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High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion

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...

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Autores principales: Shiratori, Hiromi, Feinweber, Carmen, Knothe, Claudia, Lötsch, Jörn, Thomas, Dominique, Geisslinger, Gerd, Parnham, Michael J., Resch, Eduard
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066982/
https://www.ncbi.nlm.nih.gov/pubmed/27749902
http://dx.doi.org/10.1371/journal.pone.0163184
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author Shiratori, Hiromi
Feinweber, Carmen
Knothe, Claudia
Lötsch, Jörn
Thomas, Dominique
Geisslinger, Gerd
Parnham, Michael J.
Resch, Eduard
author_facet Shiratori, Hiromi
Feinweber, Carmen
Knothe, Claudia
Lötsch, Jörn
Thomas, Dominique
Geisslinger, Gerd
Parnham, Michael J.
Resch, Eduard
author_sort Shiratori, Hiromi
collection PubMed
description 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 (r(2) = 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.
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spelling pubmed-50669822016-10-27 High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion Shiratori, Hiromi Feinweber, Carmen Knothe, Claudia Lötsch, Jörn Thomas, Dominique Geisslinger, Gerd Parnham, Michael J. Resch, Eduard PLoS One Research Article 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 (r(2) = 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. Public Library of Science 2016-10-17 /pmc/articles/PMC5066982/ /pubmed/27749902 http://dx.doi.org/10.1371/journal.pone.0163184 Text en © 2016 Shiratori et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Shiratori, Hiromi
Feinweber, Carmen
Knothe, Claudia
Lötsch, Jörn
Thomas, Dominique
Geisslinger, Gerd
Parnham, Michael J.
Resch, Eduard
High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion
title High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion
title_full High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion
title_fullStr High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion
title_full_unstemmed High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion
title_short High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion
title_sort high-throughput analysis of global dna methylation using methyl-sensitive digestion
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066982/
https://www.ncbi.nlm.nih.gov/pubmed/27749902
http://dx.doi.org/10.1371/journal.pone.0163184
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