A ligation-independent cloning method using nicking DNA endonuclease.

Using nicking DNA endonuclease (NiDE), we developed a novel technique to clone DNA fragments into plasmids. We created a NiDE cassette consisting of two inverted NiDE substrate sites sandwiching an asymmetric four-base sequence, and NiDE cleavage resulted in 14-base single-stranded termini at both ends of the vector and insert. This method can therefore be used as a ligation-independent cloning strategy to generate recombinant constructs rapidly. In addition, we designed and constructed a simple and specific vector from an Escherichia coli plasmid back-bone to complement this cloning method. By cloning cDNAs into this modified vector, we confirmed the predicted feasibility and applicability of this cloning method.