Abstract 166: Absolute quantification of DNA methylation markers using methylation-sensitive restriction enzymes and multiplex digital PCR

Abstract

Abstract Aberrant DNA methylation plays important roles in the initiation and progression of cancer. Therefore, DNA methylation markers have important diagnostic and prognostic potential. However, accurate quantification of these markers remains challenging in clinical samples of limited quality or quantity. We introduce a novel, digital approach for the absolute quantification of the amount, density and allele-specificity of DNA methylation. We designed an experimental setup combining efficient methylation-sensitive restriction enzymes with quantitative digital PCR. This allowed to measure a targeted density of DNA methylation independent of bisulphite conversion. In-silico simulations were performed to demonstrate the mathematical validity of the setup. A practical validation was carried out by quantifying RASSF1A promotor methylation in a variety of healthy and malignant reference samples and in an innovative calibration curve. Furthermore, we extended our setup to a multiplex digital PCR experiment. Hereby, methylation markers may be analyzed together with physically linked genetic markers to determine the allele-specificity of the methylation. Overall, our results show an accurate quantification of DNA methylation markers with digital PCR, independent of bisulphite conversion. Effective measurements with high precision could be obtained, even in minute amounts of DNA. Moreover, as the context-density and allele-specificity of methylation can also be determined, mechanisms in cancer biology can now be quantitatively assessed in a digital experiment. Citation Format: Rogier J. Nell, Pieter A. Van der Velden. Absolute quantification of DNA methylation markers using methylation-sensitive restriction enzymes and multiplex digital PCR [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 166.