In this study, we have optimised the comet-FISH assay to detect and compare DNA damage and repair in the p53 and hTERT gene regions of bladder cancer cell-lines RT4 and RT112, normal fibroblasts and Cockayne Syndrome (CS) fibroblasts following γ-radiation. The number and position of the fluorescent signal(s) provides information about the relative damage and subsequent repair that is occurring in the targeted gene domain(s). The alkaline single cell gel electrophoresis (comet) assay can be combined with fluorescent in situ hybridisation (FISH) methodology in order to investigate the localisation of specific gene domains within an individual cell. 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. 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. The damaged DNA (DNA strand breaks) correlates with the percentage of DNA in the tail. In damaged cells a “comet tail” is formed by fractured DNA migrating from the nucleus (head of the comet) in the electric field. 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. 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. A useful tool in the detection of overall and region-specific DNA damage is the Comet-FISH technique.

Comet fish