Strategies for construction of subtracted libraries enriched for full-length cDNAs and for preferential cloning of rare mRNAs

Marcelo Bento Soares
The University of Iowa
451 Eckstein Medical Research Building
Iowa City, IA 52242
telephone: 319-335-8250
fax: 319-335-9565
email: bento-soares@uiowa.edu
prestype: Platform
presenter: M. Bento Soares

Brian Berger, Sergey Malchenko, Irina Koroleva, Einat Snir, Tammy Kucaba, Maria de Fatima Bonaldo & Marcelo Bento Soares.

Subtracted libraries enriched for full-length cDNAs.
A major challenge of the ongoing NIH Mammalian Gene Collection Program is the identification of sufficient novel full-length cDNAs to enable achieving the yearly full-length sequencing goals of the project. In an effort to assist in the identification of novel full-length cDNAs we have constructed full-length-enriched libraries and we have developed a novel method for generation of subtracted libraries enriched for full-length cDNAs. Conventional subtractive hybridization procedures cannot be applied for full-length-enriched libraries because a truncated clone in the driver population has the potential to subtract its full-length counterpart from the library. Briefly, 100-150 bp single-stranded overhangs are generated at the 5' end of all clones in the library (tracer), for hybridization with a biotinylated driver population comprising representative clones of every sequence contig identified in the starting full-length-enriched library. The subtracted population is purified from the hybrids using streptavidin-coated magnetic beads, repaired and electroporated into bacteria for propagation of a subtracted full-length-enriched library. We have used this method successfully to generate a subtracted full-length-enriched library derived from germinal center B cells.

Preferential cloning of rare mRNAs.
Discovery of rare mRNAs in large-scale EST projects remain difficult and inefficient because of poor representation of such transcripts in cDNA libraries. In an attempt to expedite the identification of rare mRNAs, we developed a novel method for prerential cloning of rare mRNAs. Briefly, mRNA is hybridized with a driver comprising most/all already identified cDNAs and subsequently destroyed with RNAse H. THe remainder intact mRNA is linearly amplified and cloned for production of a library enriched for rare mRNAs. We have used this method to construct a mouse cDNA library enriched for rare mRNAs from hippocampus. The efficacy of our method was demonstrated by sequencing and by microarray hybridization analises.