环状定点诱变技术在载体构建中的应用

Application of circular site-directed mutagenesis in vector construction

目的探讨环状定点诱变技术在目标质粒单碱基突变,酶切位点修改和片段删除中的应用。方法设计包含目标突变,部分互补且5’端含有5~8 bp突出的引物对,环状质粒作为模板进行扩增,甲基化酶DpnI酶消化去除模板质粒。转化DH5α感受态进行缺口修复,获得定点突变的目标质粒。结果通过酶切和基因测序证明,环状定点诱变技术可高效用于单碱基突变、酶切位点修改和片段删除等突变。结论环状定点诱变技术能够简便、高效、快捷地实现目标质粒的单碱基突变、酶切位点修改和片段删除,基本满足药物开发过程中载体构建的需求。

Objective To explore the application of circular site-directed mutagenesis technology in single base mutation,restriction site modification and fragment deletion of the target plasmid.Methods The primer pairs were designed including target mutation,partial complementation,and 5’end contains 5-8 bp protruding.The circular plasmid was used as template for amplification and digested and removed by methylase enzyme DpnI.Transform DH5αfor gap repair then site-directed mutation plasmid was obtained.Results Through the validation by enzyme cutting and gene sequencing,circular site-directed mutagenesis technology could be efficiently used in single base mutation,modification of enzyme site and deletion of fragment.Conclusion The circular site-directed mutagenesis technology is simple,efficient and quick to realize single base mutation,restriction site modification and fragment deletion of the target plasmid,which can basically meet the needs of molecular construction in the process of drug development.