|Identification of rat genes by TWINSCAN gene prediction, RT-PCR, and direct sequencing.
|Year of Publication
|Wu, JQian, Shteynberg, D, Arumugam, M, Gibbs, RA, Brent, MR
|Animals, Computational Biology, Genes, Humans, Introns, Open Reading Frames, Predictive Value of Tests, Rats, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA, Software, Untranslated Regions
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.
|PubMed Central ID
|R01 HG002278 / HG / NHGRI NIH HHS / United States