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目的利用聚合酶链反应(PCR)技术对长QT综合征(LQTS)KCNQ1基因进行定点突变的研究。方法利用PCR定点突变技术,首先设计两对引物(包含预定的突变),通过3轮PCR扩增,扩增出含有所需突变位点的片段,然后将片段克隆入T载体中,通过酶切连接的方法将突变点引入到pIRES2EGFPKCNQ1中,随后用Effectene转染试剂介导转染HEK293细胞。结果在真核表达载体pIRES2EGFPKCNQ1基础上获得了KCNQ1cDNAC682T的突变体,测序结果表明在序列中发生了预期的突变。结论KCNQ1定点突变体的构建为进一步的功能研究奠定了基础。
Objective To investigate the site-directed mutagenesis of KCNQ1 gene in Long QT Syndrome (LQTS) by polymerase chain reaction (PCR). Methods Two pairs of primers (including the predetermined mutations) were designed by PCR. Two fragments containing the desired mutation were amplified by 3 rounds of PCR. The fragments were cloned into T vector and digested by restriction enzyme digestion The ligation method introduced the mutation point into pIRES2EGFPKCNQ1, followed by HEK293 cells transfected with Effectene transfection reagent. Results Based on the eukaryotic expression vector pIRES2EGFPKCNQ1, a mutant of KCNQ1cDNAC682T was obtained. The sequencing results showed that the expected mutation occurred in the sequence. Conclusion The construction of KCNQ1 site-directed mutants laid the foundation for further functional studies.