Identification of rat genes by TWINSCAN gene prediction, RT-PCR, and direct sequencing
Research output: Contribution to journal › Journal article › Research › peer-review
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.
Original language | English |
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Journal | Genome Research |
Volume | 14 |
Issue number | 4 |
Pages (from-to) | 665-71 |
Number of pages | 7 |
ISSN | 1088-9051 |
DOIs | |
Publication status | Published - 2004 |
- 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
Research areas
ID: 43976150