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DNA methylation analysis by pyrosequencing

Abstract

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.

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Figure 1: Enzymatic cascade of the pyrosequencing reaction in the example of a bisulfite-treated template sequence, including a CpG position that is methylated on approximately 50% of all molecules.
Figure 2
Figure 3: Two pyrograms analyzing 8 CpGs in the CpG island spanning the transcription start site of the DNA repair gene MLH1.
Figure 4: Potential complications during the pyrosequencing reaction.

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Acknowledgements

This work was supported by the French Ministry of Research and the European Commission under the Integrated Project 'MolPage' (contract number LSHG-CT-2004-512966).

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Correspondence to Jörg Tost or Ivo G Gut.

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Tost, J., Gut, I. DNA methylation analysis by pyrosequencing. Nat Protoc 2, 2265–2275 (2007). https://doi.org/10.1038/nprot.2007.314

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