Theory behind comet assay

The Comet Assay is based on the ability of negatively charged loops/fragments of DNA to be drawn through an agarose gel in response to an electric field. The extent of DNA migration depends directly on the DNA damage present in the cells. It should be noted that DNA lesions consisting of strand breaks after treatment with alkali either alone or in combination with certain enzymes (e.g. endonucleases) increases DNA migration, whereas DNA-DNA and DNA-protein cross-links result in retarded DNA migration compared to those in concurrent controls (Tice et al., 2000). In this assay, a suspension of cells is mixed with low melting point agarose and spread onto a microscope glass slide. Following lysis of cells with detergent at high salt concentration, DNA unwinding and electrophoresis is carried out at a specific pH. Unwinding of the DNA and electrophoresis at neutral pH (7-8) predominantly facilitates the detection of double strand breaks and cross links; unwinding and electrophoresis at pH 12.1-12.4 facilitates the detection of single and double strand breaks, incomplete excision repair sites and cross links; while unwinding and electrophoresis at a pH greater than 12.6 expresses alkali labile sites (ALS) in addition to all types of lesions listed above (Miyamae et al., 1997). When subjected to an electric field, the DNA migrates out of the cell, in the direction of the anode, appearing like a 'comet'. The size and shape of the comet and the distribution of DNA within the comet correlate with the extent of DNA damage (Fairbairn et al., 1995). Principles of image analysis are described by B Vilhar.
From-comet assay interest group