Dr. Marcelo B. Soares
451 Eckstein Medical Research Building
Iowa City, IA 52242
Phone: 319-335-8250
Fax: 319-335-9565
Email: bento-soares@uiowa.edu
Projects are approved by an IRB located at: The University of Iowa.
The approving IRB operates under a Multiple Project Assurance (MPA) recognized by DOE or by the Department of Health and Human Services (HHS).
MPA number of the IRB: M1080
Number of Human Subjects Projects reported: 1
Project Identifier: UI-97-ER61233
Project Title:
Development and Application of Subtractive Hybridization Strategies to Facilitate Gene Discovery (See: Project CU-91-2923R in FY96 Database)
Principal Investigator:
Dr. Marcelo B. Soares
Project started in: 1997
Project Funding Information:
Project received funding in Fiscal Year 1997.
Project used human subjects in Fiscal Year 1997.
Funding Sources:
Human subjects (e.g. pathological specimens) obtained for reasons not related to this project were provided by collaborators at no cost to this project
Project involves use of multiple protocols/subprojects.
Number of protocols/subprojects associated with this project: 1
Protocol/Subproject # 1
Protocol/Subproject Identifier: 9703225
IRB Review:
Type of Review: Expedited
Most Recent Approval: April 01, 1997
IRB Approval Number: 9703225
Number of Human Subjects who participated in this project/protocol during
FY 1997 (10/1/96 - 9/30/97): 5
Type of Human Subjects Involvement:
OBJECTIVES AND METHODOLOGIES
The methods we originally developed to normalize directionally cloned cDNA libraries (Soares et al., 1994; Bonaldo, Lennon & Soares, 1996) have been successfully utilized to generate a number of human, mouse and rat cDNA libraries. All human and mouse libraries (and soon the rat libraries as well) have been contributed to the I.M.A.G.E. consortium and they have been extensively used for large scale generation of expressed sequence tags (ESTs). Both the ESTs and their respective clones are publicly available. Although the use of normalized libraries has proven most advantageous to minimize the redundant identification of the mRNAs of the super-prevalent and intermediate frequency classes within a particular tissue, it cannot prevent the redundant identification of mRNAs (of any frequency class) that are expressed in multiple tissues. In other words, normalization alone cannot avoid the redundant identification of ESTs that have been obtained previously from other libraries. This problem is becoming increasingly more relevant as we approach completion of the ongoing human and mouse gene discovery efforts. Hence, we proposed to take advantage of subtractive hybridization strategies that we developed to generate libraries enriched for novel cDNAs. Briefly, pools of I.M.A.G.E. clones from which ESTs have been derived, are used as drivers in hybridizations with single or multiple normalized libraries, thus, generating subtracted libraries enriched for cDNAs not yet represented in public databases. Subtracted libraries are characterized by Southern hybridization to assess reduction in representation of clones of the driver population and are then contributed to the I.M.A.G.E. consortium for large-scale arraying and sequencing. Sequence analysis of two subtracted libraries that we have generated indicated a four-fold reduction in representation of the driver population. It is anticipated that the use of subtracted libraries will become increasingly advantageous as we strive towards the ultimate goal of identifying all human and mouse genes.
This project has two major goals: (1) to optimize the method for construction of subtracted libraries, and (2) to generate subtracted libraries to facilitate the ongoing human and mouse EST programs.
INVOLVEMENT OF HUMAN SUBJECTS Human tissues (e.g. pathological specimens) obtained for purposes not related to this project have been provided by collaborators for extraction of RNA which in turn is used for construction of cDNA libraries. Information that could lead to patient identification is not made available to us.