Oh Jung-Hwa, Kim Yong Sung, Kim Nam-Soon
Laboratory of Human Genomics, Division of Genomics and Proteomics, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-333, Korea.
Exp Mol Med. 2003 Dec 31;35(6):586-90. doi: 10.1038/emm.2003.77.
We have developed an improved method for constructing a full-length cDNA library using small quantity of material by modifying the original oligo-capping method. In our devised method, total RNAs are used in sequential oligo-capping steps directly without preliminary mRNA purification. Using this method, we constructed full- length cDNA libraries from 100 mg of total RNA. These libraries contained 8x10(5) to 8x10(6) independent clones with average insert sizes of 2.0 kb. Moreover, the number of full-length cDNAs containing the translation initiation codon ATG in the constructed libraries was estimated to 60-70%. In addition, 54% of the known cDNAs had a longer 5' end than the corresponding genes in the public database. Our results show that the method can be effectively used to construct full-length enriched cDNA libraries, especially, if starting material is limited.
我们通过改进原始的寡聚帽法，开发出一种利用少量材料构建全长cDNA文库的改进方法。在我们设计的方法中，总RNA无需预先纯化mRNA，可直接用于连续的寡聚帽步骤。使用这种方法，我们从100mg总RNA构建了全长cDNA文库。这些文库包含8×10⁵至8×10⁶个独立克隆，平均插入片段大小为2.0kb。此外，构建的文库中含有翻译起始密码子ATG的全长cDNA数量估计为60 - 70%。另外，54%的已知cDNA的5'端比公共数据库中相应基因的5'端更长。我们的结果表明，该方法可有效用于构建富含全长的cDNA文库，特别是在起始材料有限的情况下。
