Analysis of DNA Damage and Repair by Comet Fluorescence In Situ Hybridization (Comet-FISH)

Abstract

A useful tool in the detection of overall and region-specific DNA damage is the Comet-FISH technique. This method combines two well-established methods, the Comet assay (single cell gel electrophoresis), which makes it possible to detect and quantify DNA damage at the single cell level, and FISH (fluorescence in situ hybridization), a technique that allows the specific detection of selected DNA sequences. The influence of specific substances such as water pollutants or food ingredients on individual cells can be measured with the alkaline version of the Comet assay, which involves the embedding of cells in agarose on microscopic slides, lysis of cells, and separation of DNA via electrophoresis. In damaged cells a "comet tail" is formed by fractured DNA migrating from the nucleus (head of the comet) in the electric field.The damaged DNA (DNA strand breaks) correlates with the percentage of DNA in the tail. In combination with the FISH method, DNA damage or repair capacity in single cells can be measured using labelled probes, which hybridize to specific DNA sequences of interest. This protocol exemplarily provides a description of the Comet-FISH technique for the detection of DNA damage using hydrogen peroxide as a genotoxic model substance.