A simple method to construct T-vectors using XcmI cassettes amplified by nonspecific PCR

Abstract

Polymerase chain reaction (PCR) is one of the most powerful tools in cloning genes. For the direct cloning of PCR products, T-vectors, which contain complementary 3'-thymidine overhangs, are widely used. In the present study, we developed a plasmid, pNB-T, which was constructed by cloning an XcmI cassette with a sufficient length of DNA (over 500 bp long) between two XcmI restriction sites into pBluescript SK(+). An XcmI cassette was made by nonspecific PCR using a primer containing recognition sequences of XcmI so that pNB-T can easily be converted into a T-vector by restriction of the plasmid with XcmI. In addition, the recognition sequences for BamHI and NcoI were added at 5'-end of the primer in order to facilitate subcloning of the gene cloned in the T-vector. The cloning efficiency of a PCR product, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, was approximately 90%. Digestion of the recombinant plasmid containing the GAPDH gene with BamHI or NcoI liberated the DNA fragments with the expected size, demonstrating the usefulness of extra restriction sites. The method described in this report is quite simple and enables us to construct a variety of useful T-vectors.