Mapping restriction sites on large DNAs by electron microscopy.

C Moore; J Griffith

doi:10.1016/0378-1119(83)90079-3

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Mapping restriction sites on large DNAs by electron microscopy.

C Moore·J Griffith

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Abstract

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.

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Bacteriophage lambda Chromosome Mapping DNA Restriction Enzymes DNA, Viral Microscopy, Electron Journal Article Research Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.

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C Moore, J Griffith. 1983. Mapping restriction sites on large DNAs by electron microscopy.. https://doi.org/10.1016/0378-1119(83)90079-3

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