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Related Concept Videos

Single-Strand DNA Binding Proteins01:03

Single-Strand DNA Binding Proteins

For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...

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Related Experiment Video

Updated: Jun 27, 2026

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)
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Characterization of DNA-binding proteins using multiplexed competitor EMSA.

Andrew J P Smith1, Steve E Humphries

  • 1Center for Cardiovascular Genetics, Department of Medicine, University College London, 5 University Street, London WC1E 6JF, UK. Andrew.J.P.Smith@ucl.ac.uk.

Journal of Molecular Biology
|December 9, 2008
PubMed
Summary

We developed a low-cost, high-throughput method to study protein DNA interactions. This technique identified a new binding site for hepatocyte nuclear factor-3 created by a common genetic variation.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Characterizing protein DNA-binding interactions is crucial for understanding gene regulation.
  • Existing methods can be costly and time-consuming.
  • High-throughput techniques are needed for efficient analysis.

Purpose of the Study:

  • To introduce a novel, cost-effective, high-throughput method for analyzing nuclear protein DNA-binding.
  • To demonstrate the method's utility in identifying functional regulatory elements.
  • To characterize a specific protein-DNA interaction influenced by genetic variation.

Main Methods:

  • Developed Multiplexed Competitor Electrophoretic Mobility Shift Assay (MC-EMSA).
  • Utilized multiplexed oligonucleotide DNA consensus competitors.
  • Combined with standard electrophoretic mobility shift assay (EMSA) procedures.
  • Applied the method to study lipoprotein lipase gene regulation.

Main Results:

  • Successfully characterized nuclear protein DNA-binding interactions with high throughput.
  • Identified a previously unreported binding site for hepatocyte nuclear factor-3 (HNF-3).
  • This HNF-3 site was created in intron 8 of the lipoprotein lipase gene.
  • The site formation was attributed to a common single-nucleotide polymorphism (SNP) rs327.

Conclusions:

  • MC-EMSA is an efficient and low-cost method for characterizing protein-DNA binding.
  • Common genetic variations can create novel functional protein binding sites.
  • This finding provides new insights into lipoprotein lipase gene regulation.