DNA breakage detection-fish (DBD-FISH): effect of unwinding time

DBD-FISH is a new procedure that allows detection and quantification of DNA breakage in situ within specific DNA target sites. Cells embedded in an ag arose matrix on a slide are treated in an alkaline unwinding solution to tr ansform DNA breaks into single-stranded DNA (ssDNA). After removal of prote ins, DNA probes are hybridized and detected. DNA breaks increase the ssDNA and relax supercoiling of DNA loops, so more probe hybridizes, thereby incr easing the surface area and fluorescence intensity of the FISH signal. The probe selects the chromatin area to be analysed. In order to restrict the e xtension of unwound ssDNA to a region closer to the origin of the DNA break , human leukocytes were processed for DBD-FISH with a whole genome probe, a fter a 10 Gy dose of X-rays, for various unwinding times: 5, 2 min and 30 s . Two cell populations were detected after 30 s, but not with the 5 or 2 mi n unwinding times. One cell group had small to medium haloes corresponding to the relaxation of DNA supercoiling after DAPI staining, and strong DBD-F ISH labelling of induced DNA breaks, whereas the other cell group showed bi g haloes of DNA loop unfolding and an absence of DBD-FISH labelling. The la tter group was similar to cells processed by DBD-FISH without the unwinding step. Thus, they should correspond to cells unaffected by the alkaline unw inding solution, possibly because very brief unwinding times do not allow t he diffusion of the alkali into the cells deep within the gel, thus biasing the results. Taking this into account, 2 min seems to be the minimum unwin ding time required for an accurate detection of a signal by DBD-FISH. (C) 2 000 Elsevier Science B.V. All rights reserved.