Comet assay
The comet assay (also called single cell gel electrophoresis ) is a gel electrophoresis technique that enables DNA damage to be determined in individual cells . The assay was developed in 1984 by Östling and Johanson for the detection of DNA double-strand breaks. With the further development by Singh in 1988, the use of basic buffers also enabled DNA single strand breaks to be detected.
The principle of the comet assay is based on the fact that cells embedded in agarose are lysed and exposed to an electric field, known as electrophoresis . During electrophoresis , the negatively charged DNA migrates to the positive pole and, thanks to the pores in the agarose, the fragments separate according to size, as the smaller fragments cover a longer distance than the larger ones in a certain time. However, chromosomal DNA is too large to travel as a whole in the electric field. Only damaged, fragmentary DNA is able to migrate out of the cell nucleus . Under the UV microscope, the damaged cells, which were previously stained with fluorescent dyes such as ethidium bromide , now appear with a tail made of DNA fragments, which gives them the appearance of a comet.
All cells that have a cell nucleus can be used in the comet assay.
Performing the Comet assay is relatively simple and quick. The number of damaged and undamaged cell nuclei is evaluated, either by hand or using special computer programs with visual detection (densitometric).