Introduction to epigenetics and techniques
There has been a great interest in the study of epigenetic mechanisms and DNA methylation in many areas of research, including DNA repair, cell cycle control, developmental biology, cancer research, identification of biomarkers, predisposition factors and potential drug targets. Lately I’ve been immersed in this field via my close work with the Epigenetics Research Solution experts in QIAGEN. I thought that you, our readers, might also be interested in learning more about the techniques and controls that are important for epigenetic research.
DNA methylation and cancer studies
DNA methylation is an important topic in cancer research – previous studies have shown hypermethylation of tumor suppressor genes in multiple different cancer types (1). How can you detect methylation in your own DNA samples? Exposing DNA to bisulfite rapidly leads to the deamination of unmethylated cytosines which are converted to 6-sulfonyluracil. At a high pH, 6-sulfonyluracil is desulfonated to uracil, which ultimately after amplification will translate into thymidine, while methylated cytosines will not be converted. Comparing this converted DNA to the original unconverted sequence enables detailed evaluation of the location and abundance of methylated sites in CpG islands. High-resolution melting (HRM) analysis provides a rapid screening tool to accurately detect changes in the CpG methylation status of bisulfite converted DNA. Detailed quantitative measures of percent methylation at individual CpG sites are obtained by Pyrosequencing. Alternative methods for broad-scale methylation analysis include methylation-specific PCR (MSP) which is highly specific and sensitive. Commercial kits are available for all forms of analysis.
Complete details about the DNA methylation analysis based on restriction enzyme digest can be found on the QIAGEN website under our resource section.
In any methylation analysis experiment, control DNA is crucial. Below, I’ve provided a table that helps illustrate what each control will yield in your experiment.
|Expected PCR results with controls|
|Type of DNA||Primer for unmethylated target gene (PCR 1)||Primer for unmethylated target gene (bisulfite converted) (PCR 2)||Primer for methylated target gene (bisulfite converted) (PCR 3)|
|Unmethylated control DNA||PCR product||No PCR product||No PCR product|
|Unmethylated control DNA (bisulfite converted)||No PCR product||PCR product||No PCR product|
|Methylated control DNA (bisulfite converted)||No PCR product||No PCR product||PCR product|
|No-template control||No PCR product||No PCR product||No PCR product|
Webinar series on DNA methylation and Pyrosequencing
To support all your needs of we have designed some work tips and tricks in a 3-part webinar series to learn more. Check out the slides on SlideShare!
- Part I: DNA methylation: An essential element in epigenetics
- Part II: Accurate DNA methylation analysis with successful conversion
- Part III: Downstream application of pyrosequencing
More from QIAGEN
- 1. Kulis, M. and Esteller, M. (2010) DNA methylation and cancer. Adv. Genet. 70, 27. Link