CAJ | 학술논문

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 cDNA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Notch23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pcDNA3.0-3?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。
목적：채용불의뢰련접반응적극륭법，이용T7핵산외절매화류대린산화수식인물극륭Notch2장편단기인。방법장난이확증적Notch2 cDNA적편마서렬(7416 bp)인위분성3단，인물설계시대차3개편단화재체골가적인물진행감기류대린산화수식，용저사인물확증Notch2적3개편단화재체골가。연후용T7핵산외절매분별처리PCR산물，산생4개구유3′호보돌출말단적편단，병장차4개말단돌출적편단퇴화복성，완성Notch2기인극륭。결과경지당응효전영결과현시Notch23개편단화재체골가적PCR산물대소여예기대소상부，경퇴화복성획득적극륭통과PCR、매절화측서진행극륭감정，학정Notch2편마서렬이경삽입도pcDNA3.0-3?Flag재체중。결론이용T7핵산외절매화인물적감기린산화수식진행적불의뢰련접반응적극륭방법능구용우장편단기인적극륭。
Objective To clone the long coding sequence of Notch2 using T7 exonuclease and phosphorothioated primers by the ligation-independent cloning method. Methods The long coding sequence of Notch2 (7416 bp) was arti-ficially divided into three fragments because it was difficult for PCR amplification. The primers, which were used to am-plify three Notch2 fragments and the vector backbone, were phosphorothioated respectively. The three fragments of Notch2 and the vector backbone were amplified by the primers, and the PCR products were digested by T7 exonuclease, and then four fragments with 3′complementary single-stranded overhangs were produced. The complementary ends were annealed, and the Notch2 cloning was performed. Results The result of agarose Gel electrophoresis showed that PCR products of three Notch2 fragments and the vector backbone were consistent to the expectation in size. The recombinant of pcDNA3. 0-3?Flag-Notch2 was confirmed successfully by PCR, enzyme digestion and DNA sequencing. Conclusion The long fragment gene can be cloned successfully by the ligation-independent cloning method using T7 exonuclease and phosphorothioated primers.