Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes
Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes
复制标题
DOI：
10.1101/gr.145100
复制
发表时间：
2000-10-01
期刊：
影响因子：
7
通讯作者：
Hayashizaki, Y
中科院分区：
文献类型：
--
作者：
Carninci, P;Shibata, Y;Hayashizaki, Y
在努力准备小鼠全长cDNA百科全书，我们以前开发了几种技术来制备和选择全长cDNA。为了增加不同cDNA的数量，我们在这里介绍了一种策略，在一个单一的步骤中制备标准化和消减cDNA文库。该方法基于第一链全长cDNA与几种RNA驱动因子的杂交，包括作为标准化驱动因子的起始mRNA和来自含有高表达基因的微片段的流出转录物、重排克隆和作为减去驱动因子的先前测序的cDNA。我们的方法使消减/标准化文库中全长cDNA的比例保持较高。此外，我们的方法大大提高了新基因的发现相比，通过使用标准的，外壳长度的cDNA文库获得的结果。该方法也可推广到其它生物的全长cDNA文库的制备。
In the effort to prepare the mouse full-length cDNA encyclopedia, we previously developed several techniques to prepare and select full-length cDNAs. To increase the number of different cDNAs, we introduce here a strategy to prepare normalized and subtracted cDNA libraries in a single step. The method is based on hybridization of the first-strand, full-length cDNA with several RNA drivers, including starting mRNA as the normalizing driver and run-off transcripts from minilibraries containing highly expressed genes, rearrayed clones, and previously sequenced cDNAs as subtracting drivers. Our method keeps the proportion of full-length cDNAs in the subtracted/normalized library high. Moreover, our method dramatically enhances the discovery of new genes as compared to results obtained by using standard, hull-length cDNA libraries. This procedure can be extended to the preparation of full-length cDNA encyclopedias from other organisms.