Stanssens P, Opsomer C, McKeown Y M, Kramer W, Zabeau M, Fritz H J
Plant Genetic Systems N.V., Gent, Belgium.
Nucleic Acids Res. 1989 Jun 26;17(12):4441-54. doi: 10.1093/nar/17.12.4441.
An efficient method for the construction of multiple mutations in a sequential manner is described. It is based on the gapped duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) and a set of newly constructed phasmid vectors. These are characterized by the following features. Presence of the phage fl replication origin permits ready conversion to the single stranded DNA form. An amber mutation within, alternatively, the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated. By means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of mutagenesis. The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins without re-cloning. Both the mutagenesis and expression aspects were tested using the lacZ gene as a model.
本文描述了一种以序列方式构建多个突变的有效方法。它基于用于寡核苷酸定向诱变的缺口双链DNA方法（Kramer等人，1984年，《核酸研究》12卷，9441 - 9456页）以及一组新构建的噬菌粒载体。这些载体具有以下特征。噬菌体fl复制起点的存在使得能够方便地转化为单链DNA形式。bla或cat基因内的琥珀突变提供了一种方便的方法来选择已掺入诱变寡核苷酸的链。通过交替的抗生素抗性标记，可在连续的诱变轮次中构建任意数量的突变。基因表达信号的可选存在允许直接过量生产结构改变的蛋白质而无需重新克隆。使用lacZ基因作为模型对诱变和表达方面都进行了测试。
