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DNA Methylation Protocols pp 427–445Cite as

Quantitative DNA Methylation Analysis at Single-Nucleotide Resolution by Pyrosequencing®

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1708))

Abstract

Many protocols for gene-specific DNA methylation analysis are either labor intensive, not quantitative and/or limited to the measurement of the methylation status of only one or very few CpG positions. Pyrosequencing is a real-time sequencing technology that overcomes these limitations. After bisulfite modification of genomic DNA, a region of interest is amplified by PCR with one of the two primers being biotinylated. The PCR generated template is rendered single-stranded and a pyrosequencing primer is annealed to analyze quantitatively cytosine methylation. In comparative studies, pyrosequencing has been shown to be among the most accurate and reproducible technologies for locus-specific DNA methylation analyses and has become a widely used tool for the validation of DNA methylation changes identified in genome-wide studies as well as for locus-specific analyses with clinical impact such as methylation analysis of the MGMT promoter. Advantages of the Pyrosequencing technology are the ease of its implementation, the high quality and the quantitative nature of the results, and its ability to identify differentially methylated positions in close proximity.

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Acknowledgments

Work in the laboratory of JT is supported by grants from the ANR (ANR-13-EPIG-0003-05 and ANR-13-CESA-0011-05), Aviesan/INSERM (EPIG2014-01 and EPlG2014-18) and INCa (PRT-K14-049), a Sirius research award (UCB Pharma S.A.), a Passerelle research award (Pfizer), iCARE (MSD Avenir), and the institutional budget of the CNRGH.

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Authors and Affiliations

  1. Laboratory for Epigenetics and Environment, Centre National de Recherche en Génomique Humaine, CEA–Institut de Biologie Francois Jacob, Bâtiment G2, 2 rue Gaston Crémieux, 91000, Evry, France

    Florence Busato, Emelyne Dejeux, Hafida El abdalaoui & Jörg Tost

  2. Biomedical Genomics Group, Centro Nacional de Analisis Genomico, CNAG-CRG, Center for Genomic Regulation, Barcelona Institute for Science and Technology, Barcelona, Spain

    Ivo Glynne Gut

Authors
  1. Florence Busato
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  2. Emelyne Dejeux
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  3. Hafida El abdalaoui
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  4. Ivo Glynne Gut
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  5. Jörg Tost
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Corresponding author

Correspondence to Jörg Tost .

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Editors and Affiliations

  1. Laboratory for Epigenetics and Environment, Centre National de Recherche en Génomique Humaine, CEA—Institut de Biologie Francois Jacob, Evry, France

    Jörg Tost

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Busato, F., Dejeux, E., El abdalaoui, H., Gut, I.G., Tost, J. (2018). Quantitative DNA Methylation Analysis at Single-Nucleotide Resolution by Pyrosequencing®. In: Tost, J. (eds) DNA Methylation Protocols. Methods in Molecular Biology, vol 1708. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7481-8_22

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  • DOI: https://doi.org/10.1007/978-1-4939-7481-8_22

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7479-5

  • Online ISBN: 978-1-4939-7481-8

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