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2008年 第2卷 第1期

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《医学前沿（英文）》 >> 2008年 第2卷 第1期 doi: 10.1007/s11684-008-0018-x

Site-directed mutagenesis of long QT syndrome KCNQ1 gene

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;

发布日期： 2008-03-05
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To construct a polymerase chain reaction (PCR) site-directed mutagenesis of the long QT syndrome KCNQ1 gene , 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 pIRES-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, pIRES-EGFP-KCNQ1 (G983A) was transfected into HEK cells successfully. These results may shed light on further functional study of KCNQ1 gene.

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