Methylation-Specific PCR

  • Zhiqing Huang
  • Christopher F. Bassil
  • Susan K. Murphy
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1049)

Abstract

Defining DNA methylation patterns in the genome has become essential for understanding diverse biological processes including the regulation of gene expression, imprinted genes, and X chromosome inactivation and how these patterns are deregulated in human diseases. Methylation-specific (MS)-PCR is a useful tool for qualitative DNA methylation analysis with multiple advantages, including ease of design and execution, sensitivity in the ability to detect small quantities of methylated DNA, and the ability to rapidly screen a large number of samples without the need for purchase of expensive laboratory equipment. This assay requires modification of the genomic DNA by sodium bisulfite and two independent primer sets for PCR amplification, one pair designed to recognize the methylated and the other pair the unmethylated versions of the bisulfite-modified sequence. The amplicons are visualized using ethidium bromide staining following agarose gel electrophoresis. Amplicons of the expected size produced from either primer pair are indicative of the presence of DNA in the original sample with the respective methylation status.

Key words

DNA methylation Polymerase chain reaction Primer design Annealing temperature Gel electrophoresis 

Notes

Acknowledgments

We gratefully acknowledge Zack Davenport for his contributions to the artwork. We thank Allison Barratt and Carole Grenier for critical reading of the manuscript. This work was supported by Department of Defense grant W81XWH-11-1-0469, NIH grants R01DK085173 and R01CA142983, and the Gail Parkins Ovarian Cancer Awareness Fund.

References

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    Li LC, Dahiya R (2002) MethPrimer: designing primers for methylation PCRs. Bioinformatics 18:1427–1431PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Zhiqing Huang
    • 1
  • Christopher F. Bassil
    • 1
  • Susan K. Murphy
    • 1
  1. 1.Division of Gynecologic OncologyDuke University Medical CenterDurhamUSA

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