Detection of abasic sites on individual DNA molecules using atomic force microscopy.
We have developed an atomic force microscopy-based method for detecting abasic sites (AP sites) on individual DNA molecules. By using uracil and uracil DNA glycosylase, we first prepared a 250-bp DNA template consisting of two AP sites at specific locations. We then detected the AP sites by marking them with biotinylated aldehyde-reactive probes and monomeric avidin. We demonstrate here that (i) the location of monomeric avidin bound on a single DNA molecule was detectable by atomic force microscopy; (ii) the observed location 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 technique described here will provide a sensitive way of locating AP sites and contribute to screen DNA damages from individual molecules.
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Department of Mechanical Engineering, Indiana University--Purdue University Indianapolis, 46202, USA.
Qian
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Yokota
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D004247:DNA / Q000737:chemistry*; D045647:DNA Glycosylases; D018625:Microscopy, Atomic Force / Q000379:methods*; D009699:N-Glycosyl Hydrolases / Q000737:chemistry; D014498:Uracil / Q000737:chemistry; D051981:Uracil-DNA Glycosidase
D014498:Uracil; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase