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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Utilization of normalized libraries in cDNA selection experiments to identify transcribed sequences in genomic DNA

Marcelo Bento Soares[1], Maria de Fatima Bonaldo[1], Pierre Jelenc[1], Lee Lawton[1], Long Su[1] & 2Argiris Efstratiadis[2]
[1]Department of Psychiatry and [2]Genetics & Development, Columbia University and [1]The New York State Psychiatric Institute

We have further optimized the method that we developed for construction of normalized directionally cloned cDNA libraries [1] and we have used it to generate a human fetal liver and spleen library of 5 million recombinants. In order to assess the quality of this normalized library we performed a number of screenings (colony hybridization) of both starting and normalized libraries with cDNA probes representing mRNAs that occur at a wide range of frequencies in the starting library. The results that were obtained clearly indicated that normalization was successful.

We have also used this library in cDNA selection experiments to isolate transcribed sequences from within a 600 kb YAC contig spanning the Spinal Muscular Atrophy critical region [2]. Approximately 30 different cDNAs were identified and confirmed to map to this region, which corresponds to an average spacing of one gene every 20 kb. cDNA selection was carried out according to a method that we developed [3] and successfully utilized to isolate a number of chromosome 13-specific cDNAs.

As part of the quality control that we routinely perform on our libraries, we have generated a total of about 1,100 ESTs, all of which have been deposited in Genbank. These sequences were derived from a number of libraries that were constructed while we were developing this method for normalization, as well as from an infant brain and the fetal liver and spleen normalized libraries.

[1] Soares, M.B., Bonaldo, M.F., Su, L., Lawton, L. & Efstratiadis, A. Construction and characterization of a normalized cDNA library (1994). Proc. Natl. Acad. Sci. USA 91(20), 9228-9232.
[2] Kindly provided by Dr. Conrad Gilliam, Department of Genetics & Development, Columbia University.
[3]Bonaldo, M.F., Yu, M-T., Jelenc, P., Brown, S., Su, L, Lawton, L, Deaven, L., Efstratiadis, A., Warburton, D., & Soares, M.B. (1994). Selection of cDNAs using chromosome-specific genomic clones: application to Human Chromosome 13. Hum. Mol. Genet. 3 (9).