Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1

    Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlén, M. & Nyrén, P. Real-time DNA sequencing using detection of pyrophosphate release. Anal. Biochem. 242, 84–89 (1996).

    CAS  Article  PubMed  Google Scholar 

  2. 2

    Ronaghi, M., Uhlén, M. & Nyrén, P. A sequencing method based on real-time pyrophosphate. Science 281 363, 365 (1998).

    CAS  Article  PubMed  Google Scholar 

  3. 3

    Langaee, T. & Ronaghi, M. Genetic variation analyses by Pyrosequencing. Mutat. Res. 573, 96–102 (2005).

    CAS  Article  PubMed  Google Scholar 

  4. 4

    Ogino, S. et al. Sensitive sequencing method for KRAS mutation detection by Pyrosequencing. J. Mol. Diagn. 7, 413–421 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5

    Clarke, S.C. Pyrosequencing: nucleotide sequencing technology with bacterial genotyping applications. Expert Rev. Mol. Diagn. 5, 947–953 (2005).

    CAS  Article  PubMed  Google Scholar 

  6. 6

    Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7

    Rickert, A.M., Premstaller, A., Gebhardt, C. & Oefner, P.J. Genotyping of Snps in a polyploid genome by pyrosequencing. Biotechniques 32, 592–593, 596–598, 600 passim (2002).

    CAS  Article  PubMed  Google Scholar 

  8. 8

    Gruber, J.D., Colligan, P.B. & Wolford, J.K. Estimation of single nucleotide polymorphism allele frequency in DNA pools by using pyrosequencing. Hum. Genet. 110, 395–401 (2002).

    CAS  Article  PubMed  Google Scholar 

  9. 9

    Lavebratt, C. & Sengul, S. Single nucleotide polymorphism (SNP) allele frequency estimation in DNA pools using pyrosequencing. Nat. Protoc. 1, 2573–2582 (2006).

    CAS  Article  PubMed  Google Scholar 

  10. 10

    Pielberg, G., Day, A.E., Plastow, G.S. & Andersson, L. A sensitive method for detecting variation in copy numbers of duplicated genes. Genome Res. 13, 2171–2177 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11

    Deutsch, S. et al. Detection of aneuploidies by paralogous sequence quantification. J. Med. Genet. 41, 908–915 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12

    Bird, A. DNA methylation patterns and epigenetic memory. Genes Dev. 16, 6–21 (2002).

    CAS  Article  PubMed  Google Scholar 

  13. 13

    Jones, P.A. & Baylin, S.B. The epigenomics of cancer. Cell 128, 683–692 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14

    Laird, P.W. Early detection: the power and the promise of DNA methylation markers. Nat. Rev. Cancer 3, 253–266 (2003).

    CAS  Article  PubMed  Google Scholar 

  15. 15

    Brena, R.M., Huang, T.H. & Plass, C. Quantitative assessment of DNA methylation: potential applications for disease diagnosis, classification, and prognosis in clinical settings. J. Mol. Med. 84, 365–377 (2006).

    CAS  Article  PubMed  Google Scholar 

  16. 16

    Ehrich, M. et al. Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry. Proc. Natl. Acad. Sci. USA 102, 15785–15790 (2005).

    CAS  Article  PubMed  Google Scholar 

  17. 17

    Colella, S., Shen, L., Baggerly, K.A., Issa, J.P. & Krahe, R. Sensitive and quantitative universal pyrosequencing methylation analysis of CpG sites. Biotechniques 35, 146–150 (2003).

    CAS  Article  PubMed  Google Scholar 

  18. 18

    Tost, J., Dunker, J. & Gut, I.G. Analysis and quantification of multiple methylation variable positions in CpG islands by pyrosequencing. Biotechniques 35, 152–156 (2003).

    CAS  Article  PubMed  Google Scholar 

  19. 19

    Uhlmann, K., Brinckmann, A., Toliat, M.R., Ritter, H. & Nürnberg, P. Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide polymorphism analysis. Electrophoresis 23, 4072–4079 (2002).

    CAS  Article  PubMed  Google Scholar 

  20. 20

    Tost, J., El Abdalaoui, H. & Gut, I.G. Serial pyrosequencing for quantitative DNA methylation analysis. Biotechniques 40, 721–722, 724, 726 (2006).

    CAS  Article  PubMed  Google Scholar 

  21. 21

    Mirmohammadsadegh, A. et al. Epigenetic silencing of the PTEN gene in melanoma. Cancer Res. 66, 6546–6552 (2006).

    CAS  Article  PubMed  Google Scholar 

  22. 22

    Xinarianos, G. et al. Frequent genetic and epigenetic abnormalities contribute to the deregulation of cytoglobin in non-small cell lung cancer. Hum. Mol. Genet. 15, 2038–2044 (2006).

    CAS  Article  PubMed  Google Scholar 

  23. 23

    Schatz, P., Dietrich, D. & Schuster, M. Rapid analysis of CpG methylation patterns using RNase T1 cleavage and MALDI-TOF. Nucleic Acids Res. 32, e167 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  24. 24

    Yang, A.S. et al. DNA methylation changes after 5-aza-2′-deoxycytidine therapy in patients with leukemia. Cancer Res. 66, 5495–5503 (2006).

    CAS  Article  PubMed  Google Scholar 

  25. 25

    White, H.E., Durston, V.J., Harvey, J.F. & Cross, N.C. Quantitative analysis of SNRPN (correction of SNRPN) gene methylation by pyrosequencing as a diagnostic test for Prader-Willi syndrome and Angelman syndrome. Clin. Chem. 52, 1005–1013 (2006).

    CAS  Article  PubMed  Google Scholar 

  26. 26

    Wong, H.L. et al. Rapid and quantitative method of allele-specific DNA methylation analysis. Biotechniques 41, 734–739 (2006).

    CAS  Article  PubMed  Google Scholar 

  27. 27

    Yang, A.S. et al. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res. 32, e38 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  28. 28

    Karimi, M. et al. LUMA (LUminometric Methylation Assay)—a high throughput method to the analysis of genomic DNA methylation. Exp. Cell Res. 312, 1989–1995 (2006).

    CAS  Article  PubMed  Google Scholar 

  29. 29

    Li, L.C. & Dahiya, R. MethPrimer: designing primers for methylation PCRs. Bioinformatics 18, 1427–1431 (2002).

    CAS  Article  PubMed  Google Scholar 

  30. 30

    Arányi, T., Váradi, A., Simon, I. & Tusnády, G.E. The BiSearch web server. BMC Bioinformatics 7, 431 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  31. 31

    Olek, A., Oswald, J. & Walter, J. A modified and improved method for bisulphite based cytosine methylation analysis. Nucleic Acids Res. 24, 5064–5066 (1996).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32

    Boyd, V.L. & Zon, G. Bisulfite conversion of genomic DNA for methylation analysis: protocol simplification with higher recovery applicable to limited samples and increased throughput. Anal. Biochem. 326, 278–280 (2004).

    CAS  Article  PubMed  Google Scholar 

  33. 33

    Bian, Y.S., Yan, P., Osterheld, M.C., Fontolliet, C. & Benhattar, J. Promoter methylation analysis on microdissected paraffin-embedded tissues using bisulfite treatment and PCR-SSCP. Biotechniques 30, 66–72 (2001).

    CAS  Article  PubMed  Google Scholar 

  34. 34

    Kerjean, A. et al. Bisulfite genomic sequencing of microdissected cells. Nucleic Acids Res. 29, e106 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35

    Shiraishi, M. & Hayatsu, H. High-speed conversion of cytosine to uracil in bisulfite genomic sequencing analysis of DNA methylation. DNA Res. 11, 409–415 (2004).

    CAS  Article  PubMed  Google Scholar 

  36. 36

    Dupont, J.M., Tost, J., Jammes, H. & Gut, I.G. De novo quantitative bisulfite sequencing using the pyrosequencing technology. Anal. Biochem. 333, 119–127 (2004).

    CAS  Article  PubMed  Google Scholar 

  37. 37

    Warnecke, P.M. et al. Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA. Nucleic Acids Res. 25, 4422–4426 (1997).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38

    Wojdacz, T.K. & Hansen, L.L. Reversal of PCR bias for improved sensitivity of the DNA methylation melting curve assay. Biotechniques 41 274, 276, 278 (2006).

    CAS  Article  PubMed  Google Scholar 

  39. 39

    Shen, L., Guo, Y., Chen, X., Ahmed, S. & Issa, J.P. Optimizing annealing temperature overcomes bias in bisulfite PCR methylation analysis. Biotechniques 42, 48–52 (2007).

    CAS  Article  PubMed  Google Scholar 

Download references

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).

Author information

Affiliations

Authors

Corresponding authors

Correspondence to Jörg Tost or Ivo G Gut.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing