Comet assay videos 3

Comet assay videos 2

Comet assay videos

Comet assay overview

Introduction to the Comet Assay

The comet assay is a technique to measure DNA damage and other DNA alterations in individual cells. The technique aquired its name from the comet-like shape of the DNA of the cells which can be seen under the microscope after the procedure. The technique is also named single cell gel electrophoresis assay, which is more descriptive. The main steps of the procedure are preparation of a cell suspension with the cells of interest, embedding cells in an agarose gel and spreading them on an object slide, lysis of cells to remove non-DNA compounds, exposing the DNA to a neutral or alkaline buffer, subjecting the gels with the DNA to electrophoresis (which forms the "comets"), and measuring the extent of migration and stretching of DNA by microscopical analysis. As many cells are measured (e.g. 100 cells per object slide), the statistical distribution of the individual cell measurements represents the amount of DNA damage/alteration of the cell sample.
From-http://www.comet-assay.de/cometintro.php

References for comet asaay

Collins AR. Comet Assay for DNA damage and repair: principles, applications and limitations. Mol. Biotechnol. 2004; 26: 249-61.

Dixon DR, Pruski AM, Dixon LRJ, Jha AN. Marine invertebrate eco-genotoxicology: a methodological overview. Mutagenesis. 2002; 17: 495-507.

Fairbairn DW, Olive PL, O’Neill KL. The Comet Assay: A comprehensive review. Mutat. Res. 1995; 339: 37-59.

Fisher AE, Burke D, Routledge MN. Can irradiation of rectal tumour cells from patient biopsy predict outcome of radiotherapy? Proceedings of the Genome Stability network/United Kingdom Environmental Mutagen Society Joint Congress, University of Cardiff, 1 – 4 July 2007.

Gichner T, Mukherjee A, Veleminsky J. DNA staining with the fluorochromes EtBr, DAPI and YOYO-1 in the comet assay with tobacco plants after treatment with ethyl methanesulphonate, hyperthermia and DNase-I. Mutat Res. 2006; 605: 17-21.

Gichner T, Patkova Z, Szakova J, Demnerova K. Cadmium induces DNA damage in tobacco roots, but no DNA damage, somatic mutations or homologous recombination in tobacco leaves. Mutat Res. 2004; 559: 49-57.

Collins AR, Dusinská M, Horská A. Detection of alkylation damage in human lymphocyte DNA with the comet assay. Acta Biochim. Pol. 2001; 48: 611–14

Bichler J, Cavin C, Simic T, Chakraborty A, Ferk F, Hoelzl C, Schulte-Hermann R, Kundi M, Haidinger G, Angelis K, Knasmüller S. Coffee consumption protects human lymphocytes against oxidative and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trp-P-2) induced DNA-damage: Results of an experimental study with human volunteers. Food Chem. Toxicol. 2007 Epub ahead of print.

Burlinson B, Tice RR, Speit G, Agurell E, Brendler-Schwaab SY, Collins AR, Escobar P, Honma M, Kumaravel TS, Nakajima M, Sasaki YF, Thybaud V, Uno Y, Vasquez M, Hartmann A. In Vivo Comet Assay Workgroup, part of the Fourth International Workgroup on Genotoxicity Testing: results of the in vivo Comet Assay workgroup. Mutat. Res. 2007; 627: 31-5.

Collins AR, Duthie, SJ, Dobson VL. Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis. 1993; 14: 1733–735.Collins A, Dusinska M, Franklin M, Somorovska M, Petrovska H, Duthie S, Fillion L, Panayiotidis M, Raslova K, Vaughan N. Comet Assay in human biomonitoring studies: reliability, validation, and applications. Environ. Mol. Mutagen. 1997; 30: 139–46.

Bichler J, Cavin C, Simic T, Chakraborty A, Ferk F, Hoelzl C, Schulte-Hermann R, Kundi M, Haidinger G, Angelis K, Knasmüller S. Coffee consumption protects human lymphocytes against oxidative and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trp-P-2) induced DNA-damage: Results of an experimental study with human volunteers. Food Chem. Toxicol. 2007 Epub ahead of print.

Burlinson B, Tice RR, Speit G, Agurell E, Brendler-Schwaab SY, Collins AR, Escobar P, Honma M, Kumaravel TS, Nakajima M, Sasaki YF, Thybaud V, Uno Y, Vasquez M, Hartmann A. In Vivo Comet Assay Workgroup, part of the Fourth International Workgroup on Genotoxicity Testing: results of the in vivo Comet Assay workgroup. Mutat. Res. 2007; 627: 31-5.

Collins AR, Duthie, SJ, Dobson VL. Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis. 1993; 14: 1733–735.Collins A, Dusinska M, Franklin M, Somorovska M, Petrovska H, Duthie S, Fillion L, Panayiotidis M, Raslova K, Vaughan N. Comet Assay in human biomonitoring studies: reliability, validation, and applications. Environ. Mol. Mutagen. 1997; 30: 139–46.

Theory behind comet assay

The Comet Assay is based on the ability of negatively charged loops/fragments of DNA to be drawn through an agarose gel in response to an electric field. The extent of DNA migration depends directly on the DNA damage present in the cells. It should be noted that DNA lesions consisting of strand breaks after treatment with alkali either alone or in combination with certain enzymes (e.g. endonucleases) increases DNA migration, whereas DNA-DNA and DNA-protein cross-links result in retarded DNA migration compared to those in concurrent controls (Tice et al., 2000). In this assay, a suspension of cells is mixed with low melting point agarose and spread onto a microscope glass slide. Following lysis of cells with detergent at high salt concentration, DNA unwinding and electrophoresis is carried out at a specific pH. Unwinding of the DNA and electrophoresis at neutral pH (7-8) predominantly facilitates the detection of double strand breaks and cross links; unwinding and electrophoresis at pH 12.1-12.4 facilitates the detection of single and double strand breaks, incomplete excision repair sites and cross links; while unwinding and electrophoresis at a pH greater than 12.6 expresses alkali labile sites (ALS) in addition to all types of lesions listed above (Miyamae et al., 1997). When subjected to an electric field, the DNA migrates out of the cell, in the direction of the anode, appearing like a 'comet'. The size and shape of the comet and the distribution of DNA within the comet correlate with the extent of DNA damage (Fairbairn et al., 1995). Principles of image analysis are described by B Vilhar.
From-comet assay interest group

History of comet assay

This was first introduced by Ostling and Johanson in 1984. This was a neutral assay in which the lysis and electrophoresis were done under neutral conditions. Staining was done with acridine orange. The image obtained looked like a “comet” with a distinct head, comprising of intact DNA and a tail, consisting of damaged or broken pieces of DNA hence the name “Comet” Assay. The approach of Ostling and Johanson was based on previous work published by P Cook et al., 1976, who developed a method for investigating nuclear structure based on the high salt lysis of cells in the presence of non-ionic detergents.
The more versatile alkaline method of the comet assay was developed by N.P. Singh and co workers in 1988. This method was developed to measure low levels of strand breaks with high sensitivity. Several reviews have been published in recent years to highlight the procedures, advantages and limitations of this assay in genotoxicological, ecotoxicological and biomonitoring studies (Collins, 2004; Dixon et al., 2002; Fairbairn et al. 1995; Lee and Steinert, 2003). The assay has also been successfully implemented in plant cells under laboratory conditions (Gichner et al., 2004, Gichner et al., 2006).
From-comet assay interest group.

Introduction

The Single Cell Gel Electrophoresis assay (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. It was first described by Singh et al. in 1988. It has since gained in popularity as a standard technique for evaluation of DNA damage/repair, biomonitoring and genotoxicity testing. It involves the encapsulation of cells in a low-melting-point agarose suspension, lysis of the cells in neutral or alkaline (pH>13) conditions, and electrophoresis of the suspended lysed cells. 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 an imaging software.
From Wikipedia.