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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Lister, R., Pelizzola, M., Dowen, R. H., Hawkins, R. D., Hon, G., Tonti-Filippini, J., et al. (2009) Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462, 315–22.
White, H. E., Hall, V. J., and Cross, N. C. (2007) Methylation-sensitive high-resolution melting-curve analysis of the SNRPN gene as a diagnostic screen for Prader–Willi and Angelman syndromes. Clin Chem. 53, 1960–2.
Bliek, J., Verde, G., Callaway, J., Maas, S. M., De Crescenzo, A., Sparago, A., et al. (2009) Hypomethylation at multiple maternally methylated imprinted regions including PLAGL1 and GNAS loci in Beckwith–Weidemann syndrome. Eur J Hum Genet. 17, 611–9.
Zhu, W., Qin, W., Hewett, J. E., and Sauter, E. R. (2010) Quantitative evaluation of DNA hypermethylation in malignant and benign breast tissue and fluids. Int J Cancer 126, 474–82.
Toyota, M., Ahuja, N., Ohe-Toyota, M., Herman, J. G., Baylin, S. B., and Issa, J. P. (1999) CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci USA 96, 8681–6.
Issa, J. P. (2008) Colon cancer: it’s CIN or CIMP. Clin Cancer Res. 14, 5939–40.
Martin-Subero, J. I., Ammerpohl, O., Bibikova, M., Wickham-Garcia, E., Agirre, X., Alvarez, S., et al. (2009) A comprehensive microarray-based DNA methylation study of 367 hematological neoplasms. PLoS One 4, e6986.
O’Riain, C., O’Shea, D. M., Yang, Y., Le Dieu, R., Gribben, J. G., Summers, K., et al. (2009) Array-based DNA methylation profiling in follicular lymphoma. Leukemia 23, 1858–66.
Buckingham, L., Faber, L. P., Kim, A., Liptay, M., Barger, C., Basu, S., et al. (2010) PTEN, RASSF1 and DAPK site-specific hypermethylation and outcome in surgically treated stage I and II non small cell lung cancer patients. Int J Cancer 126, 1630–39.
Dufort, S., Richard, M. J., and de Fraipont, F. (2009) Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues. Anal Biochem. 391, 166–8.
Kure, S., Nosho, K., Baba, Y., Irahara, N., Shima, K., Ng, K., et al. (2009) Vitamin D receptor expression is associated with PIK3CA and KRAS mutations in colorectal cancer. Cancer Epidemiol Biomarkers Prev. 18, 2765–72.
Mikeska, T., Bock, C., El-Maarri, O., Hubner, A., Ehrentraut, D., Schramm, J., et al. (2007) Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis. J Mol Diagn. 9, 368–81.
Nosho, K., Shima, K., Irahara, N., Kure, S., Baba, Y., Kirkner, G. J., et al. (2009) DNMT3B expression might contribute to CpG island methylator phenotype in colorectal cancer. Clin Cancer Res. 15, 3663–71.
Tost, J., and Gut, I. G. (2007) DNA methylation analysis by pyrosequencing. Nat Protoc. 2, 2265–75.
Dupont, J. M., Tost, J., Jammes, H., and Gut, I. G. (2004) De novo quantitative bisulfite sequencing using the pyrosequencing technology. Anal Biochem. 333, 119–27.
Warnecke, P. M., Stirzaker, C., Melki, J. R., Millar, D. S., Paul, C. L., and Clark, S. J. (1997) Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA. Nucleic Acids Res. 25, 4422–6.
Shen, L., Guo, Y., Chen, X., Ahmed, S., and Issa, J. P. (2007) Optimizing annealing temperature overcomes bias in bisulfite PCR methylation analysis. Biotechniques 42, 48, 50, 52 passim.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Doyle, B., O’Riain, C., Appleton, K. (2011). Pyrosequencing of DNA Extracted from Formalin-Fixed Paraffin-Embedded Tissue. In: Al-Mulla, F. (eds) Formalin-Fixed Paraffin-Embedded Tissues. Methods in Molecular Biology, vol 724. Humana Press. https://doi.org/10.1007/978-1-61779-055-3_12
Download citation
DOI: https://doi.org/10.1007/978-1-61779-055-3_12
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-054-6
Online ISBN: 978-1-61779-055-3
eBook Packages: Springer Protocols