利用退火反应产生粘性末端基因克隆新方法.PDFVIP

江医学2017 年第39 卷第 14期 利用退火反应产生粘性末端的 基因克隆新方法 赵婷婷 曾静静 王启军 褚茂平 m 【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 一 高 、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 o 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 c 性将若干靶基因（P90rsk 、Nprl2、Mios 、Ragc、S6k 、Pkca ）构建在经EcoRV 和NotI 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 s 隆目标基因。 z 【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 xz A new method for gene clone producing cohesive terminals by annealingreaction y China j z 【Abstract 】 Objective 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 w and the ligation products were transformed into DH5 αcompetent cells. Results The target genes (P90rsk, Nprl2, Mios, Ragc, S6k, Pkca) were constructed on vector pcDNA3.1 (+) , which were digested with EcoRV and NotI. The constructedgenes were ver- w ified by single restriction