一种改进的快速PCR定点突变技术

A Technique of Improved Rapid Site-directed Mutagenesis

在基因工程与蛋白质工程研究中常常用到基因突变技术制备突变体,用于研究基因调控、蛋白质的结构与功能。本项研究在快速PCR定点突变技术的基础上,改进实验方法,简化实验步骤,以适用蛋白质结构改造研究的需要。建立的突变技术仅需一次PCR反应即可完成基因的定点突变,突变效率为79.6%左右。该法对1～3个连续碱基的突变和对间隔4个甚至多达15个碱基的数个密码子突变效果都很好。

Mutants prepared by mutagenesis methods are usually used to study gene regulations, gene expressions and the structure-function relationships of proteins in gene engineering and protein engineering. In this study, we focus on establishing a new gene mutation technique, improving the experimental conditions and expanding the practical extents for meeting the demands of engineered protein structures. The target genes were inserted into the cloning vectors--plasmids. The mutation was completed only by one step PCR reaction using a double-stranded DNA template and double-mutation primers catalyzed by Taq plus DNA polymerase. After the DNA template was digested by a restriction endonuclease, the vectors were introduced into host cells and the target genes with mutations were expressed by using host cells＇ transcription and translation mechanism. The mutation rate was 79.6%. This method was effective for the mutation of 1～3 continuous bases and of a few codons spaced by several bases.