The comet assay: mechanisms and technical considerations.

The comet assay is frequently used to measure DNA damage in individual cells. In order to better understand the mechanisms behind the technique, we have studied the behaviour of DNA under different electrophoresis conditions in mammalian cells exposed to gamma radiation. The comet tails obtained after neutral electrophoresis seem to consist of DNA loops which are attached to structures in the nucleus, since the DNA cannot move in the second direction after two-dimensional electrophoresis. When the DNA is labelled by a short pulse, microautoradiography reveals that all label appear in the head of the comets when neutral electrophoresis is applied. After chase incubation, the label moves out into the tails. This gives further support to the view that the DNA loops are fixed to some structure in the nucleus where also the DNA synthesis takes place. Under alkaline electrophoresis conditions, however, the entire comet tails move in the new electrophoresis direction. Thus, it appears that the alkaline comet tails consist of free DNA fragments. Further, the effects of alkaline concentration and sodium chloride during unwinding and electrophoresis are discussed. Throughout the study, a protocol for drying and fixation of the comets has been used.