Vector-capping: A simple method for preparing a high-quality full-length cDNA library
Vector-capping: A simple method for preparing a high-quality full-length cDNA library
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DOI：
10.1093/dnares/12.1.53
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发表时间：
2005-02-28
期刊：
影响因子：
4.1
通讯作者：
Kimura, T
中科院分区：
文献类型：
--
作者：
Kato, S;Ohtoko, K;Kimura, T
全长cDNA在鉴定基因和确定其启动子区域中起着重要作用。在这里，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）它既不包含产生突变的PCR过程，也不包含引起cDNA截短的限制性酶处理，（iii）由于在其5 ′端存在额外的dGMP，确保了cDNA的完整性，（iv）当使用培养的细胞或新鲜组织时，约95%的cDNA克隆是全长的，（v）几微克的总RNA而不进行mRNA纯化足以制备含有>10（5）个独立克隆的文库，和（vi）可以克隆高达9.5kbp的长尺寸全长cDNA。这种方法将加速各种真核生物的全面基因分析。
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.