Mapping restriction sites on large DNAs by electron microscopy.
We have developed a novel technique to map restriction sites on large duplex DNAs by electron microscopy. In this method, the sample DNA is first cut with a restriction enzyme. The resulting fragments are briefly digested with Escherichia coli exonuclease III, and treated with wheat germ RNA polymerase II to fill-in with RNA the resulting gaps. These small RNAs, complementary to sequences immediately adjacent to either side of the restriction site, are isolated from the DNA template and R-looped to the full-length DNA. When this material is prepared by the formamide-cytochrome spreading technique, small bubbles are visible wherever there is a restriction site on the DNA. Improved methods of mapping are outlined.
D010582:Bacteriophage lambda / Q000235:genetics; D002874:Chromosome Mapping / Q000379:methods*; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000235:genetics; D008854:Microscopy, Electron / Q000379:methods
D004279:DNA, Viral; D004262:DNA Restriction Enzymes