Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA.
Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA.
复制标题
使用 M13 衍生载体进行寡核苷酸定向诱变：在任何 DNA 片段中产生点突变的有效且通用的程序。
DOI：
10.1093/nar/10.20.6487
复制
发表时间：
1982
影响因子：
14.9
通讯作者：
M. Smith
中科院分区：
文献类型：
--
作者：
M. Zoller;M. Smith
本文介绍了一种多用途和高效的方法，用于在克隆到M13衍生载体的DNA片段中构建寡脱氧核糖核苷酸定向的位点特异性突变。作为一个例子，在酵母MATa1基因的克隆中产生过渡突变被描述。将寡核苷酸与模板DNA杂交，用大肠杆菌DNA聚合酶（大片段）延伸寡核苷酸引物，并与T4 DNA连接酶连接，生成共价封闭的双链分子。通过碱性蔗糖梯度离心分离得到的双链闭合环状DNA （CC-DNA）与未连接和不完全延伸的分子分离。这种纯化对于高效生产突变体至关重要。用CC-DNA片段转化大肠杆菌JM101细胞，从单个斑块中分离单链DNA。通过限制性内切酶筛选靶区hif - 1位点缺失的突变分子，以及以诱变寡核苷酸为探针进行点杂交，筛选突变分子。从测序中分离出双链DNA。突变体产生的效率在10-45%之间，不需要预防错配修复。
This paper presents a versatile and efficient procedure for the construction of oligodeoxyribonucleotide directed site-specific mutations in DNA fragments cloned into M13 derived vectors. As an example, production of a transition mutation in a clone of the yeast MATa1 gene is described. The oligonucleotide is hybridized to the template DNA and covalently closed closed double stranded molecules are generated by extension of the oligonucleotide primer with E. coli DNA polymerase (large fragment) and ligation with T4 DNA ligase. The resulting double stranded closed circular DNA (CC-DNA) is separated from unligated and incompletely extended molecules by alkaline sucrose gradient centrifugation. This purification is essential for production of mutants at high efficiency. Competent E. coli JM101 cells are transformed with the CC-DNA fraction and single stranded DNA is isolated from individual plaques. The recombinants are screened for mutant molecules by 1) restriction endonuclease screening for the loss of the Hinf I site in the target region, and 2) by dot blot hybridization using the mutagenic oligonucleotide as probe. Double stranded DNA is isolated from the sequencing. Efficiency of mutant production is in the range of 10-45% and no precautions to prevent mismatch repair are required.