Insert restriction enzyme cutting-free cloning strategy for expression plasmid construction

Abstract

Plasmids are important tools for producing biological reagents and performing molecular biological investigations. They are often constructed by the traditional restriction digestion/ligation cloning method. However, there are some drawbacks of this method due to its nature. It is time-consuming; sometimes there are no suitable restrictive digestion sites and the efficiency of digestion of the polymerase chain reaction (PCR) products is low. To address these problems, we employed an insert cutting-free cloning (ICFC) method in this work. Two kinds of DNA fragments containing the same gene but different terminal sequences were amplified by PCR. Inserts with desired cohesive ends were generated by successively mixing, denaturing and re-annealing these DNA fragments. Finally, they were ligated to the restriction-endonuclease digested vectors. Using this method, we successfully constructed a STAT3 (signal transducer and activator of transcription 3) expression plasmid. The method proved to be simple and efficient. It made the selection of restriction endonucleases and vectors easier, as well as simplified and shortened the cloning process.