改进重叠延伸PCR克隆tau基因6种同工异构体

采用一种改进的重叠延伸PCR技术,通过2次PCR的方法,对人tau基因进行剪切和拼接,实现了tau基因的6种同工异构体的克隆;然后将6种tau基因克隆到真核载体pcDNA4上,通过western blot在蛋白水平验证了tau蛋白的表达. 结果表明,这种改进重叠延伸PCR方法能够对基因进行多个位置的剪切和连接,由于该法在基因重组过程中无需利用限制性内切酶,因此具有很大的灵活性和简便性. 

Cloning of Six Isoforms of Tau by Modified Overlap Extension PCR Technique

Here we describe a new modified overlap extension PCR method for cloning human tau isoforms which can cut and splice gene to make inserts for expression vectors easily. The isoforms could be simply cloned to eukaryotic expression vector pcDNA4 and the tau protein expression be detected by western blot. Our data implicate that this method is much convenience and easier to cut and splice the multiple cloning site without restriction enzyme. It possesses great flexibility, valuablity and may provide broad applications in molecular cloning.