Cohen B, Carmichael G G
DNA. 1986 Aug;5(4):339-43. doi: 10.1089/dna.1986.5.339.
We describe a new method for constructing tandem repeats of DNA fragments that allows one to control the number of tandem copies appearing in a final recombinant DNA clone. The principle of the method is to prepare DNA fragments with predetermined cohesive ends and to ligate them together in such a way that unique multimers are generated. First, the fragment of interest is inserted into a vector with a multisite cloning region. Clones are picked with the fragment in both orientations, and the fragments are excised using different pairs of restriction enzymes. The resulting fragments with preprogrammed cohesive ends are mixed, ligated, and digested with one enzyme whose site appears in the cloning region of the vector. From this material multimeric fragments containing specific ends can be isolated and recloned into appropriate vectors. As an example of the method, we show the construction of clones containing either three or six tandem copies of the origin of DNA replication of the mouse polyoma virus. The prospect of using this technique to construct inverted repeat structures is discussed. As part of this work we have constructed a modified M13mp8 vector which contains a unique Xho I cloning site.
我们描述了一种构建DNA片段串联重复序列的新方法，该方法能让人控制最终重组DNA克隆中出现的串联拷贝数。该方法的原理是制备具有预定粘性末端的DNA片段，并以产生独特多聚体的方式将它们连接在一起。首先，将感兴趣的片段插入具有多克隆位点区域的载体中。挑选片段以两种方向插入的克隆，并用不同的限制性酶对组合切除片段。将产生的具有预编程粘性末端的片段混合、连接，并用一种其位点出现在载体克隆区域的酶进行消化。从这种材料中可以分离出含有特定末端的多聚体片段，并重新克隆到合适的载体中。作为该方法的一个例子，我们展示了构建含有小鼠多瘤病毒DNA复制起点三个或六个串联拷贝的克隆。讨论了使用该技术构建反向重复结构的前景。作为这项工作的一部分，我们构建了一个改良的M13mp8载体，它含有一个独特的Xho I克隆位点。
