title: 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.
authors.affiliation: Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: Jia Qian
authors.initials: JQ
authors.lastname: Wu
authors.forename: David
authors.initials: D
authors.lastname: Shteynberg
authors.forename: Manimozhiyan
authors.initials: M
authors.lastname: Arumugam
authors.forename: Richard A
authors.initials: RA
authors.lastname: Gibbs
authors.forename: Michael R
authors.initials: MR
authors.lastname: Brent
mesh_terms: D000818:Animals; D019295:Computational Biology / Q000379:methods; D005796:Genes / Q000235:genetics*; D006801:Humans; D007438:Introns / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D011237:Predictive Value of Tests; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software; D020506:Untranslated Regions / Q000235:genetics
chemical_list: D020506:Untranslated Regions