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Research Paper
Improved Quantification of DNA Methylation Using Methylation-Sensitive Restriction Enzymes and Real-Time PCR
Ko Hashimoto, Shoichi Kokubun, Eiji Itoi and Helmtrud I. Roach
volume 2 | issue 2
april/may/june 2007Pages: 86 - 91
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Heterogeneity of cells with respect to the DNA methylation status at a specific CpG site is a problem when assessing methylation status. We have developed a simple two-step method for the quantification of the percent of cells that display methylation at a specific CpG site in the promoter of a specific gene. The first step is overnight digestion of genomic DNA (optimal conc. 20ng/5μl) with a relevant methylation-sensitive restriction enzyme (optimal 2 units). This is followed by real time PCR, using the SYBRŪ Green method, with primers that bracket the site cleaved by the enzyme. By including fully methylated and fully non-methylated DNA in each PCR plate, the errors caused by non-specific digestion or incomplete digestion can be measured and used to adjust the raw results and thus increase specificity. The method can detect differences in methylation status if these are more than 10%. No specialized equipment is required beyond the real-time PCR system and the method can be adapted for any of the 53 commercially available methylation-sensitive restriction enzymes.
Authors
Ko Hashimoto
Tohoku University Graduate School of Medicine, Sendai, Japan
Shoichi Kokubun
Tohoku University Graduate School of Medicine, Sendai, Japan
Eiji Itoi
Tohoku University Graduate School of Medicine, Sendai, Japan
Helmtrud I. Roach
University of Southampton, Southampton, UK
We now provide open access to journal articles published online for one year or more. This article may be downloaded at the following link:
Download PDF If the document does not open, please right-click on the link (control-click on a Macintosh) and select the option to save the file to disk.