[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 PCR2.1. 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, 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.
Li
Wei
W
Institute of Cardiology, Tongji Medical College, Huazhong Science and Technology University, Wuhan 430 022, China. liwei3911@sina.com
Yang
Jun-Guo
JG
Ren
Fa-Xin
FX
Kang
Cai-Lian
CL
Zhang
Shou-Yan
SY
D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004622:Embryo, Mammalian; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008133:Long QT Syndrome / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D017422:Sequence Analysis, DNA; D014162:Transfection
D018076:DNA, Complementary