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

In the effort to prepare the mouse full-length cDNA encyclopedia, we previo usly 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 meth od is based on hybridization of the first-strand, full-length cDNA with sev eral RNA drivers, including starting mRNA as the normalizing driver and run -off transcripts from minilibraries containing highly expressed genes, rear rayed clones, and previously sequenced cDNAs as subtracting drivers. Our me thod 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 cD NA libraries. This procedure can be extended to the preparation of full-len gth cDNA encyclopedias from other organisms.