Enrichment of methylated molecules using enhanced-ice-co-amplification at lower denaturation temperature-PCR (E-ice-COLD-PCR) for the sensitive detection of disease-related hypermethylation

• Published in: Epigenomics Vol. 10; no. 5; pp. 525 - 537
• Main Authors: Mauger, Florence, Kernaleguen, Magali, Lallemand, Céline, Kristensen, Vessela N, Deleuze, Jean-François, Tost, Jörg
• Format: Journal Article
• Language: English
• Published: England Future Medicine Ltd 01.05.2018; Future Medicine
• Subjects: Amplification; Biochemistry, Molecular Biology; Biomarkers; Bisulfite; Breast cancer; Cancer; Cold; Cold starts; Denaturation; DNA methylation; DNA probes; Enrichment; Epigenetics; Genetics; Genomics; Ice; Life Sciences; Mutation; Nucleic acids; Polymerase chain reaction; Precision medicine; United States > US; Germany; multiplex and circulating cell-free DNA; quantification; E-ice-COLD-PCR; methylation; bisulfite converted DNA
• ISSN: 1750-1911; 1750-192X
• DOI: 10.2217/epi-2017-0166

The detection of specific DNA methylation patterns bears great promise as biomarker for personalized management of cancer patients. Co-amplification at lower denaturation temperature-PCR (COLD-PCR) assays are sensitive methods, but have previously only been able to analyze loss of DNA methylation. Enhanced (E)-ice-COLD-PCR reactions starting from 2 ng of bisulfite-converted DNA were developed to analyze methylation patterns in two promoters with locked nucleic acid (LNA) probes blocking amplification of unmethylated CpGs. The enrichment of methylated molecules was compared to quantitative (q)PCR and quantified using serial dilutions. E-ice-COLD-PCR allowed the multiplexed enrichment and quantification of methylated DNA. Assays were validated in primary breast cancer specimens and circulating cell-free DNA from cancer patients. E-ice-COLD-PCR could prove a useful tool in the context of DNA methylation analysis for personalized medicine.