解放军医药杂志Issue(8)：51-55,5.DOI:10.3969/j.issn.2095-140X.2015.08.013
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
Research of Long Fragment Gene Cloning Using T7 Exonuclease and Phosphorothioated Primers
兰泓 1张玉祥1
作者信息
- 1. 100069 北京，首都医科大学生物化学与分子生物学系
- 折叠
摘要
Abstract
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.关键词
Notch2基因/长片段/不依赖连接反应/T7核酸外切酶/引物硫代磷酸化修饰Key words
Notch2 gene/Long fragment/Ligation-independent cloning/T7 exonuclease/Phosphorothioated primer分类
医药卫生引用本文复制引用
兰泓,张玉祥..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[J].解放军医药杂志,2015,(8):51-55,5.
