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武景阳, 李朝翠, 孔清华, 毛炳宇. 2008: 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. 动物学研究, 29(4): 368-372. DOI: 10.3724/SP.J.1141.2008.04368
引用本文: 武景阳, 李朝翠, 孔清华, 毛炳宇. 2008: 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. 动物学研究, 29(4): 368-372. DOI: 10.3724/SP.J.1141.2008.04368
WU Jing-yang, LI Chao-cui, KONG Qing-hua, MAO Bing-yu. 2008: Construction and Analysis of A Directional Xenopus laevis Embryonic cDNA Library for Yeast Two-hybrid Using Modified Random Primers. Zoological Research, 29(4): 368-372. DOI: 10.3724/SP.J.1141.2008.04368
Citation: WU Jing-yang, LI Chao-cui, KONG Qing-hua, MAO Bing-yu. 2008: Construction and Analysis of A Directional Xenopus laevis Embryonic cDNA Library for Yeast Two-hybrid Using Modified Random Primers. Zoological Research, 29(4): 368-372. DOI: 10.3724/SP.J.1141.2008.04368

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

  • 摘要: 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,连接子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。

     

    Abstract: Here we describe a new strategy for cDNA library construction, in which the random primers directing the first strand cDNA synthesis contain additional d (AC) at their 5′-ends, and the linkers which will be added to the double strand cDNA contain d(GTCG) at the 5′-ends. When the linker is added to the 3′-end of the cDNA, a complete SalI site d(GTCGAC) will form at the 3′-end but not at the 5′-end of the cDNA fragment. After SalI digestion, the 3′-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 cDNA 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 cDNA library construction was successful.

     

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