A Ligation-Independent Cloning Method using Nicking DNA Endonuclease | Zendy

Jie Yang | Zendy; Zhihong Zhang | Zendy; Xin A. Zhang | Zendy; Qingming Luo | Zendy

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A Ligation-Independent Cloning Method using Nicking DNA Endonuclease

Author(s) -

Jie Yang,

Zhihong Zhang,

Xin A. Zhang,

Qingming Luo

Publication year - 2010

Publication title -

biotechniques

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.617

H-Index - 131

eISSN - 1940-9818

pISSN - 0736-6205

DOI - 10.2144/000113520

Subject(s) - cloning vector , restriction enzyme , plasmid , cloning (programming) , insert (composites) , molecular cloning , multiple cloning site , ligation , recombinant dna , in vitro recombination , microbiology and biotechnology , biology , endonuclease , dna , vector (molecular biology) , genetics , gene , peptide sequence , computer science , materials science , programming language , composite material

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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