Kato Seishi, Ohtoko Kuniyo, Ohtake Hideki, Kimura Tomoko
Department of Rehabilitation Engineering, Research Institute, National Rehabilitation Center for Persons with Disabilities, 4-1 Namiki, Tokorozawa, Saitama 359-8555, Japan.
DNA Res. 2005 Feb 28;12(1):53-62. doi: 10.1093/dnares/12.1.53.
Full-length cDNAs play an essential role in identifying genes and determining their promoter regions. Here we describe a simple method for constructing a full-length cDNA library, which has the following advantages: (i) it consists of only three steps including direct ligation between a vector and a cDNA strand using T4 RNA ligase, (ii) it contains neither a PCR process generating mutations nor restriction enzyme treatment causing truncation of cDNA, (iii) the intactness of cDNA is assured due to the presence of an additional dGMP at its 5' end, (iv) approximately 95% of cDNA clones are full-length when cultured cells or fresh tissues are used, (v) several micrograms of total RNA without mRNA purification is sufficient for preparation of a library containing >10(5) independent clones, and (vi) a long-sized full-length cDNA up to 9.5 kbp can be cloned. This method will accelerate comprehensive gene analysis in a variety of eukaryotes.
全长cDNA在基因鉴定及其启动子区域的确定中发挥着重要作用。在此，我们描述了一种构建全长cDNA文库的简单方法，该方法具有以下优点：（i）它仅由三个步骤组成，包括使用T4 RNA连接酶在载体和cDNA链之间直接连接；（ii）它既不包含会产生突变的PCR过程，也不包含会导致cDNA截短的限制酶处理；（iii）由于在其5'端存在额外的dGMP，确保了cDNA的完整性；（iv）当使用培养细胞或新鲜组织时，约95%的cDNA克隆是全长的；（v）无需纯化mRNA，几微克的总RNA就足以制备一个包含>10⁵个独立克隆的文库；（vi）可以克隆长达9.5 kbp的长链全长cDNA。该方法将加速对各种真核生物的全面基因分析。
