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Pyrosequencing of DNA Extracted from Formalin-Fixed Paraffin-Embedded Tissue

  • Brendan DoyleEmail author
  • Ciarán O’Riain
  • Kim Appleton
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 724)

Abstract

Gene promoter hypermethylation is recognised as an important mechanism by which genes may be silenced both physiologically and in disease states. This mechanism of gene silencing has been shown to play a role in many common human tumours. A number of methods are available for the detection of promoter hypermethylation, including the methylation-specific polymerase chain reaction (PCR), bisulphite sequencing, and pyrosequencing. Pyrosequencing is a reproducible method for obtaining data on the methylation status of DNA. It also has the advantage of providing quantitative data regarding the amount of methylation present in multiple CpGs in a given sample. The technique is based on the bisulphite conversion of unmethylated cytosine to uracil and subsequent amplification by PCR. The technique is also appropriate for use on DNA extracted from formalin-fixed paraffin-embedded tissue.

Key words

Pyrosequencing Methylation Bisulphite modification 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Brendan Doyle
    • 1
    Email author
  • Ciarán O’Riain
    • 2
  • Kim Appleton
    • 3
  1. 1.Beatson Institute for Cancer ResearchGlasgowUK
  2. 2.John Vane Science CentreLondonUK
  3. 3.Centre for Oncology and Applied PharmacologyUniversity of GlasgowGlasgowUK

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