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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2008, Vol. 2 Issue (1) : 100-104     DOI: 10.1007/s11684-008-0018-x
Site-directed mutagenesis of long QT syndrome KCNQ1 gene
LI Wei1, WANG Bin1, XU Qiumei1, KE Qinmei1, YANG Junguo2, DU Rong2, TIAN Li2, WANG Qing3
1.Geriatric Department, Union Hospital, Tongji Medical College, Huazhong Science and Technology University; 2.Institute of Cardiology, Union Hospital, Tongji Medical College, Huazhong Science and Technology University; 3.Center of Cardiovascular Genetics, the Cleveland Clinic Foundation;
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Abstract  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.
Issue Date: 05 March 2008
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