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Quantification of DNA methylation independent of sodium bisulfite conversion using methylation‐sensitive restriction enzymes and digital PCR
Author(s) -
Nell Rogier J.,
Steenderen Debby,
Menger Nino V.,
Weitering Thomas J.,
Versluis Mieke,
Velden Pieter A.
Publication year - 2020
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.24111
Subject(s) - dna methylation , digital polymerase chain reaction , sodium bisulfite , epigenetics , biology , bisulfite , methylation , bisulfite sequencing , illumina methylation assay , context (archaeology) , dna , microbiology and biotechnology , in silico , methylated dna immunoprecipitation , polymerase chain reaction , computational biology , genetics , gene , gene expression , chemistry , organic chemistry , paleontology
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.