Setzer D R, Hmiel R M, Liao S Y
Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, OH 44106.
Nucleic Acids Res. 1990 Jul 25;18(14):4175-8. doi: 10.1093/nar/18.14.4175.
We describe a simple modification of commonly used single-stranded cloning vectors that permits the efficient recovery of mutant DNA molecules in oligonucleotide-directed mutagenesis experiments, even when the absolute efficiency of mutagenesis is very low. The modification consists of the insertion of a short synthetic DNA fragment into the vector's polylinker and permits the identification of mutant clones based on a standard chromogenic plate assay for bacterial colonies or phage plaques producing functional beta-galactosidase. Other useful properties of the original vector are retained in the modified version. In vitro mutagenesis reactions are carried out with two oligonucleotides, one to introduce the mutation of interest, and the second to correct a frameshift mutation introduced into the beta-galactosidase gene of the modified vector. We have found that these two sequence changes are closely linked following transformation of an appropriate E. coli strain with the products of the in vitro mutagenesis reaction, and have thereby recovered desired mutations at a frequency of about 50% even when the overall mutagenesis efficiency is less than 1%. By alternately correcting and re-introducing the beta-galactosidase frameshift mutation, we have shown that multiple rounds of mutagenesis can be carried out on the same template with a high efficiency of mutant recovery in each step. Modifications similar or identical to those we describe here should be feasible for most commonly used single-stranded cloning vectors and should increase the usefulness of these vectors by providing an additional option for oligonucleotide-directed mutagenesis to be used in conjunction with or in lieu of other commonly used approaches.
我们描述了一种对常用单链克隆载体的简单修饰方法，该方法能在寡核苷酸定向诱变实验中高效回收突变DNA分子，即便诱变的绝对效率非常低。这种修饰是在载体的多克隆位点插入一段短的合成DNA片段，并能基于用于检测产生功能性β-半乳糖苷酶的细菌菌落或噬菌斑的标准显色平板检测法来鉴定突变克隆。修饰后的载体保留了原始载体的其他有用特性。体外诱变反应使用两条寡核苷酸进行，一条用于引入感兴趣的突变，另一条用于校正引入到修饰载体β-半乳糖苷酶基因中的移码突变。我们发现，用体外诱变反应产物转化合适的大肠杆菌菌株后，这两个序列变化紧密相连，因此即使总体诱变效率低于1%，也能以约50%的频率回收所需突变。通过交替校正和重新引入β-半乳糖苷酶移码突变，我们表明可以在同一模板上高效进行多轮诱变，且每一步都能高效回收突变体。对于大多数常用的单链克隆载体，与我们在此描述的修饰类似或相同的修饰应该是可行的，并且通过提供寡核苷酸定向诱变的额外选项，可与其他常用方法结合使用或替代其他常用方法，从而增加这些载体的实用性。
