Protocol

Comet Fluorescence In Situ Hybridization (Comet-FISH): Detection of DNA Damage

¹ Department of Nutritional Toxicology, Institute for Nutrition, Friedrich-Schiller-University Jena, 07743 Jena, Germany

²Corresponding author (michael.glei{at}uni-jena.de)

INTRODUCTION

The Comet-FISH technique is a useful tool for detection of overall and region-specific DNA damage and repair in individual cells. It combines two well-established methods, the Comet assay (single-cell gel electrophoresis) and fluorescence in situ hybridization (FISH). The Comet assay serves as the basis of Comet-FISH and is a relatively simple and fast method that allows separation of fragmented from nonfragmented DNA and quantification of DNA damage and repair. FISH enables detection of specifically labeled DNA sequences of interest, including whole chromosomes. The combined technique of Comet-FISH is a modification of the Comet assay that inserts a hybridization step after unwinding and electrophoresis and permits the labeling of specific gene sequences or telomeres. Comet-FISH has been applied for detection of site-specific breaks in DNA regions that are relevant for development of various diseases, and has also been used to study the distribution of DNA damage and repair in the complete genome. Moreover, DNA sequence modifications can be detected in individual cells using Comet-FISH. Whereas results from the Comet assay alone reflect only the level of overall DNA damage, the addition of the FISH technique allows the assignment of the probed sequences to the damaged or undamaged part of the comet (tail or head, respectively).