Florence Mauger1, Magali Kernaleguen1, Céline Lallemand1, Vessela N Kristensen2,3,4, Jean-François Deleuze1,5,6, Jörg Tost1. 1. Laboratory for Epigenetics & Environment, Centre National de Recherche en Génomique Humaine, CEA-Institut de Biologie François Jacob, Evry, France. 2. Department of Cancer Genetics, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway. 3. Department of Clinical Molecular Biology & Laboratory Science (EpiGen), Akershus University Hospital, Division of Medicine, 1476 Lørenskog, Norway. 4. Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway. 5. Centre d'Etudes du Polymorphisme Humain, CEPH-Fondation Jean Dausset, Paris, France. 6. Laboratoire d'Excellence GenMed, France.
Abstract
AIM: 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. MATERIALS & METHODS: 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. RESULTS: 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. CONCLUSION: E-ice-COLD-PCR could prove a useful tool in the context of DNA methylation analysis for personalized medicine.
AIM: The detection of specific DNA methylation patterns bears great promise as biomarker for personalized management of cancerpatients. 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. MATERIALS & METHODS: 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. RESULTS: 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 cancerpatients. CONCLUSION: E-ice-COLD-PCR could prove a useful tool in the context of DNA methylation analysis for personalized medicine.
Authors: Warwick J Locke; Dominic Guanzon; Chenkai Ma; Yi Jin Liew; Konsta R Duesing; Kim Y C Fung; Jason P Ross Journal: Front Genet Date: 2019-11-14 Impact factor: 4.599