Jung R, Scott M P, Oliveira L O, Nielsen N C
United States Department of Agriculture, Purdue University, West Lafayette, IN 47907.
Gene. 1992 Nov 2;121(1):17-24. doi: 10.1016/0378-1119(92)90157-k.
A method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded DNA without the necessity for phenotypic selection is described. Plasmids denatured with alkali and purified by adsorption to and elution from nitrocellulose have single-stranded regions where primers can hybridize and serve as templates for a T7 DNA polymerase-catalyzed synthesis of complementary mutant DNA strands. When this procedure was carried out such that the original nonmutant strand contained uracil [method of Kunkel, Proc. Natl. Acad. Sci. USA 82(1985)488-492], mutation frequencies of between 30% and 40% were obtained. The technique has been used to generate mutant genes in plasmids of a wide variety of sizes. The largest plasmid manipulated and successfully mutagenized was 22 kb. The method is rapid and efficient and is not dependent upon either f1 phage vectors or the presence of restriction sites in the vicinity of the sequence targeted for mutation.
本文描述了一种无需表型选择即可对双链DNA进行寡脱氧核糖核苷酸定向诱变的方法。用碱变性并通过吸附到硝酸纤维素上然后洗脱进行纯化的质粒具有单链区域，引物可在该区域杂交并作为T7 DNA聚合酶催化合成互补突变DNA链的模板。当按照原始非突变链含有尿嘧啶的方式进行该程序时[昆克尔方法，《美国国家科学院院刊》82(1985)488 - 492]，获得了30%至40%的突变频率。该技术已用于在各种大小的质粒中产生突变基因。操作并成功诱变的最大质粒为22 kb。该方法快速有效，不依赖于f1噬菌体载体，也不依赖于靶向突变序列附近的限制性位点的存在。
