[Multiplex mapping of human cDNAs]. Technical progress report
J. Craig Venter, National Institute of Neurological Disorders and Stroke, has begun to identify genes expressed in the human brain by partially sequences cDNA clones. We are collaborating with the Venter group and using their sequence data to develop methods for rapid localization of newly identified cDNAs to human chromosomes. We are applying the ABI automated DNA sequencer to the analysis of fluorescently-tagged PCR products for assigning sequences to individual human chromosomes. The steps in our mapping protocol are (1) to design PCR primers from the Venter laboratory-generated sequence data, (2) to test the primers for specific amplification from human genomic DNA, (3) to use the primers for PCR amplification from a somatic cell hybrid cell mapping panel, (4) to determine the presence or absence of the specific amplification products from each cell line DNA by electrophoretic analysis using the ABI sequencer, and (5) to analyze the pattern of amplification results from the hybrid panel to identify the chromosomal origin of the cDNA sequence. We have demonstrated the principle by mapping 12 sequences or ``Expressed Sequence Tags`` (ESTs), providing primer sequence data for subsequent subchromosomal localizations. We will now concentrate on developing methodology to allow multiplexing the amplification reactions and analysis of the reaction products, to achieve a high throughput with a minimum allocation of resources. This project will generate a data set from which to evaluate strategies to identify functional primer sequences from cDNA sequence data.
- Research Organization:
- American Type Culture Collection, Rockville, MD (United States)
- Sponsoring Organization:
- USDOE, Washington, DC (United States)
- DOE Contract Number:
- FG05-91ER61232
- OSTI ID:
- 10113038
- Report Number(s):
- DOE/ER/61232--1; ON: DE92005523
- Country of Publication:
- United States
- Language:
- English
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(Multiplex mapping of human cDNAs)
[Multiplex mapping of human cDNAs]. Technical progress report