【摘要】 利用聚合酶链反应(PCR)技术对长QT综合征(LQTS)KCNQ1基因进行定点突变的研究。首先设计两对引物(包含预定的突变),通过3轮PCR扩增,扩增出含有所需突变位点的片段,然后将片段克隆入T载体中,通过酶切连接的方法将突变点引入到pIRES2 EGFP KCNQ1中,随后用Effectene转染试剂介导转染HEK293细胞。结果在真核表达载体pIRES2 EGFP KCNQ1基础上获得了KCNQ1cDNAC934T的突变体,测序表明在序列中发生了预期的突变。将含突变点的pIRES2 EGFP KCNQ1转染HEK293细胞后,在荧光显微镜下观察到被转染的HEK293细胞发出绿色荧光,表明含突变点的pIRES2 EGFP KCNQ1得到了表达。更多还原

【Abstract】 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 pIRES₂-EGFP-KCNQ1.With Effectene Transfection Reagent,pIRES₂-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 pIRES₂-EGFP-KCNQ1 containing the mutation site was expressed correctly.更多还原