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Related Experiment Videos

Surface plasmon resonance-based sensors to identify cis-regulatory elements.

Limin Lin1, Joseph W Harris, H Garrett R Thompson

  • 1Department of Biomedical Engineering, University of California-Irvine, Irvine, California 92697-2715, USA.

Analytical Chemistry
|November 13, 2004
PubMed
Summary

Researchers developed a surface plasmon resonance (SPR) assay to identify DNA binding proteins. This high-throughput method successfully detected physiologically significant cis-regulatory elements in nuclear cell lysates.

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Area of Science:

  • Functional genomics
  • Molecular biology
  • Biophysical techniques

Background:

  • Transcription regulation in eukaryotes involves multiprotein complexes binding to cis-regulatory elements.
  • Comprehensive identification of cis-regulatory elements is crucial for functional genomics.
  • A high-throughput assay is needed for efficient identification of these elements.

Purpose of the Study:

  • To develop and demonstrate a high-throughput surface plasmon resonance (SPR)-based assay for identifying cis-regulatory elements.
  • To determine if SPR can detect protein binding to specific DNA sequences in raw nuclear lysates.

Main Methods:

  • Immobilized a 16-basepair double-stranded DNA region of the SQSTM1 promoter onto a surface plasmon resonance (SPR) sensor (Texas Instruments Spreeta).

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  • Used a control DNA sequence differing by a single base pair.
  • Exposed immobilized DNA probes to raw nuclear lysate from NIH/3T3 cells and measured SPR signal changes.
  • Validated findings by measuring transcriptional activity using a luciferase-reporter vector in transfected NIH/3T3 cells.

    • Main Results:

    • Observed a significant difference in SPR signal between the promoter DNA and the single-base pair variant when exposed to nuclear lysate.
    • Demonstrated a significant difference in transcriptional activity between the two DNA sequences in live cells.
    • The SPR-based assay effectively distinguished between functional and non-functional DNA elements.

    Conclusions:

    • A surface plasmon resonance (SPR)-based sensor is capable of identifying physiologically significant cis-regulatory elements.
    • This SPR assay provides a high-throughput method for functional genomics research.
    • The technique can detect specific DNA-protein interactions relevant to gene regulation.