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Related Experiment Videos

ORF-FINDER: a vector for high-throughput gene identification.

Irene T Rombel1, Kathryn F Sykes, Simon Rayner

  • 1Center for Biomedical Inventions, Department of Internal Medicine, University of Texas-Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9185, USA. irene.rombel@utsouthwestern.edu

Gene
|January 30, 2002
PubMed
Summary
This summary is machine-generated.

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We created ORF-FINDER, a system to efficiently identify open reading frames (ORFs) in fragmented DNA. This method enriches for gene-coding sequences, aiding genome analysis and gene discovery.

Area of Science:

  • Molecular Biology
  • Genomics
  • Bioinformatics

Background:

  • Identifying open reading frames (ORFs) is crucial for genome annotation and gene discovery.
  • Existing methods for ORF selection can be inefficient or lack specificity.
  • Fragmented genomic DNA presents challenges for accurate ORF identification.

Purpose of the Study:

  • To develop a simple and efficient system for selecting ORFs from fragmented genomic DNA.
  • To create accompanying software for optimizing insert size and minimizing non-coding sequence selection.
  • To demonstrate the system's effectiveness in enriching for ORFs from complex genomes.

Main Methods:

  • Developed ORF-FINDER vectors with a translational start site out of frame with Green Fluorescent Protein (GFP).

Related Experiment Videos

  • Insertion of DNA fragments in frame with GFP, lacking stop codons, results in ORF-GFP fusion protein expression.
  • Utilized GeneWorks and GenomeAnalyzer software to predict optimal insert sizes for ORF selection.

    • Main Results:

    • Achieved a 25-fold enrichment of ORFs from cloned yeast genomic DNA.
    • Demonstrated successful isolation of ORFs from complex eukaryotic parasite genomes.
    • Validated the system's ability to maximize gene-coding ORFs and minimize non-coding ones.

    Conclusions:

    • The ORF-FINDER system provides a simple and efficient method for selecting ORFs from fragmented genomic DNA.
    • ORF-FINDER facilitates genome sequencing projects, gene building, and the construction of ORF-enriched expression libraries.
    • This technology enhances the ability to identify and analyze coding sequences in diverse genomic contexts.