Kim S C, Szybalski W
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Gene. 1988 Nov 15;71(1):1-8. doi: 10.1016/0378-1119(88)90071-6.
By generating totally asymmetric and complementary cohesive ends it is possible to amplify any cloned DNA fragment, while assuring that all repeating units are ligated in the same orientation. Starting with plasmid pUC18, which contains a unique BspMI site, an amplification plasmid, pSK3, was constructed in which a multiple cloning site (MCS) is flanked by two BspMI recognition sites with identical cut sites, creating the complementary 5'-ATGC and 5'-GCAT single-stranded ends. Any DNA fragment cloned into the MCS could be amplified by (i) excision with BspMI, (ii) fragment isolation, (iii) self-ligation of the fragments using T4 DNA ligase, (iv) selection of multimers of desired length, and (v) cloning them into the BspMI-digested original plasmid. Using this procedure, plasmids carrying either 30 copies of the 60-bp MCS fragment (a control experiment) or ten copies of the 1.2-kb luxA gene fragment were constructed. The plasmids were stable since all the repeat units were in the same orientation, as determined by restriction analysis. Potentially, not only BspMI but other class-IIS restriction enzymes (with recognition sites separated by a fixed distance from the staggered cut points) could be applied, preferably those that create 4-to-5-nucleotide-long cohesive ends and utilize rather rare recognition sites.
通过产生完全不对称且互补的粘性末端，可以扩增任何克隆的DNA片段，同时确保所有重复单元以相同方向连接。从含有独特BspMI位点的质粒pUC18开始，构建了一个扩增质粒pSK3，其中多克隆位点（MCS）两侧是两个具有相同切割位点的BspMI识别位点，产生互补的5'-ATGC和5'-GCAT单链末端。克隆到MCS中的任何DNA片段都可以通过以下步骤进行扩增：（i）用BspMI切割，（ii）片段分离，（iii）使用T4 DNA连接酶将片段进行自身连接，（iv）选择所需长度的多聚体，以及（v）将它们克隆到用BspMI消化的原始质粒中。使用该程序，构建了携带60-bp MCS片段30个拷贝的质粒（一个对照实验）或携带1.2-kb luxA基因片段10个拷贝的质粒。经限制性分析确定，由于所有重复单元都处于相同方向，这些质粒是稳定的。潜在地，不仅BspMI，而且其他IIS类限制性内切酶（其识别位点与交错切割点相隔固定距离）也可以应用，优选那些产生4至5个核苷酸长粘性末端并利用相当罕见识别位点的酶。
