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A nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins.

T H Tan1

  • 1PRI/DynCorp, NCI-Frederick Cancer Research and Development Center, Maryland 21702.

Analytical Biochemistry
|January 1, 1991
PubMed
Summary
This summary is machine-generated.

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A new nonradioactive screening method effectively clones genes for DNA binding proteins. This digoxigenin-based technique replaces radioactive probes, enabling efficient gene discovery.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Traditional cloning of DNA binding proteins often relies on radioactive probes, posing safety and disposal concerns.
  • Previous methods utilized radioactively labeled DNA probes to identify proteins with specific DNA recognition sequences.

Purpose of the Study:

  • To develop a novel, nonradioactive screening method for cloning genes that encode sequence-specific DNA binding proteins.
  • To adapt existing protocols by replacing radioactive labeling with a digoxigenin-based hapten system.

Main Methods:

  • Developed a nonradioactive strategy using digoxigenin-labeled DNA probes.
  • Expressed fusion proteins from recombinant bacteriophage lambda gt11/lambda ZAP.
  • Immobilized fusion proteins on nitrocellulose filters and probed with digoxigenin-labeled DNA.

Related Experiment Videos

  • Detected specifically bound DNA probes via antibody-enzyme conjugate and enzyme substrates.

    • Main Results:

    • Successfully implemented a nonradioactive screening technique for identifying DNA binding proteins.
    • Demonstrated the efficacy of digoxigenin labeling as a substitute for radioactive probes.
    • Isolated several cDNA clones encoding DNA binding proteins using this novel method.

    Conclusions:

    • The developed nonradioactive method is a viable and effective alternative for cloning DNA binding proteins.
    • This technique enhances safety and simplifies the screening process in molecular cloning.
    • The method facilitates the discovery of novel DNA binding proteins and their corresponding genes.