Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes

作者：Carninci P^*; Shibata Y; Hayatsu N; Sugahara Y; Shibata K; Itoh M; Konno H; Okazaki Y; Muramatsu M; Hayashizaki Y

来源：Genome Research, 2000, 10(10): 1617-1630.

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, hull-length cDNA libraries. This procedure can be extended to the preparation of full-length cDNA encyclopedias from other organisms.

• 出版日期2000-10