对帽式捕获法筛选的cDNA进行标准化和扣除，以制备全长cDNA文库，用于快速发现新基因。
Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes.
作者信息
Carninci P, Shibata Y, Hayatsu N, Sugahara Y, Shibata K, Itoh M, Konno H, Okazaki Y, Muramatsu M, Hayashizaki Y
机构信息
Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center, Tsukuba, Japan.
出版信息
Genome Res. 2000 Oct;10(10):1617-30. doi: 10.1101/gr.145100.
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, full-length cDNA libraries. This procedure can be extended to the preparation of full-length cDNA encyclopedias from other organisms.
在致力于构建小鼠全长cDNA文库的过程中，我们之前开发了多种制备和筛选全长cDNA的技术。为了增加不同cDNA的数量，我们在此引入一种一步制备标准化和消减cDNA文库的策略。该方法基于第一链全长cDNA与多种RNA驱动子的杂交，包括作为标准化驱动子的起始mRNA以及来自包含高表达基因的微型文库、重排克隆和先前测序的cDNA的延伸转录本作为消减驱动子。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，与使用标准全长cDNA文库获得的结果相比，我们的方法显著提高了新基因的发现率。该程序可扩展到从其他生物体制备全长cDNA文库。
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