High-efficiency full-length cDNA cloning by biotinylated CAP trapper
High-efficiency full-length cDNA cloning by biotinylated CAP trapper
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DOI：
10.1006/geno.1996.0567
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发表时间：
1996-11-01
期刊：
影响因子：
4.4
通讯作者：
Schneider, C
中科院分区：
文献类型：
--
作者：
Carninci, P;Kvam, C;Schneider, C
我们已经设计了一种方法，用于有效地构建高含量的全长cDNA文库的基础上化学引入生物素基团的二醇残基的帽结构的真核mRNA，然后通过RNase I处理，以选择全长cDNA。通过使用链霉亲和素包被的磁珠在帽位点捕获生物素残基进行选择，从而消除不完全合成的cDNA。当用这种方法构建小鼠脑全长cDNA文库时，我们的评估表明，总克隆的95%以上是全长的，并且可以以高效率（1.2 × 10（7）/10 μ g起始mRNA）产生重组克隆。对120个随机挑选的克隆的分析表明起始mRNA群体的无偏表示。(C)出版社：Academic Press，Inc.
We have devised a method for efficiently constructing high-content full-length cDNA libraries based on chemical introduction of a biotin group into the diol residue of the cap structure of eukaryotic mRNA, followed by RNase I treatment to select full-length cDNA. The selection occurs by trapping the biotin residue at the cap sites using streptavidin-coated magnetic beads, thus eliminating incompletely synthesized cDNAs. When this method was used to construct a mouse brain full-length cDNA library, our evaluation showed that more than 95% of the total clones were of full length, and recombinant clones could be produced with high efficiency (1.2 x 10(7)/10 mu g starting mRNA). The analysis of 120 randomly picked clones indicates an unbiased representation of the starting mRNA population. (C) 1996 Academic Press, Inc.