Open AccessA 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/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) , multiple cloning site , molecular cloning , insert (composites) , ligation , recombinant dna , in vitro recombination , biology , microbiology and biotechnology , endonuclease , dna , genetics , vector (molecular biology) , gene , peptide sequence , computer science , materials science , composite material , programming language
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.