OLIGONUCLEOTIDE-DIRECTED MUTAGENESIS - A SIMPLE METHOD USING 2 OLIGONUCLEOTIDE PRIMERS AND A SINGLE-STRANDED-DNA TEMPLATE
OLIGONUCLEOTIDE-DIRECTED MUTAGENESIS - A SIMPLE METHOD USING 2 OLIGONUCLEOTIDE PRIMERS AND A SINGLE-STRANDED-DNA TEMPLATE
复制标题
DOI：
10.1089/dna.1.1984.3.479
复制
发表时间：
1984-01-01
期刊：
影响因子：
--
通讯作者：
SMITH, M
中科院分区：
文献类型：
--
作者：
ZOLLER, MJ;SMITH, M
本文介绍了一种简单有效的利用单链噬菌体载体进行寡核苷酸定向突变的方法。对我们先前发表的方法（Zoller和Smith，1982）进行的修改的特点是使用两种引物，其中一种是标准M13测序引物，另一种是诱变寡核苷酸。两个引物同时退火到单链模板DNA上，通过DNA聚合酶I（大片段）延伸，并连接在一起形成突变的野生型有缺口的异源双链体。大肠杆菌直接用这种DNA转化;不需要像我们以前的报告中那样分离共价闭合的环状DNA。使用诱变寡核苷酸作为探针，通过噬斑提升杂交鉴定突变体。作为该方法的一个例子，使用十七核苷酸在酵母菌MATa基因中产生T → G颠换，该基因克隆到载体M13 mp5中。诱变效率约为50%。通过DNA测序验证所需突变的产生。相同的程序已被用于不加修改地产生限制性位点的插入以及500个碱基的特异性缺失。
This paper presents a simple and efficient method for oligonucleotide-directed mutagenesis using vectors derived from single-stranded phage. This modification of our previously published procedure (Zoller and Smith, 1982) features the use of two primers, one of which is a standard M13 sequencing primer and the other is the mutagenic oligonucleotide. Both primers are simultaneously annealed to single-stranded template DNA, extended by DNA polymerase I (large fragment), and ligated together to form a mutant wild-type gapped heteroduplex.Escherichia coliis transformed directly with this DNA; the isolation of covalently closed circular DNA as in our previous report is not necessary. Mutants are identified by plaque lift hybridization using the mutagenic oligonucleotide as a probe. As an example of the method, a heptadecanucleotide was used to create a T → G transversion in theMATa gene ofSaccharomyces cerevisiaecloned into the vector M13mp5. The efficiency of mutagenesis was approximately 50%. Production of the desired mutation was verified by DNA sequencing. The same procedure has been used without modification to create insertions of restriction sites as well as specific deletions of 500 bases.