Modelling the comet assay

The single-cell gel electrophoresis technique or comet assay is widely regarded as a quick and reliable method of analysing DNA damage in individual cells. It has a proven track record from the fields of biomonitoring to nutritional studies. The assay operates by subjecting cells that are fixed in agarose to high salt and detergent lysis, thus removing all the cellular content except the DNA. By relaxing the DNA in an alkaline buffer, strands containing breaks are released from supercoiling. Upon electrophoresis, these strands are pulled out into the agarose, forming a tail which, when stained with a fluorescent dye, can be analysed by fluorescence microscopy. The intensity of this tail reflects the amount of DNA damage sustained. Despite being such an established and widely used assay, there are still many aspects of the comet assay which are not fully understood. The present review looks at how the comet assay is being used, and highlights some of its limitations. The protocol itself varies among laboratories, so results from similar studies may vary. Given such discrepancies, it would be attractive to break the assay into components to generate a mathematical model to investigate specific parameters. The technique The single-cell gel electrophoresis assay has been used for over 20 years to assess DNA damage. The assay is able to measure both singleand double-strand breaks with versatility and sensitivity [1]. It has been used successfully over the years in key areas from human nutrition to biomonitoring [2,3]. In a typical assay, cells are fixed in agarose on a frosted slide and lysed to remove cellular proteins, leaving a cavity containing DNA with sufficient salt (2.5 M NaCl) and detergent to disrupt and remove membranes, histones and soluble cell constituents [3]. The DNA is then left to unwind in a buffer solution before being electrophoresed to pull the negatively charged, extended and relaxed loops containing strand breaks out of the cavity, forming the tail [4]. Intact DNA remains in the head and supercoiled. Comets are visualized using an intercalating dye such as ethidium bromide or DAPI (4′,6-diamidino2-phenylindole), which binds in the minor groove, and is normally analysed using comet-specific software [1]. The comet technique has been adapted over the years into different protocols such as the alkaline and neutral versions which were thought to discriminate between doubleand single-strand breaks. This theory was revealed by Collins et al. [1] at the most recent International Comet Assay conference to be a misconception as alkaline conditions are not specific to detecting single-strand breaks. FISH (fluorescent in situ hybridization) techniques were also adapted for application to the comet where the DNA probe can be hybridized to a specific gene sequence or even to a whole chromosome [5,6]. Comet-FISH has been used to