Abstract
Methylation-specific polymerase chain reaction (MSP) is a technique that has facilitated the detection of promoter hypermethylation at CpG islands in cell lines and clinical samples, including fresh/frozen tissues. The ability of MSP to differentiate methylated from unmethylated cytosine is dependent upon sodium bisulfite treatment of DNA which retains the methylation marks of cytosines together with the specific amplification of this modified DNA using primer sets complimentary only to the formerly methylated or unmethylated alleles. Nested-MSP (MN-MSP) is an alternative method that overcomes the limitations of MSP, especially when it comes to analyzing samples with low quality/quantity of starting DNA (e.g., paraffin-embedded specimens). MN-MSP includes a first round of amplification using primers unbiased toward the methylation status of a single (MN-MSP) or multiple (multiplex MN-MSP) genes followed by conventional MSP. Although MSP and NM-MSP are simple techniques that can easily be incorporated in most molecular biology laboratories, the ability to accurately determine the promoter methylation status of genes largely depends upon the careful design of MSP primers as well as other steps outlined in this chapter.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bird, A.P. (1986) CpG-rich islands and the function of DNA methylation. Nature 321, 209–213.
Belinsky, S.A., Nikula, K. J., Palmisano, W.A., et al. (1998) Aberrant methylation of p16(INK4a) is an early event in lung cancer and a potential biomarker for early diagnosis. Proc Natl Acad Sci USA 95, 11891–11896.
Hanahan, D., Weinberg, R. A. (2000) The hallmarks of cancer. Cell 100, 57–70.
Herman, J. G., Baylin, S. B. (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349, 2042–2054.
Herman, J. G., Graff, J. R., Myohanen, S., et al. (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 93, 9821–9826.
Clark, S. J., Harrison, J., Paul, C. L. et al. (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res 22, 2990–2997.
Frommer, M., McDonald, L. E., Millar, D. S., et al. (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.
House, M. G., Guo, M., Iacobuzio-Donahue, C., et al. (2003) Molecular progression of promoter methylation in intraductal papillary mucinous neoplasms (IPMN) of the pancreas. Carcinogenesis 24, 193–198.
van Engeland, M., Weijenberg, M. P., Roemen, G. M., et al. (2003) Effects of dietary folate and alcohol intake on promoter methylation in sporadic colorectal cancer: the Netherlands cohort study on diet and cancer. Cancer Res 63, 3133–3137.
Hegi, M. E., Diserens, A. C., Gorlia, T., et al. (2005) MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 352, 997–1003.
Belinsky, S. A., Liechty, K. C., Gentry, F. D., et al. (2006) Promoter hypermethylation of multiple genes in sputum precedes lung cancer incidence in a high-risk cohort. Cancer Res 66, 3338–3344.
Brandes, J. C., Carraway, H., Herman, J. G. (2007) Optimal primer design using the novel primer design program: MSPprimer provides accurate methylation analysis of the ATM promoter. Oncogene 26, 6229–6237.
Acknowledgement
The authors would like to acknowledge members of the Herman laboratory, especially Dr Johan C. Brandes, Dr Joshi J. Alumkal, and Dr Mingzhou Guo for contributions to some parts of these protocols.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Licchesi, J.D.F., Herman, J.G. (2009). Methylation-Specific PCR. In: Tost, J. (eds) DNA Methylation. Methods in Molecular Biology, vol 507. Humana Press. https://doi.org/10.1007/978-1-59745-522-0_22
Download citation
DOI: https://doi.org/10.1007/978-1-59745-522-0_22
Publisher Name: Humana Press
Print ISBN: 978-1-934115-61-9
Online ISBN: 978-1-59745-522-0
eBook Packages: Springer Protocols