Jo C, Jo S A
Division of Degenerative Diseases, Department of Biomedical Sciences and Biomedical Brain Research Center, National Institute of Health, Seoul, Korea.
Plasmid. 2001 Jan;45(1):37-40. doi: 10.1006/plas.2000.1500.
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.
聚合酶链反应（PCR）是克隆基因最强大的工具之一。对于PCR产物的直接克隆，含有互补3'-胸腺嘧啶突出端的T载体被广泛使用。在本研究中，我们开发了一种质粒pNB-T，它是通过将一个具有足够长度DNA（超过500 bp长）的XcmI盒在两个XcmI限制位点之间克隆到pBluescript SK(+)中构建而成。通过使用含有XcmI识别序列的引物进行非特异性PCR制备XcmI盒，这样通过用XcmI限制该质粒，pNB-T可以很容易地转化为T载体。此外，为了便于在T载体中克隆的基因进行亚克隆，在引物的5'-末端添加了BamHI和NcoI的识别序列。PCR产物甘油醛-3-磷酸脱氢酶（GAPDH）基因的克隆效率约为90%。用BamHI或NcoI消化含有GAPDH基因的重组质粒，释放出预期大小的DNA片段，证明了额外限制位点的有用性。本报告中描述的方法非常简单，使我们能够构建各种有用的T载体。
