DNA Methylation pp 189-205

Part of the Methods in Molecular Biology book series (MIMB, volume 507)

Identification and Quantification of Differentially Methylated Loci by the Pyrosequencing™ Technology

  • Emelyne Dejeux
  • Hafida El abdalaoui
  • Ivo Glynne Gut
  • Jörg Tost

Abstract

Most available protocols for gene-specific DNA methylation analysis are either labor intensive, not quantitative, 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 polymerase chain reaction (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 CpGs within 120 bases. 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. A minimum amount of 10 ng of bisulfite-treated DNA is necessary to obtain high reproducibility and avoid random amplification. The required DNA amount can be provided by an individual sample or a pool of samples to rapidly investigate the presence of variable DNA methylation patterns. The use of pools and serial pyrosequencing, that is, the successive use of several pyrosequencing primers on the same DNA template, significantly reduces cost, labor, and analysis time as well as saving precious DNA samples for the analysis of gene-specific DNA methylation patterns.

Keywords

Serial pyrosequencing pooled DNA real-time synthesis bisulfite epigenotyping quantification 

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Emelyne Dejeux
    • 1
  • Hafida El abdalaoui
    • 1
  • Ivo Glynne Gut
    • 2
  • Jörg Tost
    • 1
  1. 1.Laboratory for EpigeneticsCentre National de Génotypage, CEA – Institut de GénomiqueEvryFrance
  2. 2.Department of Technology DevelopmentCentre National de Génotypage, CEA – Institut de GénomiqueEvryFrance

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