title: High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion.
abstract: DNA methylation is a major regulatory process of gene transcription, and aberrant DNA methylation is associated with various diseases including cancer. Many compounds have been reported to modify DNA methylation states. Despite increasing interest in the clinical application of drugs with epigenetic effects, and the use of diagnostic markers for genome-wide hypomethylation in cancer, large-scale screening systems to measure the effects of drugs on DNA methylation are limited. In this study, we improved the previously established fluorescence polarization-based global DNA methylation assay so that it is more suitable for application to human genomic DNA. Our methyl-sensitive fluorescence polarization (MSFP) assay was highly repeatable (inter-assay coefficient of variation = 1.5%) and accurate (r2 = 0.99). According to signal linearity, only 50-80 ng human genomic DNA per reaction was necessary for the 384-well format. MSFP is a simple, rapid approach as all biochemical reactions and final detection can be performed in one well in a 384-well plate without purification steps in less than 3.5 hours. Furthermore, we demonstrated a significant correlation between MSFP and the LINE-1 pyrosequencing assay, a widely used global DNA methylation assay. MSFP can be applied for the pre-screening of compounds that influence global DNA methylation states and also for the diagnosis of certain types of cancer.
authors.affiliation: Project Group Translational Medicine and Pharmacology TMP, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Frankfurt am Main, Germany.
authors.forename: Hiromi
authors.identifier: http://orcid.org/0000-0002-5826-1401
authors.initials: H
authors.lastname: Shiratori
authors.affiliation: Project Group Translational Medicine and Pharmacology TMP, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Frankfurt am Main, Germany.
authors.forename: Carmen
authors.initials: C
authors.lastname: Feinweber
authors.affiliation: Institute of Clinical Pharmacology, Goethe - University, Frankfurt am Main, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Knothe
authors.affiliation: Project Group Translational Medicine and Pharmacology TMP, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Frankfurt am Main, Germany.|Institute of Clinical Pharmacology, Goethe - University, Frankfurt am Main, Germany.
authors.forename: Jörn
authors.initials: J
authors.lastname: Lötsch
authors.affiliation: Institute of Clinical Pharmacology, Goethe - University, Frankfurt am Main, Germany.
authors.forename: Dominique
authors.initials: D
authors.lastname: Thomas
authors.affiliation: Project Group Translational Medicine and Pharmacology TMP, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Frankfurt am Main, Germany.|Institute of Clinical Pharmacology, Goethe - University, Frankfurt am Main, Germany.
authors.forename: Gerd
authors.initials: G
authors.lastname: Geisslinger
authors.affiliation: Project Group Translational Medicine and Pharmacology TMP, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Frankfurt am Main, Germany.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Parnham
authors.affiliation: Project Group Translational Medicine and Pharmacology TMP, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Frankfurt am Main, Germany.
authors.forename: Eduard
authors.initials: E
authors.lastname: Resch
mesh_terms: D010582:Bacteriophage lambda / Q000235:genetics; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D018899:CpG Islands; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D019175:DNA Methylation; D004279:DNA, Viral / Q000032:analysis / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods*; D015894:Genome, Human; D006801:Humans; D053719:Tandem Mass Spectrometry
chemical_list: D004279:DNA, Viral; D004247:DNA