Normalization and Subtraction of Cap-Trapper-Selected cDNAs to Prepare Full-Length cDNA Libraries for Rapid Discovery of New Genes
Abstract
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.
Footnotes
-
↵1 Corresponding author.
-
E-MAIL rgscerg{at}rtc.riken.go.jp or carninci{at}rtc.riken.go.jp; FAX 81-298-369098.
-
Article and publication are at www.genome.org/cgi/doi/10.1101/gr.145100.
-
- Received April 20, 2000.
- Accepted July 24, 2000.
- Cold Spring Harbor Laboratory Press