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Updated: Jun 6, 2026

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage
Published on: May 6, 2020
A ligation-independent cloning method using nicking DNA endonuclease
Jie Yang1, Zhihong Zhang, Xin A Zhang
1Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong, University of Science and Technology, Wuhan, China.
Abstract:
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.
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