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

  • Florence Busato
  • Emelyne Dejeux
  • Hafida El abdalaoui
  • Ivo Glynne Gut
  • Jörg Tost
Part of the Methods in Molecular Biology book series (MIMB, volume 1708)


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.

Key words

Pyrosequencing Real-time synthesis Bisulfite Epigenotyping Quantification Heterogeneous DNA methylation Biomarker 



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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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Florence Busato
    • 1
  • Emelyne Dejeux
    • 1
  • Hafida El abdalaoui
    • 1
  • Ivo Glynne Gut
    • 2
  • Jörg Tost
    • 1
  1. 1.Laboratory for Epigenetics and EnvironmentCentre National de Recherche en Génomique Humaine, CEA–Institut de Biologie Francois JacobEvryFrance
  2. 2.Biomedical Genomics Group, Centro Nacional de Analisis Genomico, CNAG-CRG, Center for Genomic RegulationBarcelona Institute for Science and TechnologyBarcelonaSpain

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