Lee J H, Skowron P M, Rutkowska S M, Hong S S, Kim S C
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Kusong-dong, Taejon.
Genet Anal. 1996 Dec;13(6):139-45. doi: 10.1016/s1050-3862(96)00164-7.
In order to make high-copy-number multimers of DNA fragments in a tandem unit, two different gene amplification vectors (pSK9 and pBBS1) were developed. Two identical class-IIS restriction enzyme sites (BspMI for pSK9 and BbsI for pBBSI) were inversely oriented in each vector with the same cut site, creating asymmetric and complementary cohesive ends (5'-CCCC and 5'-GGGG). Multimers were made by: (i) cloning a target DNA into the class-IIS restriction enzyme cut site of each vector; (ii) excision of the monomeric insert by digestion with the class-IIS restriction enzyme; (iii) isolation of the fragments; (iv) self-ligation of the fragments; (v) cloning into the original vector digested with the class-IIS restriction enzyme; and (vi) repeating steps (i) through (v) to generate higher-order multimers. Various-sized multimers of a 93-bp DNA fragment encoding magainin, an antimicrobial peptide, were obtained with the gene amplification vector, pBBS1. Larger multimers, up to about 108 copies, were constructed from the monomer by the sequential amplification procedure. Of six different Escherichia coli hosts examined for the stability of multimers, the multimers were the most stable in E. coli D1210. The gene amplification vector system described here is very efficient and can be applied in the construction of tandem multimers of any kind of DNA, as long as the cloned DNA does not contain the cut site of the class-IIS restriction enzyme to be utilized.
为了在串联单元中制备高拷贝数的DNA片段多聚体，开发了两种不同的基因扩增载体（pSK9和pBBS1）。在每个载体中，两个相同的IIS类限制性酶切位点（pSK9的BspMI和pBBSI的BbsI）以相同的切割位点反向排列，产生不对称且互补的粘性末端（5'-CCCC和5'-GGGG）。多聚体的制备方法如下：（i）将目标DNA克隆到每个载体的IIS类限制性酶切位点；（ii）用IIS类限制性酶消化切除单体插入片段；（iii）分离片段；（iv）片段自身连接；（v）克隆到用IIS类限制性酶消化的原始载体中；（vi）重复步骤（i）至（v）以产生更高阶的多聚体。使用基因扩增载体pBBS1获得了编码抗菌肽马盖宁的93bp DNA片段的各种大小的多聚体。通过连续扩增程序从单体构建了更大的多聚体，最多可达约108个拷贝。在所检测的六种不同大肠杆菌宿主中，多聚体在大肠杆菌D1210中最稳定。本文所述的基因扩增载体系统非常高效，只要克隆的DNA不包含要使用的IIS类限制性酶的切割位点，就可应用于构建任何类型DNA的串联多聚体。
