An improved method of alkaline sucrose density gradient sedimentation to detect less than one lesion per 1 Mb DNA

We improved alkaline sucrose density gradient sedimentation to detect very long single-strand DNA at the megabase level (from less than 1 to about 4 Mb). Hitherto, these have not sedimented correctly due to some artifacts. One artifact was aggregation of sticky DNA and proteins formed in the gradient. Then, in some gradients, biphasic distribution was observed, the major peak of which was reasonable as a result of random scission by X-rays, but the minor, fast-sedimenting population was another artifact resulting from incomplete denaturation of the DNA. We mainly reduced the centrifugal force and used a solution for cell lysis with a high concentration of salt. By means of this procedure, DNA single-strand breaks induced by relatively low doses of X-rays and subsequent repair processes can be measured in human fibroblasts. The protocol is also applicable to the study of DNA damage accompanied by strand scission, such as by UV or dimethyl sulfate as well as their repair. The technique is sensitive enough to detect even single-strand breaks induced by 0.1 J/m2 UV and sufficiently reproducible that breaks induced by increasing UV dosages were dose dependent. Thus, this technique was proven to be very sensitive, reliable and simple to perform. Therefore, this improvement will be extremely useful to investigators studying DNA repair.