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Related Experiment Video

Updated: May 29, 2026

DNA Methylation: Bisulphite Modification and Analysis
12:34

DNA Methylation: Bisulphite Modification and Analysis

Published on: October 21, 2011

Analysing DNA methylation using bisulphite pyrosequencing.

Thomas Mikeska1, Jörg Felsberg, Chelsee A Hewitt

  • 1Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia. Thomas.Mikeska@petermac.org

Methods in Molecular Biology (Clifton, N.J.)
|September 14, 2011
PubMed
Summary

Bisulphite pyrosequencing quantitatively analyzes DNA methylation in short sequences up to 100-bp. This method detects methylation at individual CpG sites with high accuracy, suitable for formalin-fixed samples.

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Last Updated: May 29, 2026

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Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

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Area of Science:

  • Molecular Biology
  • Epigenetics

Background:

  • DNA methylation is a crucial epigenetic modification regulating gene expression.
  • Accurate quantification of DNA methylation is essential for understanding various biological processes and diseases.

Purpose of the Study:

  • To describe the methodology of bisulphite pyrosequencing for DNA methylation analysis.
  • To highlight its quantitative capabilities, detection limits, and applications.

Main Methods:

  • Bisulphite treatment of DNA followed by PCR amplification.
  • Biotin-labeled single-stranded DNA templates used for pyrosequencing.
  • Sequential nucleotide addition and light emission detection to quantify methylation at CpG sites.

Main Results:

  • Quantitative DNA methylation analysis of sequences up to 100-bp.
  • Detection limit of approximately 5% methylation at individual CpG sites.
  • Identification of heterogeneous DNA methylation patterns without single allele resolution.

Conclusions:

  • Bisulphite pyrosequencing is a robust quantitative method for DNA methylation analysis.
  • The technique is well-suited for short DNA sequences, including those from archival formalin-fixed paraffin-embedded specimens.
  • Serial bisulphite pyrosequencing enables analysis of longer DNA fragments.