Identification of rat genes by TWINSCAN gene prediction, RT-PCR, and direct sequencing

Genome Res. 2004 Apr;14(4):665-71. doi: 10.1101/gr.1959604.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Computational Biology / methods
  • Genes / genetics*
  • Humans
  • Introns / genetics
  • Open Reading Frames / genetics
  • Predictive Value of Tests
  • Rats
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Sequence Analysis, DNA / methods*
  • Software
  • Untranslated Regions / genetics


  • Untranslated Regions