Skip to main content

Detection and Analysis of DNA Methylation by Pyrosequencing

Part of the Methods in Molecular Biology book series (MIMB,volume 863)

Abstract

Pyrosequencing is a “sequencing by synthesis” technique which can be used to quantify DNA methylation at specific CpG sites within the target region of interest. Biotin labelled polymerase chain reaction (PCR) products form the template for base-pair nucleotide incorporation causing a light emitting cascade reaction resulting in the formation of a pyrogram and the calculation of the percentage methylation for each site. Prior to pyrosequencing, it is essential to bisulphite-convert the DNA sample and then perform locus-specific PCR for the region of interest. One of the PCR primers needs to be biotinylated and a separate sequencing primer is required for the pyrosequencing itself.

Key words

  • Pyrosequencing
  • DNA
  • Methylation
  • Epigenetics
  • Bisulphite
  • Pyrogram
  • CpG

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-61779-612-8_17
  • Chapter length: 12 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   149.00
Price excludes VAT (USA)
  • ISBN: 978-1-61779-612-8
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   189.00
Price excludes VAT (USA)
Hardcover Book
USD   189.00
Price excludes VAT (USA)
Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Nyren, P. (2007) The history of pyrosequencing, Methods Mol Biol 373, 1–14.

    PubMed  CAS  CrossRef  Google Scholar 

  2. Tost, J., and Gut, I. G. (2007) DNA methylation analysis by pyrosequencing, Nat Protoc 2, 2265–2275.

    PubMed  CAS  CrossRef  Google Scholar 

  3. Tost, J., and Gut, I. G. (2007) Analysis of gene-specific DNA methylation patterns by pyrosequencing technology, Methods Mol Biol 373, 89–102.

    PubMed  CAS  Google Scholar 

  4. Frommer, M., McDonald, L. E., Millar, D. S., Collis, C. M., Watt, F., Grigg, G. W., Molloy, P. L., and Paul, C. L. (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands, Proc Natl Acad Sci USA 89, 1827–1831.

    PubMed  CAS  CrossRef  Google Scholar 

  5. Clark, S. J., Statham, A., Stirzaker, C., Molloy, P. L., and Frommer, M. (2006) DNA methylation: bisulphite modification and analysis, Nat Protoc 1, 2353–2364.

    PubMed  CAS  CrossRef  Google Scholar 

  6. Gusev, N. B. (2001) Some properties of caldesmon and calponin and the participation of these proteins in regulation of smooth muscle contraction and cytoskeleton formation, Biochemistry (Mosc) 66, 1112–1121.

    CAS  CrossRef  Google Scholar 

  7. Hsiung, D. T., Marsit, C. J., Houseman, E. A., Eddy, K., Furniss, C. S., McClean, M. D., and Kelsey, K. T. (2007) Global DNA methylation level in whole blood as a biomarker in head and neck squamous cell carcinoma, Cancer Epidemiol Biomarkers Prev 16, 108–114.

    PubMed  CrossRef  Google Scholar 

  8. Sriraksa, R., Chaopatchayakul, P., Jearanaikoon, P., Leelayuwat, C., and Limpaiboon, T. Verification of complete bisulfite modification using Calponin-specific primer sets, Clin Biochem 43, 528–530.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Hilary A. A. Colyer .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Colyer, H.A.A., Armstrong, R.N., Sharpe, D.J., Mills, K.I. (2012). Detection and Analysis of DNA Methylation by Pyrosequencing. In: Dumitrescu, R., Verma, M. (eds) Cancer Epigenetics. Methods in Molecular Biology, vol 863. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-612-8_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-612-8_17

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-611-1

  • Online ISBN: 978-1-61779-612-8

  • eBook Packages: Springer Protocols