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A sensitive method for the determination of protein-DNA binding specificities.
1Imperial Cancer Research Fund Laboratories, Lincoln's Inn Fields, London, UK.
Nucleic Acids Research
|November 11, 1990
Summary
This study presents a sensitive method to identify DNA binding protein sequences using limited protein amounts. The technique employs Polymerase Chain Reaction (PCR) and immunoprecipitation for efficient DNA site recovery.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Determining DNA sequence specificity of proteins is crucial for understanding gene regulation.
- Existing methods may require large amounts of purified protein, limiting their application.
Purpose of the Study:
- To develop a sensitive and rapid method for identifying DNA sequence specificity of DNA binding proteins.
- To enable the recovery of specific DNA binding sites from limited protein quantities, such as crude cell extracts or cell-free reactions.
Main Methods:
- Incubation of cell extracts or cell-free translated proteins with random sequence oligonucleotides.
- Purification of protein-DNA complexes via immunoprecipitation.
- Amplification of bound DNA using Polymerase Chain Reaction (PCR) in iterative rounds.
Main Results:
- Successfully recovered authentic high-affinity binding sites for the transcription factor SRF.
- Demonstrated the utility of epitope tagging for site recovery when specific antibodies are unavailable.
- Efficiently recovered DNA binding sites for the Fos protein, which requires complex formation for high-affinity binding.
Conclusions:
- The described method is sensitive and rapid for determining DNA binding protein sequence specificity.
- It allows for the recovery of specific DNA sites even with small amounts of protein.
- The technique is versatile, accommodating various protein sources and binding complexities.