A Ligation-Independent Cloning Method using Nicking DNA Endonuclease
2010; Future Science Ltd; Volume: 49; Issue: 5 Linguagem: Inglês
10.2144/000113520
ISSN1940-9818
AutoresJie Yang, Zhihong Zhang, Xin A. Zhang, Qingming Luo,
Tópico(s)RNA Interference and Gene Delivery
ResumoUsing 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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