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COBRA: a sensitive and quantitative DNA methylation assay

Z Xiong1, P W Laird

  • 1Department of Surgery, University of Southern California School of Medicine, The Norris Comprehensive Cancer Center, Room 6415, Mailstop #73, 1441 Eastlake Avenue, Los Angeles, CA 90033, USA.

Nucleic Acids Research
|June 15, 1997
PubMed
Summary

We developed COBRA, a quantitative DNA methylation analysis method. This technique accurately measures DNA methylation levels in small samples, even from paraffin-embedded tissues.

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Area of Science:

  • Molecular Biology
  • Epigenetics
  • Genomics

Background:

  • DNA methylation is a critical epigenetic modification influencing gene expression.
  • Accurate quantification of DNA methylation is essential for understanding various biological processes and diseases.
  • Existing methods may require large DNA quantities or are not suitable for archival samples.

Purpose of the Study:

  • To introduce and validate COBRA ( ما-dependent restriction enzyme digestion analysis), a novel quantitative technique for DNA methylation analysis.
  • To assess the applicability of COBRA for analyzing small amounts of genomic DNA, including those from paraffin-embedded tissues.

Main Methods:

  • Utilized sodium bisulfite treatment to convert unmethylated cytosines to uracils.
  • Employed restriction enzyme digestion to differentiate between methylated and unmethylated DNA sequences in PCR products.

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  • Quantified DNA methylation levels by analyzing the ratio of digested to undigested PCR products.

    • Main Results:

    • Demonstrated that COBRA provides a linear quantitative representation of DNA methylation levels across a broad spectrum.
    • Showcased the technique's reliability when applied to DNA extracted from microdissected paraffin-embedded tissue samples.
    • Confirmed the ease of use and quantitative accuracy of the COBRA method.

    Conclusions:

    • COBRA is a robust and quantitative method for determining DNA methylation levels at specific gene loci.
    • The technique is suitable for analyzing limited DNA samples, including challenging archival materials like paraffin sections.
    • COBRA offers a valuable tool for epigenetic research, combining accuracy, ease of use, and versatility.