DNA methylation analysis by pyrosequencing

Nat Protoc. 2007;2(9):2265-75. doi: 10.1038/nprot.2007.314.


Pyrosequencing is a sequencing-by-synthesis method that quantitatively monitors the real-time incorporation of nucleotides through the enzymatic conversion of released pyrophosphate into a proportional light signal. Quantitative measures are of special importance for DNA methylation analysis in various developmental and pathological situations. Analysis of DNA methylation patterns by pyrosequencing combines a simple reaction protocol with reproducible and accurate measures of the degree of methylation at several CpGs in close proximity with high quantitative resolution. After bisulfite treatment and PCR, the degree of each methylation at each CpG position in a sequence is determined from the ratio of T and C. The process of purification and sequencing can be repeated for the same template to analyze other CpGs in the same amplification product. Quantitative epigenotypes are obtained using this protocol in approximately 4 h for up to 96 DNA samples when bisulfite-treated DNA is already available as the starting material.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / chemistry
  • Adaptor Proteins, Signal Transducing / metabolism
  • Base Composition
  • CpG Islands
  • DNA Methylation*
  • Genomic Imprinting
  • Humans
  • MutL Protein Homolog 1
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / metabolism
  • Polymerase Chain Reaction
  • Sequence Analysis, DNA / methods*
  • Sulfites / chemistry


  • Adaptor Proteins, Signal Transducing
  • MLH1 protein, human
  • Nuclear Proteins
  • Sulfites
  • MutL Protein Homolog 1
  • hydrogen sulfite