AN EFFICIENT SITE-DIRECTED MUTAGENESIS USING POLYMERASE CHAIN REACTION

AN EFFICIENT SITE-DIRECTED MUTAGENESIS USING POLYMERASE CHAIN REACTION

作者：杨香娇;陈常庆;王德宝;杨胜利;;;;;

作者机构：Shanghai Institute of Biochemistry;Academia Sinica;Shanghai 200031;PRC;PRC;Shanghai Center of Biotechnology;Shanghai 200233;PRC; 来源：中国科学 年：1991 卷：000 期：006

页码：P.712-718 页数：7 中图分类：O6 正文语种：CHI

关键词：polymerase chain reaction aite-directed mutagenesis on the EcoRI restriction gene DNA sequencing Taq DNA polymerase 摘要：We report here a simple and efficient method for site-directed mutagenesis using polymer-ase chain reaction (PCR). In constructing a new expression plasmid for the EcoRI restrictiongene, we made two point mutations. While one created a new Sall site prior to the SDsequence, the other replaced Glu144 with Lys. A 1.5 kb Sall-PstI fragment isolated frompER101 was used as the template. Two 25 mer oligonucleotide primers containing the de-sired mutations were synthesized and used to direct PCR amplification with Taq DNA poly-merase. About 0.5μg of the 0.49 kb fragment was obtained from 0.05 μ of the 1.5 kb frag-ment by carrying out polymerase chain reaction for