Construction of mutant and chimeric genes using the polymerase chain reaction.
Construction of mutant and chimeric genes using the polymerase chain reaction.
复制标题
DOI：
10.1093/nar/17.2.723
复制
发表时间：
1989-01
影响因子：
14.9
通讯作者：
F. Vallette;Emmanuelle Mege;A. Reiss;M. Adesnik
中科院分区：
文献类型：
--
作者：
F. Vallette;Emmanuelle Mege;A. Reiss;M. Adesnik
在聚合酶链反应（PCR）中，复杂DNA样本中一小段DNA的特异性扩增受到DNA变性和酶促合成的重复循环的影响，酶促合成是由两个与DNA相反链内区域互补的寡核苷酸引发的。在本报告中，描述了一种简单而有效的方法，其中PCR方法用于将特定突变引入双链DNA分子。在这个过程中，一个超螺旋质粒DNA作为PCR的模板，其中携带突变序列的引物被纳入扩增产物中。诱变引物和扩增DNA中方便的限制性内切位点的存在允许用适当的限制性内切酶和DNA连接酶处理PCR混合物，将野生型DNA片段直接替换为突变片段。采用该方法，在大鼠肝细胞色素P450b （P450IIB1）的氨基末端信号段中引入单个氨基酸替换、16个氨基酸缺失和4个氨基酸替换为来自另一个膜蛋白的12个氨基酸片段。
In the polymerase chain reaction (PCR) the specific amplification of a small segment of DNA within a complex DNA sample is effected by repeated cycles of DNA denaturation and enzymatic synthesis primed by two oligonucleotides complementary to regions within opposite strands of the DNA. In this report a simple and efficient method is described in which PCR methodology is used to introduce specific mutations into a double stranded DNA molecule. In this procedure a supercoiled plasmid DNA serves as template for a PCR in which a primer bearing the mutated sequence is incorporated into the amplified product. The presence of convenient restriction sites in the mutagenic primer and in the amplified DNA permit direct replacement of a wild type DNA segment with the mutated segment by treating the PCR mixture with the appropriate restriction endonucleases followed by DNA ligase. Using this procedure, a single amino acid replacement, a 16 amino acid deletion and a replacement of four amino acids with a twelve amino acid segment from another membrane protein were introduced into the amino terminal signal segment of rat hepatic cytochrome P450b (P450IIB1).