Carninci P, Kvam C, Kitamura A, Ohsumi T, Okazaki Y, Itoh M, Kamiya M, Shibata K, Sasaki N, Izawa M, Muramatsu M, Hayashizaki Y, Schneider C
Genome Science Laboratory, Tsukuba Life Science Center, Ibaraki, Japan.
Genomics. 1996 Nov 1;37(3):327-36. doi: 10.1006/geno.1996.0567.
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 micrograms starting mRNA). The analysis of 120 randomly picked clones indicates an unbiased representation of the starting mRNA population.
我们设计了一种方法，通过将生物素基团化学引入真核mRNA帽结构的二醇残基，然后用核糖核酸酶I处理以选择全长cDNA，从而高效构建高含量全长cDNA文库。选择过程是通过使用链霉亲和素包被的磁珠捕获帽位点的生物素残基来实现的，从而消除未完全合成的cDNA。当使用该方法构建小鼠脑全长cDNA文库时，我们的评估表明，总克隆中超过95%是全长的，并且可以高效产生重组克隆（每10微克起始mRNA产生1.2×10⁷个）。对120个随机挑选的克隆进行分析表明，起始mRNA群体得到了无偏差的呈现。
