定点突变技术是指通过聚合酶链式反应(PCR)等方法对DNA片段中特定位置碱基进行替换,删除或添加新的碱基的技术。体外定点突变技术是当前生物,医学各领域研究中的一种重要的实验手段,能够用来探究蛋白质结构与功能之间的关系,优化基因表达,改造载体等目的。小编今天为大家介绍一种常用的体外定点突变的方法,QuikChange Site-directed Mutagenesis。
QuikChange方法以质粒作为模板,经过PCR反应,Dpn I 酶切和转化三个步骤,能够在一天内完成定点突变实验。当然在实验以前,我们需要设计包含有目的突变点的引物。下面小编将简单介绍引物设计,PCR反应,Dpn I 酶切以及转化这几个步骤。详细内容可参考Agilent QuikChange II XL Site-Directed Mutagenesis Kit 手册。
引物设计
引物应该是一对互补的寡核苷酸链,其中含有目的突变位点。引物长度在25-45碱基之间, 引物的Tm值要大于78 oC,长于45个碱基的引物将有可能形成引物二聚体,从而影响突变反应的效率。目的突变点应该位于引物的中间位置,突变位点两侧各有10-15个与模板一致碱基。另外引物的GC含量至少应为40%, 并且有至少一个G或C碱基位于末端。小编推荐利用Agilent官网中的软件来设计引物,www.agilent.com/genomics/qcpd
PCR 反应
PCR反应需要考虑两个方面:一个是PCR反应体系,另一个是PCR反应条件。在设计PCR反应体系时,需要注意的是引物的浓度。反应体系中引物需要过量,通常一个50 μL的反应体系内应该含有超过125g的引物。在首次尝试设计反应体系时,可以加入10 ng的质粒模板,如果反应没有成功,需要优化模板的用量,范围应该在5 ng-50 ng之间。设计PCR反应条件时,常选择5 min的延伸时间,18个循环。循环数大于18, 将有可能引入非特异突变。
Dpn I 酶切
Dpn I限制性内切酶用来降解甲基化的模板DNA以避免后续转化可能导致的假阳性克隆。通常情况下,50 μL反应体系在中加入1 μL Dpn I酶,37 oC放置1 h 即可消除模板DNA。
转化
Quikchange方法的好处是不需要进行再次克隆,可以直接将Dpn I酶切的产物转化进入大肠杆菌的感受态细胞中。转化采用常规的热激方法即可。转化后平板需要在培养箱内放置不少于16 h。最后挑选阳性克隆,测序进行验证。在实验过程中,可能会遇到各种问题,比如转化效率很低,突变效率不高,出现假阳性克隆等等,详细解决对策可点击本链接。
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“通常一个50 μL的反应体系内应该含有超过125g的引物”这句话中的125g 是125克吗,是不是单位错了呀