Generally, restriction endonucleases bind to their recognition sequences in DNA and 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. Techniques have been developed to alter the action of restriction enzymes to hydrolyze only one strand of the duplex, to produce DNA molecules that are “nicked”, rather than cleaved. Restriction endonucleases that have asymmetric recognition sequences are potential subjects for the development of strand specific nicking endonucleases. 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), or the creation of small gaps (2).
Protocols for DNA Nicking
- Cloning with USER Enzyme
- Comet Assay - Modified for Detection of Oxidized Bases Using the Repair Endonucleases Fpg, hOGG1 and Endonuclease III (Nth)
- Determining Genome Targeting Efficiency using T7 Endonuclease I
- Double Digest Protocol with Standard Restriction Enzymes
- Optimizing Restriction Endonuclease Reactions
- Nicking Endonucleases: The Discovery and Engineering of Restriction Enzyme Variants
- Why Choose Recombinant Enzymes?
- Troubleshooting Guide for Cloning
- NEBuffer Activity/Performance Chart with Restriction Enzymes
Other Tools & Resources
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Chromatin conformation capture (3C) techniques allow study of the spatial organization of eukaryotic chromosomes in a 3D context. Learn more about this and other applications of restriction enzymes.