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Related Experiment Video

Updated: Jun 4, 2026

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
06:07

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

Published on: August 5, 2022

Methylation interference assay.

Michael Carey, Stephen T Smale

    CSH Protocols
    |March 2, 2011
    PubMed
    Summary
    This summary is machine-generated.

    Methylation interference is a high-resolution technique to map protein-DNA interactions by identifying critical bases. Modifications that block protein binding are revealed through DNA cleavage and gel electrophoresis.

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    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Genetics

    Background:

    • Proteins recognize specific DNA sequences through interactions with bases.
    • Understanding these interactions is crucial for deciphering gene regulation and cellular processes.
    • High-resolution methods are needed to pinpoint the exact bases involved in sequence-specific recognition.

    Purpose of the Study:

    • To describe methylation interference as a key technique for studying protein-DNA interactions.
    • To explain how this method identifies bases critical for sequence-specific protein binding.
    • To highlight its utility in mapping protein-DNA interfaces.

    Main Methods:

    • Methylation of DNA (32)P-end-labeled DNA with dimethyl sulfate (DMS).
    • Incubation with the target protein to allow binding.

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    Related Experiment Videos

    Last Updated: Jun 4, 2026

    Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
    06:07

    Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

    Published on: August 5, 2022

    Methylated DNA Immunoprecipitation
    21:24

    Methylated DNA Immunoprecipitation

    Published on: January 2, 2009

    Antibody-Free Assay for RNA Methyltransferase Activity Analysis
    08:31

    Antibody-Free Assay for RNA Methyltransferase Activity Analysis

    Published on: July 9, 2019

  • Separation of bound and unbound DNA fractions using electrophoretic mobility shift assay (EMSA).
  • Piperidine-induced DNA backbone cleavage at methylated sites.
  • Analysis of cleaved fragments on a sequencing gel alongside chemical sequencing ladders.

    • Main Results:

    • Methylation at critical contact points interferes with protein binding.
    • DNA modifications that do not interfere with binding remain in the bound fraction.
    • The unbound fraction is enriched for DNA with interfering methylation.
    • Analysis reveals specific bases involved in sequence-specific protein recognition.

    Conclusions:

    • Methylation interference is a powerful, high-resolution method for mapping protein-DNA interfaces.
    • It precisely identifies bases critical for sequence-specific recognition by proteins.
    • The technique is valuable for studying major-groove guanines, minor-groove adenines, and single-stranded cytosines.