Abstract
MethyLight is a sodium-bisulfite-dependent, quantitative, fluorescence-based, real-time PCR method to sensitively detect and quantify DNA methylation in genomic DNA. MethyLight relies on methylation-specific priming combined with methylation-specific fluorescent probing. This combination of methylation-specific detection principles results in a highly methylation-specific detection technology, with an accompanying ability to sensitively detect very low frequencies of hypermethylated alleles. The high sensitivity and specificity of MethyLight make it uniquely well suited for detection of low-frequency DNA methylation biomarkers as evidence of disease. At the same time, the quantitative accuracy of real-time PCR and the flexibility to design bisulfite-dependent, methylation-independent control reactions allows for a quantitative assessment of these low-frequency methylation events. We describe the experimental steps of MethyLight analysis in detail. Furthermore, we present here principles and design examples for three types of quality-control reactions. QC-1 reactions are methylation-independent reactions to monitor sample quantity and integrity. QC-2 reactions are bisulfite-independent reactions to monitor recovery efficiencies of the bisulfite-conversion methodology used. QC-3 reactions are bisulfite-independent primed reactions with variable bisulfite-dependent probing to monitor completeness of the sodium bisulfite treatment. We show that these control reactions perform as expected in a time-course experiment interrupting sodium bisulfite conversion at various timepoints.
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
Eads, C. A., Danenberg, K. D., Kawakami, K., et al. (1999) CpG island hypermethylation in human colorectal tumors is not associated with DNA methyltransferase overexpression. Cancer Res 59, 2302′2306.
Eads, C. A., Danenberg, K. D., Kawakami, K., et al. (2000) MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 28, e32.
Trinh, B. N., Long, T. I., Laird, P. W. (2001) DNA methylation analysis by MethyLight technology. Methods 25, 456′462.
Ogino, S., Kawasaki, T., Brahmandam, M., et al. (2006) Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis. J Mol Diagn 8, 209′217.
Weisenberger, D. J., Campan, M., Long, T. I., et al. (2005) Analysis of repetitive element DNA methylation by MethyLight. Nucleic Acids Res 33, 6823′6836.
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.
Laird, P. W. (2003) The power and the promise of DNA methylation markers. Nat Rev Cancer 3, 253′266.
Fiegl, H., Gattringer, C., Widschwendter, A., et al. (2004) Methylated DNA collected by tampons – a new tool to detect endometrial cancer. Cancer Epidemiol Biomarkers Prev 13, 882′888.
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
Campan, M., Weisenberger, D.J., Trinh, B., Laird, P.W. (2009). MethyLight. In: Tost, J. (eds) DNA Methylation. Methods in Molecular Biology, vol 507. Humana Press. https://doi.org/10.1007/978-1-59745-522-0_23
Download citation
DOI: https://doi.org/10.1007/978-1-59745-522-0_23
Publisher Name: Humana Press
Print ISBN: 978-1-934115-61-9
Online ISBN: 978-1-59745-522-0
eBook Packages: Springer Protocols