|Function and cDNA Resources Section
DOE Human Genome Program Contractor-Grantee
97. Novel Approaches to Facilitate Gene Discovery and the Development of a Non-Redundant Arrayed Collection of Full-Length cDNAs
Sergey Malchenko1, Brian Berger1, Vera Da Costa Soares1, Maria De Fatima Bonaldo1, and Marcelo Bento Soares1,2
Departments of 1Pediatrics and 2Physiology and Biophysics, The University of Iowa, Iowa City, IA 52242
(A) Gene Discovery. Serial subtraction of normalized cDNA libraries has proven powerful to expedite gene discovery in large-scale EST programs. This strategy enabled us to generate large non-redundant collections of rat (41,000), mouse brain (22,000), and human (15,700) cDNAs within a two-year time frame, with minimal sequencing effort. This process, however, can only facilitate the identification of mRNAs that are represented in starting libraries. Since the number of primary recombinants needed to guarantee representation of rare mRNAs exceeds those typically attained in standard libraries, it is anticipated that a fraction of such transcripts will not be represented. Furthermore, mRNAs whose expression is limited to a small number of cells within a tissue may also not be appropriately represented in a bulk tissue library regardless of their level of expression. To address this problem, we developed a method aimed at the cloning of mRNAs that are either under- or not-represented in standard normalized libraries. We have applied this procedure to construct a mouse hippocampus cDNA library significantly enriched for rare mRNAs. Enrichment was documented by analysis of over 1,000 ESTs as well as by Southern hybridization of library DNA with a number of cDNA probes that were under-represented in the non-normalized or normalized mouse hippocampus libraries.
(B) Development of non-redundant collections of full-length cDNAs. Full-length-enriched libraries have been and continue to be constructed and made available to the Mammalian Gene Collection program, a trans-NIH initiative to generate high accuracy sequence of large numbers of full-length cDNAs. However, given that enrichments are typically of the order of 50%, some screening strategy is necessary for en masse selection of the full-length clones in these libraries. We are developing novel methods and strategies to address this problem with the goal of generating comprehensive non-redundant collections of arrayed full-length cDNAs.
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