【摘要】 目的:利用结合单酶切位点的融合PCR技术对癫痫相关基因SCN1A进行定点突变。方法:首先设计两对引物PF1/PR1和PF2/PR2,PF1和PR2均位于突变位点最近的单酶切位点处,而突变位点设计在第一对反向引物(PR1)和第二对正向引物上(PF2)。通过重叠延伸法两次PCR扩增:第一次用PF1/PR1和PF2/PR2分开扩增,以扩增产物作模板,PF1/PR2作引物进行融合PCR,得到的扩增产物即含有所需要的突变位点,最后将扩增片段克隆入pMD18-T载体,经测序筛选阳性克隆。结果:DNA测序表明SCN1A基因所编码的第946位密码子由精氨酸(Arg)突变为组氨酸(His),再通过酶切和连接反应将重组质粒上的突变片段替换SCN1A表达质粒上的对应片段,成功构建了SCN1A突变载体。结论:与现在常用的长距离PCR定点诱导突变相比较,结合单酶切位点的融合PCR定点突变技术具备扩增距离短的优点,大大降低了自发突变的概率,适合于大肠杆菌中易自发突变的较大载体的定点诱变。更多还原

【Abstract】 Objective:Fusion PCR coupling monocloning sites is used to make site-directed mutagenesis of epilepsy-related gene SCN1A in vitro.Methods: Two sets of primers PF1/PR1 and PF2/PR2 were designed to make mutation: There were two monocloning sites on primer PF1 and PR2 and the mutation site on primer PR1 and PF2.Mutagenesis was performed in a two-step PCR.The first step was performed in two reactions with PF1/PR1 and PF2/PR2 respectively and the Fusion PCR was performed with PF1/PR2 using the first two PCR products as templates.The Fusion PCR fragment which contained the mutation site was subcloned into the pMD18-T vector.The correct clones were screened by sequencing analyses.Results:The sequencing results showed R946H mutation was obtained,and then,the fragment from SCN1A-expressing construct was replaced by mutant fragment by a series of enzyme-digesting and ligation reactions.We successfully constructed the vector expressing SCN1A mutant R946H.Conclusion:Comparing to long distance PCR,Fusion PCR coupling monocloning sites is suitable for site-directed mutagenesis of unstable and large plasmids,because short fragment amplified by PCR will reduce efficiency of unexpected mutations.更多还原