The alkaline single-cell gel electrophoresis/comet assay: a way to study DNA repair in radicle cells of germinating Vicia faba.

ABSTRACT

Dry seeds are known to accumulate DNA damage with time of storage. Repair of DNA lesions during germination of Vicia faba seeds was followed in the radicles using the alkaline single-cell gel electrophoresis/comet assay. In this assay nuclei were liberated, mixed with agarose and spread out over a microscope slide. After lysis of the nuclear membrane and unwinding of the DNA duplex, DNA was stretched during electrophoresis, giving a comet-like migration pattern. The more DNA was damaged, the higher its mobility. DNA repair took place rapidly the first hours of imbibition and more slowly until ca 33 h after onset of germination. A small amount of heavily damaged cells remained present. Labelling with BrdU provided the possibility to localize repair patches and replicated sites in the comet migration pattern. At 15 h of germination, incorporation of BrdU in radicle DNA was situated at random over the entire comet. At 33 h, DNA repair was more or less accomplished and BrdU was mainly localized in the 'heads' of most comets.