Identification of Rat Genes by TWINSCAN Gene Prediction, RT–PCR, and Direct Sequencing
Abstract
The publication of a draft sequence of a third mammalian genome—that of the rat—suggests a need to rethink genome annotation. New mammalian sequences will not receive the kind of labor-intensive annotation efforts that are currently being devoted to human. In this paper, we demonstrate an alternative approach: reverse transcription-polymerase chain reaction (RT–PCR) and direct sequencing based on dual-genome de novo predictions from TWINSCAN. We tested 444 TWINSCAN-predicted rat genes that showed significant homology to known human genes implicated in disease but that were partially or completely missed by methods based on protein-to-genome mapping. Using primers in exons flanking a single predicted intron, we were able to verify the existence of 59% of these predicted genes. We then attempted to amplify the complete predicted open reading frames of 136 genes that were verified in the single-intron experiment. Spliced sequences were amplified in 46 cases (34%). We conclude that this procedure for elucidating gene structures with native cDNA sequences is cost-effective and will become even more so as it is further optimized.
Footnotes
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Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.1959604.
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↵3 Corresponding author. E-MAIL brent{at}cse.wustl.edu; FAX (314) 935-7302.
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- Accepted November 17, 2003.
- Received September 11, 2003.
- Cold Spring Harbor Laboratory Press
