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篇目详细内容 |
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【篇名】 |
Site-directed mutagenesis of long QT syndrome KCNQ1 gene in vitro |
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【刊名】 |
Frontiers of Medicine in China |
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【刊名缩写】 |
Front. Med. China |
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【ISSN】 |
1673-7342 |
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【EISSN】 |
1673-7458 |
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【DOI】 |
10.1007/s11684-008-0018-x |
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【出版社】 |
Higher Education Press and Springer-Verlag |
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【出版年】 |
2008 |
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【卷期】 |
2
卷1期 |
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【页码】 |
100-104
页,共
5
页 |
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【作者】 |
LI Wei;
YANG Junguo;
DU Rong;
TIAN Li;
WANG Bin;
XU Qiumei;
KE Qinmei;
WANG Qing;
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【关键词】 |
long QT syndrome, KCNQ1, polymerase chain reaction, site-directed mutagenesis |
【摘要】 |
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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. |
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