一种快捷有效的构建cDNA文库的方法

A Rapid and Efficient Method for Construction of cDNA Library

构建cDNA文库是研究基因组功能基因一种有效的手段，传统的建库方法通常需要在双链cDNA两端分别加上带限制性酶切位点的接头序列，通过酶切使之产生与质粒匹配的粘性末端，然后与质粒连接构成文库。本文介绍一种简单而且不需要核酸内切酶的构建cDNA文库的方法。该方法的主要步骤包括：（1）通过反转录合成cDNA第一条链，通过置换底物的方式在cDNA3’末端合成接头序列。（2）利用cDNA两端的接头引物，通过TaqDNA合成酶PER扩增合成cDNA第二条链。（3）将PCR产生的双链cDNA直接与T-载体连接。（4）将连接物转化大肠杆菌，得到cDNA文库。利用本方法，我们成功构建红树植物红海榄的cDNA文库。PCR结果显示插入的片段分布在O．5～3．0kb之间，大部分cDNA的长度在500—1000bp之间，表明cDNA具有较好的完整性。本方法具有操作简单、高效率、低成本、RNA模板需要量少的特点，而且适用于任何高等生物RNA样品。

Construction of cDNA library is an efficient tool to study functional genes in genomic width. The traditional method of construction of eDNA library requires adding adaptors to each end of eDNA, which provides a specific site for restriction nuclease. In this work, a rapid and efficient method is introduced, which does not required restriction nuclease and DNA ligase. This method includes the following processes： （1） first strand of eDNA was synthesed through reverse transcription, meanwhile, adaptors were added to both end of eDNA; （2） the second strand of eDNA was synthesed by PCR using Taq polymerase, two adaptor sequences served as primers; （3） ds eDNA were directly ligated into T- vectors; （4） the ligated vectors were transformed into E. coli and generated a eDNA library. By this method, cDNA library of Rhizophora stylosa was constructed. PCR showed that the fragment length of eDNA library ranged from 500bp to 3kb and the lengths of the most inserts were 500-1000bp, indicating that the library is highly qualified. The method shown in this work is in simple operation, high efficiency, low cost, and less RNA template. Additionally, it is suitable for all kinds of eukaryotie RNA samples.