A ligation-independent cloning method using nicking DNA endonuclease

作者：Yang, Jie; Zhang, Zhihong^*; Zhang, Xin A.; Luo, Qingming

来源：Biotechniques, 2010, 49(5): 817-+.

DOI：10.2144/000113520

• ligation-independent cloning (LIC)
• nicking DNA endonuclease (NiDE)
• plasmid construction

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 backbone to complement this cloning method. By cloning cDNAs into this modified vector, we confirmed the predicted feasibility and applicability of this cloning method.

• 出版日期2010-11
• 单位华中科技大学