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pBluescriptR Library Method Overview

Dr. Michael Brownstein, National Institute of Mental Health, NIH, Bethesda, MD and by Drs. Shiraki Toshiyuki and Piero Carninci (RIKEN) constructed libraries in the vector pBluescriptR. The protocols used were based on the papers referenced below. Briefly, normalized and subtracted cDNA libraries were prepared in a single step, to increase the number of different cDNAs. This method:
  • Was based on hybridization of the first-strand, full-length cDNA with several RNA drivers including:
    • starting mRNA as the normalizing driver
    • run-off transcripts from mini-libraries containing highly expressed genes, rearrayed clones, and previously sequenced cDNAs as subtracting drivers
  • Kept the proportion of full-length cDNAs in the subtracted/normalized library high.
  • Enhanced the discovery of new genes as compared to results obtained by using standard, full-length cDNA libraries.

References:

  1. Margolin J. Of Mice, Men, and the Genome. Genome Res. 2000 Oct;10(10):1431-2.
  2. Carninci P, Shibata Y, Hayatsu N, Itoh M, Shiraki T, Hirozane T, Watahiki A, Shibata K, Konno H, Muramatsu M, Hayashizaki Y. Balanced-size and long-size cloning of full-length, cap-trapped cDNAs into vectors of the novel lambda-FLC family allows enhanced gene discovery rate and functional analysis. Genomics 2001 Sep;77(1/2):79-90.
  3. Shibata Y, Carninci P, Watahiki A, Shiraki T, Konno H, Muramatsu M, Hayashizaki Y. Cloning full-length, cap-trapper-selected cDNAs by using the single-strand linker ligation method. Biotechniques 2001 Jun;30(6):1250-4.