利用T7核酸外切酶和硫代磷酸化修饰引物克隆长片段基因的研究

发布时间：2018-06-25 00:56

本文选题：Notch基因 + 长片段　；参考：《解放军医药杂志》2015年08期

【摘要】：目的采用不依赖连接反应的克隆法,利用T7核酸外切酶和硫代磷酸化修饰引物克隆Notch2长片段基因。方法将难以扩增的Notch2 c DNA的编码序列(7416 bp)人为分成3段,引物设计时对此3个片段和载体骨架的引物进行碱基硫代磷酸化修饰,用这些引物扩增Notch2的3个片段和载体骨架。然后用T7核酸外切酶分别处理PCR产物,产生4个具有3'互补突出末端的片段,并将此4个末端突出的片段退火复性,完成Notch2基因克隆。结果琼脂糖凝胶电泳结果显示Notch2 3个片段和载体骨架的PCR产物大小与预期大小相符,经退火复性获得的克隆通过PCR、酶切和测序进行克隆鉴定,确定Notch2编码序列已经插入到pc DNA3.0-3*Flag载体中。结论利用T7核酸外切酶和引物的碱基磷酸化修饰进行的不依赖连接反应的克隆方法能够用于长片段基因的克隆。
[Abstract]:Objective to clone the Notch2 long fragment gene using T7 nucleic acid exonuclease and thiophosphorylation modified primers. Methods the coding sequence of Notch2c DNA (7416 BP) which was difficult to amplify was divided into three segments. The primers were designed to modify the three fragments and the primer of the vector skeleton. The primers were used to amplify the three fragments of Notch2 and the framework of the vector. Then the PCR products were treated with T7 nucleic acid exonuclease to produce four fragments with 3 'complementary protruding ends. The four fragments were annealed and renatured to complete the cloning of Notch2 gene. Results the results of agarose gel electrophoresis showed that the PCR products of Notch23 fragments and vector skeleton were the same size as expected size. The clones obtained by annealing renaturation were identified by PCR, restriction endonuclease digestion and sequencing. It was confirmed that the Notch2 coding sequence had been inserted into the PC DNA 3.0-3 Flag vector. Conclusion the ligation-independent cloning method using the base phosphorylation modification of T7 exonuclease and primer can be used for the cloning of long fragment genes.
【作者单位】：首都医科大学生物化学与分子生物学系;
【分类号】：R3416

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