Open AccessEnrichment 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
Author(s) -
Florence Mauger,
Magali Kernaleguen,
Céline Lallemand,
Vessela Kristensen,
JeanFrançois Deleuze,
Jörg Tost
Publication year - 2018
Publication title -
epigenomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.265
H-Index - 60
eISSN - 1750-1911
pISSN - 1750-192X
DOI - 10.2217/epi-2017-0166
Subject(s) - biology , microbiology and biotechnology , dna methylation , bisulfite sequencing , dna , cold pcr , nucleic acid , locked nucleic acid , methylation , real time polymerase chain reaction , bisulfite , polymerase chain reaction , context (archaeology) , biochemistry , oligonucleotide , gene expression , gene , point mutation , paleontology , mutation
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.