To construct a polymerase chain reaction(PCR)site-directed mutagenesis of the long QT syndrome KCNQ1 gene in vitro,two sets of primers were designed according to the sequence of KCNQ1 cDNA and a mismatch was introduce...To construct a polymerase chain reaction(PCR)site-directed mutagenesis of the long QT syndrome KCNQ1 gene in vitro,two sets of primers were designed according to the sequence of KCNQ1 cDNA and a mismatch was introduced into primers.Mutagenesis was performed in a two-step PCR.The amplified fragments from the third PCR which contained the mutation site were sub-cloned into the T-vector pCR2.1.Then,the fragments containing the mutation site was obtained from pCR2.1 using restriction enzymes digestion and inserted into the same restriction site of pIRES2-EGFP-KCNQ1.The sequencing analysis shows that the mutation site was correct.Mutation from A to G in site 983 of KCNQ1 cDNA was found.Using the Effectene transfection reagent,pIRES2-EGFP-KCNQ1(G983A)was transfected into HEK cells successfully.These results may shed light on further functional study of KCNQ1 gene.展开更多
文摘To construct a polymerase chain reaction(PCR)site-directed mutagenesis of the long QT syndrome KCNQ1 gene in vitro,two sets of primers were designed according to the sequence of KCNQ1 cDNA and a mismatch was introduced into primers.Mutagenesis was performed in a two-step PCR.The amplified fragments from the third PCR which contained the mutation site were sub-cloned into the T-vector pCR2.1.Then,the fragments containing the mutation site was obtained from pCR2.1 using restriction enzymes digestion and inserted into the same restriction site of pIRES2-EGFP-KCNQ1.The sequencing analysis shows that the mutation site was correct.Mutation from A to G in site 983 of KCNQ1 cDNA was found.Using the Effectene transfection reagent,pIRES2-EGFP-KCNQ1(G983A)was transfected into HEK cells successfully.These results may shed light on further functional study of KCNQ1 gene.
