E comet assay
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This document discusses the comet assay, a gel electrophoresis technique used to detect DNA damage at the single-cell level. It was developed in 1984 as a way to measure single and double-strand DNA breaks, base damage, and apoptotic nuclei. The comet assay involves embedding cells in agarose on a microscope slide, lysing them, and running an electric current to pull negatively charged DNA fragments toward the anode. Broken DNA migrates from the nucleus, resembling a comet with a bright head and tail, allowing measurement of DNA damage. The assay is widely used to evaluate genotoxicity of chemicals, pharmaceuticals, and environmental contaminants.
E comet assay
- 1. DR. P. SUGANYA ASSISTANT PROFESSOR SRI KALISWARI COLLEGE (AUTONOMOUS) SIVAKASI E- COMETE ASSAY
- 2. INTRODUCTION (SINGLE GEL ELECTROPHORESIS) Assay was developed – ostling & Johansson in 1984 Later revised by Singh et al in 1988 This assay based on the gel electrophoresis That can be measure DNA damage in individual eukaryotic cells It’s versatile, relatively simple to perform and sensitive.
- 3. Make to use –its ability to measure –single strand breaks, detection of DNA double strand breaks links, base damage & apoptic nuclei The limit of sensitivity is approximately – strand as per diploid mammalian cell. DNA damage and its repair in single cell suspensions prepared from yeast, protozoa, plants, invertebrates and animals also be studied using this assay.
- 4. Originally developed to measure – variation in DNA damage and repair capacity within a population of mammalian cells 1. Applications of the comet assay 2. Popular assay to DNA damage
- 5. REVIEW OF THIS TECHNIQUE E- comet assay is gel electrophoresis method To visualize & measure DNA strands breaks in individual cell microscopy. Cells are embedded in agarose on a microscope – immersed in a lysis solution & then exposed to an electric field to attrate- ve charged fragments of a towards the anode. After the electrophoresis- DNA – stained- using fluorescence dyes – viewed using a fluorescence microscope
- 6. Broken DNA migrates- nucleus- in the electric field- & then preparation – then resembles a “comet” – brightly- fluorescent head (the nucleus) & a “tail” (fragmented DNA) The relative size of the tail is an index of DNA damage The comet assay – measure – single, double strands breaks- damage to DNA bases, DNA inter strand cross- link & apoptotic fragments.
- 7. Comet assay can be used to evaluate- genotoxicity- many test chemicals, pharmaceuticals & nanoparticles, consumer products, medical devices, environmental alaminantets, complex mixture, impurities. ADME (Absorption, Distribution, Metabolism & Excretion) Comet – used to detect – DNA – DNA cross- linkage- useful – early predicting – mechanism of
- 8. aneuploidy induction – even a cytostatic to cytotoxic doses