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

Genome Res. 2000 Oct;10(10):1617-30. doi: 10.1101/gr.145100.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cloning, Molecular / methods*
  • Cricetinae
  • DNA, Complementary / biosynthesis
  • DNA, Complementary / isolation & purification*
  • Female
  • Gene Library*
  • Male
  • Mice
  • Pregnancy
  • RNA Caps*
  • Reproducibility of Results
  • Sensitivity and Specificity

Substances

  • DNA, Complementary
  • RNA Caps