title: A ligation-independent cloning method using nicking DNA endonuclease.
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.
authors.affiliation: Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong, University of Science and Technology, Wuhan, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Zhang
authors.forename: Xin A
authors.initials: XA
authors.lastname: Zhang
authors.forename: Qingming
authors.initials: Q
authors.lastname: Luo
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry; D053904:DNA Breaks, Single-Stranded; D018076:DNA, Complementary / Q000235:genetics; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008164:Luminescent Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction
chemical_list: D001426:Bacterial Proteins; D018076:DNA, Complementary; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D004247:DNA; D003850:Deoxyribonuclease I