As a rule, when restriction endonucleases bind to their recognition sequences in DNA, they hydrolyze both strands of the duplex at the same time. Two independent hydrolytic reactions proceed in parallel, most often driven by the presence of two catalytic sites within each enzyme, one for hydrolyzing each strand. We have begun to engineer altered restriction enzymes that hydrolyze only one strand of the duplex, to produce DNA molecules that are “nicked”, rather than cleaved. These conventional nicks (3´-hydroxyl, 5´-phosphate) can serve as initiation points for a variety of further enzymatic reactions such as replacement DNA synthesis, strand-displacement amplification (1), exonucleolytic degradation, or the creation of small gaps (2).
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