PCR Site-Directed Mutagenesis of Long QT Syndrome KCNQ1 Gene in vitro
应用PCR技术对先天性长QT综合征KCNQ1 基因进行定点突变的研究PCR Site-Directed Mutagenesis of Long QT Syndrome KCNQ1 Gene in vitro

To study PCR site-directed mutagenesis of long QT syndrome KCNQ1 gene in vitro.The site-directed mutagenesis of LQTS gene KCNQ1 was made by PCR.Two sets of primers were designed according to the sequence of KCNQ1 cDNA,and mismatch was introduced into primers.Mutagenesis was performed in a three-step PCR.The amplified fragments from the third PCR which contained the mutation site were subcloned into the T-vecor PCR 2.1.Then the fragments containing the mutation site was obtained from PCR2.1 with restriction enzyme digestion and was inserted into the same restriction site of pIRES_2-EGFP-KCNQ1.With Effectene Transfection Reagent,pIRES_2-EGFP-KCNQ1 was transfected into HEK293 cell.The sequencing analysis showed that the mutation site was correct.Mutation from T to C in 934 site of KCNQ1 cDNA was found.Under the fluorescence microscope,the green fluorescence was spread in the transfected HEK293 cell,meaning the pIRES_2-EGFP-KCNQ1 containing the mutation site was expressed correctly.