Identification and quantification of heterogeneously-methylated DNA fragments using epiallele-sensitive droplet digital polymerase chain reaction (EAST-ddPCR)

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

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.

Details

Original languageEnglish
Pages (from-to)299-312
Number of pages14
JournalCancer Genomics and Proteomics
Volume15
Issue number4
Publication statusPublished - 1 Jul 2018
Peer-reviewedYes

External IDs

PubMed 29976635
ORCID /0000-0002-3549-2477/work/148145340

Keywords

Sustainable Development Goals

Keywords

  • Digital PCR, DNA methylation, Heterogeneously methylated epialleles, Leukemia, Prostate cancer cells