Cleaving DNA at any predetermined site with adapter-primers and class-IIS restriction enzymes.
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.
Kim
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SC
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Podhajska
A J
AJ
Szybalski
W
W
D004247:DNA; D004262:DNA Restriction Enzymes; D004277:DNA, Single-Stranded; D015252:Deoxyribonucleases, Type II Site-Specific; D005818:Genetic Engineering; D009838:Oligodeoxyribonucleotides; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D004247:DNA; D004262:DNA Restriction Enzymes; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific