【摘要】 目的:改进重叠延伸PCR法,实现一种引入DNA定点突变的准确简便方法。方法:通过应用不同的扩增酶和反应体系,以重叠延伸PCR的方法产生引入突变位点的DNA片断,然后再亚克隆到载体中。该文以人cyclin D1启动子的NF-κB位点(-39/-30)为例。结果:通过DNA测序证明定点突变成功引入。一次引入4个突变碱基。突变引入率为100%。更多还原

【Abstract】 Objective:To develop a new method for generation of site-directed mutation by modifying overlap extension PCR.Method: mutation generation by overlap extension PCR using different DNA polymerase and reaction buffer.The mutation fragment is subcloned into T-vector.In this study,we selected the NF-κB site(-39/-30)of human cyclin D1 promoter for example.Result: The mutation is verified by DNA sequencing.Four point-mutations were intruduced by one-step.The overall rate of obtaining the multiple mutant sites was 100%.Conclusion: An effective and cheap method of DNA mutation by modifying overlap extension PCR had developed.更多还原