A qPCR technology for direct quantification of methylation in untreated DNA

Kamilla Kolding Bendixen (), Maria Mindegaard, Samantha Epistolio, Giulia Dazio, Francesco Marchi, Paolo Spina, Eva C. Arnspang, Mette Soerensen, Ulf Bech Christensen, Milo Frattini and Rasmus Koefoed Petersen
Additional contact information
Kamilla Kolding Bendixen: PentaBase A/S
Maria Mindegaard: PentaBase A/S
Samantha Epistolio: Institute of Pathology, Ente Ospedaliero Cantonale (EOC)
Giulia Dazio: Institute of Pathology, Ente Ospedaliero Cantonale (EOC)
Francesco Marchi: Neurocenter of the Southern Switzerland, Regional Hospital of Lugano
Paolo Spina: Institute of Pathology, Ente Ospedaliero Cantonale (EOC)
Eva C. Arnspang: University of Southern Denmark
Mette Soerensen: University of Southern Denmark
Ulf Bech Christensen: PentaBase A/S
Milo Frattini: Institute of Pathology, Ente Ospedaliero Cantonale (EOC)
Rasmus Koefoed Petersen: PentaBase A/S

Nature Communications, 2023, vol. 14, issue 1, 1-11

Abstract: Abstract DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases. Analysis of DNA methylation patterns has until now been dependent on either a chemical or an enzymatic pre-treatment, which are both time consuming procedures and potentially biased due to incomplete treatment. We present a qPCR technology, EpiDirect®, that allows for direct PCR quantification of DNA methylations using untreated DNA. EpiDirect® is based on the ability of Intercalating Nucleic Acids (INA®) to differentiate between methylated and unmethylated cytosines in a special primer design. With this technology, we develop an assay to analyze the methylation status of a region of the MGMT promoter used in treatment selection and prognosis of glioblastoma patients. We compare the assay to two bisulfite-relying, methyl-specific PCR assays in a study involving 42 brain tumor FFPE samples, revealing high sensitivity, specificity, and the clinical utility of the method.

Date: 2023
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DOI: 10.1038/s41467-023-40873-y

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