利用修饰的随机引物构建非洲爪蟾酵母双杂交定向cDNA文库

Construction and Analysis of A Directional Xenopus laevis Embryonic cDNA Library for Yeast Two-hybrid Using Modified Random Primers

描述了一种新的构建cDNA文库的方法,其中用来合成cDNA第一链的随机引物5′-端被加上碱基d(AC),在cDNA双链合成后所添加的连接头的末端含有碱基d(GTCG)。在cDNA双链3′-端,连接头连接上后会形成一个完整的SalI酶切位点d(GTCGAC),而在5′-端几乎不会形成SalI位点。在SalI酶切后利用形成的3′-粘性末端与5′-EcoRI粘性末端一起将cDNA双链定向导入线性化的质粒载体,再通过转化细菌获得cDNA文库。利用此方法构建了一个不同发育时期非洲爪蟾胚胎酵母双杂交cDNA文库。检测了文库中空载体的比例,插入片段大小和不同基因的表达水平几个指标,都符合预期,但是同时发现定位于mRNA的3′-端的插入片段比例比较低。这种倾向性符合文献报道,应该是实验系统的倾向性,不影响进一步的酵母双杂交实验。这些数据证明成功地构建了定向非洲爪蟾酵母双杂交cDNA文库。

Here we describe a new strategy for eDNA library construction, in which the random primers directing the first strand eDNA synthesis contain additional d （AC） at their 5＇-ends, and the linkers which will be added to the double strand eDNA contain d（GTCG） at the 5＇-ends. When the linker is added to the 3＇-end of the eDNA, a complete SalI site d（GTCGAC） will form at the 3＇-end but not at the 5＇-end of the eDNA fragment. After Sall digestion, the T-cohesive and the pre-existing 5＇-EcoRI cohesive ends are used to introduce the double stranded cDNA into the linearized plasmid vectors. The ligation products were used to transform E.coli to produce a cDNA library. Using this method, we constructed a directional Xenopus laevis embryonic eDNA library for yeast two-hybrid. We checked the ratio of empty vectors, the size of inserts and existence of several genes, the results showed that eDNA library construction was successful.