Identification and Quantification of Heterogeneously-methylated DNA Fragments Using Epiallele-sensitive Droplet Digital Polymerase Chain Reaction (EAST-ddPCR)

Abstract

Background/aim: DNA methylation plays an important role in the initiation and propagation of carcinogenesis; however, the role of heterogeneously methylated epialleles is currently not well studied, also due to the lack of sensitive, unbiased and high throughput methods. Here, a newly developed droplet digital PCR (ddPCR)-based method was evaluated regarding its ability to quantify such heterogeneously methylated epialleles with sufficient analytical sensitivity and specificity.

Materials and methods: Genomic DNA from blood leukocytes and bone marrow aspirate of an 8-year old male with B-cell acute lymphoblastic leukemia (B-ALL) and from normal and malignant prostate cell lines were analysed using ddPCR.

Results: By using these DNA samples, the specificity of an applied set of fluorescence-labeled probes was demonstrated as a proof of concept.

Conclusion: All individual heterogeneously-methylated epialleles were quantifiable by a set of fluorescence-labeled probes with complementary sequences to epialleles in a closed-tube and high-throughput manner. The new method named epiallele-sensitive droplet digital PCR (EAST-ddPCR) may give new insights in the generation and regulation of epialleles and may help in finding new biomarkers for the diagnosis of benign und malignant diseases.

Keywords: DNA methylation; Digital PCR; heterogeneously methylated epialleles; leukemia; prostate cancer cells.