usage of the standard and modified comet assay in assessment. The sensitivity of the alkaline comet assay in detecting dna lesions induced by xrays, gamma rays and alpha particles. The damage usually detected are single strand breaks and double strand breaks.
Dna damage and repair in arabidopsis thaliana as measured. The purpose of the comet assay is to identify substances that cause dna damage. Dna damage and repair measured by comet assay in cancer. Dna damage in the liver and kidney cells of adult male wistar rats was studied using the comet assay after a 28day oral administration of tembotrione at doses of 0. The spreadsheet generator is provided to help generate graphs and tables in excel from the raw data produced by the comet assay iv program.
Experiences with the in vivo and in vitro comet assay in. 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. 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. The oxiselect 96well comet assay is a fast and sensitive kit for the measurement of cellular dna. 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. Heparinized blood samples were obtained by venipuncture from a nonsmoking, healthy male donor aged 3040 years. Dna damage is commonly measured at the level of individual cells using the socalled comet. Assay principle the cell biolabs oxiselect comet assay is a single cell gel electrophoresis assay scge for simple evaluation of cellular dna damage. 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. Assessing dna damage in hemocytes of the freshwater mussel. Ap biology 2017 frq 6 student samples college board. Dna damage is commonly measured at the level of individual cells using the socalled comet assay singlecell gel electrophoresis. Comet assay dna damage was determined by alkaline single cell microgel electrophoresis comet assay assay following the method proposed by 18 with minor modifications as suggested by 19.
This information can provide a great deal of useful information regarding the diagnosis and progression of diseases, the. Dna damage, due to environmental factors and normal metabolic processes inside the cell, occurs at a rate of 1,000 to 1,000,000 molecular lesions per cell per. The aim of this study was to investigate the induction of dna damage by bpa using the comet assay in male mice tissues after oral gavage exposure according to. Evaluating in vitro dna damage using comet assay protocol. 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. Pdf comet assay for dna damage and repair researchgate. Mutation research 493 2001 8793 dna damage and repair in arabidopsis thaliana as measured by the comet assay after treatment with different classes of genotoxins merten menkea, ipeng chena, karel j.
Dna damage and repair detected by the comet assay in lymphocytes of african petrol attendants. 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. The in vitro comet assay conducted in cultured cells can be used for screening large number of compounds and. During the past two decades the comet based in vitro dna repair assay has been used regularly to measure base excision repair ber related. Limitations of the comet assay, and misunderstandings it is wise to point out the limitations of the comet assay at this stage, if only to emphasise that many of these limitations are perceived but not real.
Dna damage in the brain cells assessed by the tailing ratio percent was 24. 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. Under an electrophoretic field, damaged cellular dna containing fragments and strand breaks is separated from intact dna, yielding a classic comet tail shape under the microscope. This unit describes alkaline comet assay procedures for assessing dna strand breaks and oxidative base alterations. The comet assay is an extremely sensitive dna damage assay. A sensitive method for detecting dna damage in individual cells. Abstract the comet assay single cell gel electropho resis is the most common. Follow the instructions to install and launch opencomet. Comet assay measures dna breaks, induced by different factors. 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. This has led to increased pollution of ecosystems as well as deterioration of the air, water and soil quality. 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. The comet assay is a single cell gel electrophoresis assay scge for simple evaluation of cellular dna damage.
The most widely used method for assessment of dna damage is the alkaline comet assay. The comet assay is a quantitative method for measuring dna damage primarily strand breaks. The comet assay is also used to monitor dna repair by living cells. Differences in quantification of dna doublestrand breaks assessed by 53bp1 h2ax focus formation assays and the comet assay in mammalian cells treated with irradiation and nacetyllcysteine. The fpgmodified comet assay showed significantly higher values already at dose of 0. Comet assay software for measuring dna damage and repair. Regulatory genotoxicity testing aims to detect dna alterations in form of gene mutations, larger scale chromosomal damage and recombination and aneuploidy. Based on the dna damage results determined by the single cell gel electrophoresis comet method, it was found that the% tail dna values of the brain, kidney, liver, and skin tissues of the rats in the experimental group increased more than those in the control group. Pathophysiology 16 2009 7988 such as albumin and sucrose, or spintrap molecules such as phenyltertbutyl nitrone pbn, should be added during dispersion of tissues into single cells.
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