摘要
目的利用聚合酶链反应技术介导PRKAG2基因cDNA上第994和995位碱基的体外定点突变。方法以PRKAG2基因为模板,利用PCR技术进行定点突变,设计2对引物,将突变位点设计在引物上,通过PCR扩增引物,扩增片段上含有所需要的突变位点,并将其克隆到TA载体,通过序列分析进行确证。结果序列分析提示,994处碱基由A→C,995处碱基由G→A,使PRKAG2基因第302位密码子由精氨酸(Arg)突变为谷氨酰胺(Gin)。结论PCR技术诱导定点突变准确、高效,成功完成了PRKAG2基因的体外定点突变,为进一步研究该突变位点的功能奠定了基础。
Objective To investigate the site-directed mutagenesis at 994bp and 995bp of the PRKAG2 induced by PCR eDNA in vitro. Methods The site-directod mutagenesis of PRKAG2 gene was made by PCR. Two sets of primers were designed according to the sequence of the PRKAG2 eDNA, and mismatch was introduced into primers. Mutagenesis was performed in a two-step PCR. The amplified fragment contained the mutation site. The PCR product was cloned into TA cloning vector. Results The sequencing analysis showed that the mutation site was correct. Mutations from A to C 994 and G to A 995 sites of PRKAG2 eDNA were found. Conclusion PCR site-diroeted mutagenesis method is accurate and highly efficient. The construction of PRKAG2 site-directed mutant establishes the base of its further functional study.
出处
《中国心血管病研究》
CAS
2009年第5期381-383,共3页
Chinese Journal of Cardiovascular Research
关键词
心脏遗传病
PRKAG2基因
聚合酶链反应
定点突变
Cardiac inherited disease
PRKAG2
Polymerase chain reaction
Site-directed mutagenesis