We are proposing to construct a reference normalized human cDNA catalogue in which the great majority of the expressed genes should be represented. The catalogue will comprise a number of different normalized cDNA libraries from a variety of human tissues and stages of development. In this catalogue, each library component will have a characteristic sequence identifier (tissue/temporal-specific IDs), provided by the oligonucleotide utilized to prime first strand cDNA synthesis, which will be unique to each library. All cDNA libraries will be constructed by directional cloning into a phagemid vector according to an established protocol (Soares, 1993) and normalized individually. This collection of normalized libraries will then be pooled and re-normalized to generate the catalogue. Because each library will have a characteristic sequence identifier, the origin of any clone within the catalogue will be known. A series of subtractive hybridization experiments involving each individual normalized library (or combinations of them) and the cDNA catalogue will be performed to subdivide the catalogue into a number of sublibraries according to the tissue and/or temporal-specificity of their components. It is noteworthy that because each library will have a specific sequence identifier, it will be possible to assess the tissue (or temporal)-specificity of any subtracted library by single pass sequencing of a random sampling of clones.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
1R01HG000980-01
Application #
2209220
Study Section
Genome Study Section (GNM)
Project Start
1994-06-10
Project End
1997-05-31
Budget Start
1994-06-10
Budget End
1995-05-31
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Psychiatry
Type
Schools of Medicine
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10027
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Bonaldo, M F; Lennon, G; Soares, M B (1996) Normalization and subtraction: two approaches to facilitate gene discovery. Genome Res 6:791-806
Yoon, S J; LeBlanc-Straceski, J; Ward, D et al. (1994) Organization of the human keratin type II gene cluster at 12q13. Genomics 24:502-8