Quantification of DNA methylation using methylation-sensitive restriction enzymes and multiplex digital PCR

Abstract

Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one well-studied aspect of epigenetic regulation, but is challenging to quantify accurately. In this study, we introduce a digital approach for the absolute quantification of the amount, density and allele-specificity of DNA methylation. Combining the efficiency of methylation-sensitive restriction enzymes with the quantitative power of digital PCR, DNA methylation is measured accurately without the need to treat the DNA samples with sodium bisulphite. Moreover, as the combination of PCR amplicon and restriction enzyme is flexible, the context and density of DNA methylation can be taken into account. Additionally, by extending the experimental setup to a multiplex digital PCR, methylation markers may be analysed together with physically linked genetic markers to determine the allele-specificity of the methylation. In-silico simulations demonstrated the mathematical validity of the experimental setup. Next the approach was validated in a variety of healthy and malignant reference samples in the context of RASSF1A promotor methylation. RASSF1A is an established tumour suppressor gene, that is aberrantly methylated in many human cancers. A dilution series of well-characterized reference samples cross-validated the sensitivity and dynamic range of the approach. Compared to conventional PCR based methods, digital PCR provides a more accurate and more sensitive approach to quantify DNA methylation. As no sodium bisulphite conversion is needed, also analysis of minute amounts of DNA could be carried out efficiently.