Detection of Specific DNA Lesions by a Combination of Comet Assay and FISH in Plants

Menke M., Angelis K.J., Schubert I.

Keywords: comet assay, Vicia faba, endonucleases, single-/double-strand breaks, fluorescence in situ hybridization, chromosomal domains
Abstract: We have studied comet formation on Vicia faba nuclei embedded in agarose and treated with the endonucleases DNase I (to produce SSBs or DSBs at random sites), FokI (to produce DSBs preferentially within FokI repeats), or EcoRI (to produce DSBs at random sites but not within FokI elements). DNase I–induced SSBs were detected when enzyme treatment was followed by alkaline denaturation. DSBs efficiently mediated comet formation using neutral conditions. FISH with DNA probes, detecting specific chromosomal domains such as FokI element-containing heterochromatin, NORs, or telomeres, was done on comets. The distribution of FISH signals between the head and tail of comets indicated to which degree these domains were damaged and reflected the distribution of cleavage sites for the applied restriction endonucleases within these domains. The data confirmed the expectation that the observed comet formation was based on enzyme-specific DNA breakage.
IEB authors: Karel J. Angelis