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Science 22 April 1988:
Vol. 240. no. 4851, pp. 504 - 506
DOI: 10.1126/science.2833816
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Articles

Science, Vol 240, Issue 4851, 504-506
Copyright © 1988 by American Association for the Advancement of Science

articles

Cleaving DNA at any predetermined site with adapter-primers and class-IIS restriction enzymes

SC Kim, AJ Podhajska, and W Szybalski

McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.

A four-component system has been designed that makes it possible to prepare a double-stranded (ds) DNA fragment; one fragment end is predesigned (by the use of a class-IIS restriction enzyme and adapter-primer), and the other end corresponds to any normal restriction cut. The system is composed of the phage M13mp7 single-stranded (ss) target DNA; the Fok I restriction enzyme; an oligodeoxynucleotide adapter-primer, which permits one to introduce Fok I cuts at any specified site in the target DNA; and DNA polymerase, which converts the ss target into a ds form ready for cloning. In this system, the oligodeoxynucleotide adapter-primer serves several purposes. The 5' hairpin ds domain of the adapter-primer contains the Fok I recognition site. Its 3' ss domain selects a complementary site on the target ss DNA, hybridizes with it to form the ds cleavage site, and serves as a primer to convert the ss M13mp7 target to ds DNA.


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Science. ISSN 0036-8075 (print), 1095-9203 (online)