Quantification of DNA methylation independent of sodium bisulfite conversion using methylation-sensitive restriction enzymes and digital PCR.

ABSTRACT

Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one epigenetic aspect but is challenging to quantify. In this study, we introduce a digital approach for the quantification of the amount and density of DNA methylation. We designed an experimental setup combining efficient methylation-sensitive restriction enzymes with digital polymerase chain reaction (PCR) to quantify a targeted density of DNA methylation independent of bisulfite conversion. By using a stable reference and comparing experiments treated and untreated with these enzymes, copy number instability could be properly normalized. In silico simulations demonstrated the mathematical validity of the setup and showed that the measurement precision depends on the amount of input DNA and the fraction methylated alleles. This uncertainty could be successfully estimated by the confidence intervals. Quantification of RASSF1 promoter methylation in a variety of healthy and malignant samples and in a calibration curve confirmed the high accuracy of our approach, even in minute amounts of DNA. Overall, our results indicate the possibility of quantifying DNA methylation with digital PCR, independent of bisulfite conversion. Moreover, as the context-density of methylation can also be determined, biological mechanisms can now be quantitatively assessed.