Detection of abasic sites an individual DNA molecules using atomic force microscopy

We have developed an atomic force microscopy-based method for detecting aba sic sites (AP sites) on individual DNA molecules. By using uracil and uraci l DNA glycosylase, we first prepared a 250-bp DNA template consisting of tw o AP sites at specific locations. We then detected the AP sites by marking them with biotinylated aldehyde-reactive probes and monomeric avidin. We de monstrate here that (i) the location of monomeric avidin bound on a single DNA molecule was detectable by atomic force microscopy; (ii) the observed l ocation of avidin was in good agreement to the predicted AP sites at a few nanometer resolution; and (iii) by end-labeling the 5'-terminus of one DNA strand, the AP sites were determined without directional ambiguity. The tec hnique described here will provide a sensitive way of locating AP sites and contribute to screen DNA damages from individual molecules.