作　　者： (赵婷婷）; (曾静静）; (王启军）; (褚茂平）;

机构地区： 温州医科大学附属第二医院/育婴儿童医院儿科,325027

出　　处： 《浙江医学》 2017年第14期1166-1169,I0002,共5页

摘　　要： 目的通过引物设计和退火反应产生内切酶的粘性末端,旨在开发一种高效、简单、可靠的分子克隆方法。方法选定限制性内切酶(平末端或者粘性末端)设计3条或4条引物,同时进行两次独立的聚合酶链式反应(PCR),随后把两次PCR产物回收后混在一起经过解链退火,再将其与双酶切的载体质粒进行连接反应,最后转化即可得到目的克隆。结果利用此方法成功一次性将若干靶基因(P90rsk、Nprl2、Mios、Ragc、S6k、Pkca)构建在经Eco RV和Not I双酶切后的pcDNA3.1(+)载体上,经酶切及测序验证。结论该方法无需考虑靶基因内部是否存在酶切位点,可省去PCR产物酶切及之后的纯化回收过程,可快速并准确地克隆目标基因。 jective To develop a new effective gene clone method by designing three or four primers to produce cohesive terminals.Methods Two independent PCRs were run based on these designed primers,mixed the two PCR products together,and then went through denaturation and annealing.After that,the mixed products were ligated with the digested vector and the ligation products were transformed into DH5 α competent cells.Results The target genes （P90rsk,Nprl2,Mios,Ragc,S6k,Pkca） were constructed on vector pcDNA3.1 （＋）,which were digested with EcoRV and Notl.The constructedgenes were verified by single restriction digestion and sequencing.Conclusion The developed method overcomes the limitation of restriction endonuclease sites present in the target genes and can also omit the digestion process of the PCR products and subsequent purification.

关 键 词： 分子克隆 聚合酶链反应 退火 限制性酶切位点 粘性末端