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Methods

Identification of Rat Genes by TWINSCAN Gene Prediction, RT–PCR, and Direct Sequencing

¹ Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA ² Laboratory for Computational Genomics, Washington University, St. Louis, Missouri 63130, USA

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.

³ Corresponding author.
E-MAIL brent{at}cse.wustl.edu; FAX (314) 935-7302.

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