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Selecting open reading frames from DNA.

Paola Zacchi1, Daniele Sblattero, Fiorella Florian

  • 1SISSA, Trieste, Italy.

Genome Research
|May 3, 2003
PubMed
Summary
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This study presents a novel method for selecting functional open reading frames (ORFs) from DNA using a phage display system. This technique efficiently identifies protein-coding sequences for various applications.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Selecting functional DNA sequences from complex genetic material is crucial for various biological applications.
  • Existing methods may lack efficiency or specificity in identifying open reading frames (ORFs).

Purpose of the Study:

  • To develop and validate a method for selecting DNA encoding functional ORFs from noncoding DNA.
  • To demonstrate the utility of this method in a phage display system and its potential for other applications like the yeast two-hybrid system.

Main Methods:

  • Cloning DNA fragments upstream of a fusion gene (beta-lactamase and fd phage gene 3) within a specific vector.
  • Utilizing ampicillin resistance conferred by functional ORFs for selection.
  • Employing Cre recombinase to remove the beta-lactamase gene post-selection.

Related Experiment Videos

  • Testing the vector with a plasmid containing tissue transglutaminase and analyzing surviving clones via sequencing and immunological detection.

    • Main Results:

    • All sequenced surviving clones contained functional ORFs.
    • 83% of identified ORFs were localized to known genes.
    • At least 80% of selected ORFs produced immunologically detectable polypeptides.
    • Specific monoclonal antibodies successfully identified clones with the correct epitope (anti-tTG).

    Conclusions:

    • The described method efficiently selects functional ORFs from DNA libraries.
    • This approach is adaptable for various systems beyond phage display, including the yeast two-hybrid system.
    • The method facilitates the selection of random ORFs for applications such as identifying the coding potential of whole organisms.