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

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          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 (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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          Most cited references51

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          DNA Methylation Analysis: Choosing the Right Method

          In the burgeoning field of epigenetics, there are several methods available to determine the methylation status of DNA samples. However, choosing the method that is best suited to answering a particular biological question still proves to be a difficult task. This review aims to provide biologists, particularly those new to the field of epigenetics, with a simple algorithm to help guide them in the selection of the most appropriate assay to meet their research needs. First of all, we have separated all methods into two categories: those that are used for: (1) the discovery of unknown epigenetic changes; and (2) the assessment of DNA methylation within particular regulatory regions/genes of interest. The techniques are then scrutinized and ranked according to their robustness, high throughput capabilities and cost. This review includes the majority of methods available to date, but with a particular focus on commercially available kits or other simple and straightforward solutions that have proven to be useful.
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            DNA methylation analysis by pyrosequencing.

            Pyrosequencing is a sequencing-by-synthesis method that quantitatively monitors the real-time incorporation of nucleotides through the enzymatic conversion of released pyrophosphate into a proportional light signal. Quantitative measures are of special importance for DNA methylation analysis in various developmental and pathological situations. Analysis of DNA methylation patterns by pyrosequencing combines a simple reaction protocol with reproducible and accurate measures of the degree of methylation at several CpGs in close proximity with high quantitative resolution. After bisulfite treatment and PCR, the degree of each methylation at each CpG position in a sequence is determined from the ratio of T and C. The process of purification and sequencing can be repeated for the same template to analyze other CpGs in the same amplification product. Quantitative epigenotypes are obtained using this protocol in approximately 4 h for up to 96 DNA samples when bisulfite-treated DNA is already available as the starting material.
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              DNA methyltransferase inhibitors and their emerging role in epigenetic therapy of cancer.

              The DNA methyltransferase (DNMT) inhibitors azacytidine and decitabine are the most successful epigenetic drugs to date and are still the most widely used as epigenetic modulators, even though their application for oncological diseases is restricted by their relative toxicity and poor chemical stability. Zebularine (1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one), a more stable and less toxic cytidine analog, is another inhibitor of DNMT with concomitant inhibitory activity towards cytidine deaminase. Unfortunately, there is no new information related to the possible clinical applications of zebularine. Although many new inhibitors of DNMT have been identified, none of them can so far replace azacytidine, decitabine and, to a lesser degree, zebularine. This review summarizes the current data and knowledge about azacytidine, decitabine and zebularine, and their role in present and possible future epigenetic cancer therapy. We also discuss the molecular modes of action of these agents with consideration of their different toxicities and demethylation profiles, reflecting their complex and partially overlapping biological effects.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                17 October 2016
                2016
                : 11
                : 10
                : e0163184
                Affiliations
                [1 ]Project Group Translational Medicine and Pharmacology TMP, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Frankfurt am Main, Germany
                [2 ]Institute of Clinical Pharmacology, Goethe - University, Frankfurt am Main, Germany
                Inc, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: HS ER MJP GG.

                • Data curation: HS.

                • Formal analysis: HS CK JL.

                • Funding acquisition: GG JL MJP HS.

                • Investigation: HS CF CK DT.

                • Methodology: HS CF ER CK.

                • Project administration: HS.

                • Resources: ER CK JL DT GG.

                • Supervision: JL GG.

                • Validation: HS CF.

                • Visualization: HS.

                • Writing – original draft: HS.

                • Writing – review & editing: ER MJP.

                Author information
                http://orcid.org/0000-0002-5826-1401
                Article
                PONE-D-16-19626
                10.1371/journal.pone.0163184
                5066982
                27749902
                54704378-0b32-4f5b-a28a-fd97fc609ca2
                © 2016 Shiratori et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 15 May 2016
                : 2 September 2016
                Page count
                Figures: 4, Tables: 1, Pages: 16
                Funding
                Funded by: Landesoffensive zur Entwicklung wissenschaftlich-ökonomischer Exzellenz (LOEWE), LOEWE-Zentrum für Translationale Medizin und Pharmakologie
                Funded by: Deutsche Forschungsgemeinschaft Excellence Cluster 147 Cardiopulmonary Systems (ECCPS)
                Award Recipient : Gerd Geißlinger
                Funded by: Nakajima Foundation
                Award Recipient :
                Funded by: Else Kröner-Fresenius Foundation Research Training Group Translational Research Innovation - Pharma (TRIP)
                Funded by: funder-id http://dx.doi.org/10.13039/501100004963, Seventh Framework Programme;
                Award ID: 602919
                Award Recipient :
                This work was supported by Landesoffensive zur Entwicklung wissenschaftlich-ökonomischer Exzellenz (LOEWE), LOEWE-Zentrum für Translationale Medizin und Pharmakologie - recipients (GG, JL, MJP), http://www.proloewe.de/en/loewe-research-initiatives/loewe-researchinitiatives/pharmaceutical-research.html; Deutsche Forschungsgemeinschaft Excellence Cluster 147 Cardiopulmonary Systems (ECCPS; GG), http://www.dfg.de/en/research_funding/programmes/list/projectdetails/index.jsp?id=24676099&sort=nr_asc&prg=EXC&wb=2; Nakajima Foundation (HS), http://www.nakajimafound.or.jp/; Else Kröner-Fresenius Foundation Research Training Group Translational Research Innovation – Pharma (TRIP) – recipients (GG, JL), http://www.ekfs.de/de/. Additional support of the analytical environment was gained from the European Union Seventh Framework Programme (FP7/2007 - 2013) under grant agreement no. 602919 (“GLORIA”; JL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and life sciences
                Cell biology
                Chromosome biology
                Chromatin
                Chromatin modification
                DNA methylation
                Biology and life sciences
                Genetics
                Epigenetics
                Chromatin
                Chromatin modification
                DNA methylation
                Biology and life sciences
                Genetics
                Gene expression
                Chromatin
                Chromatin modification
                DNA methylation
                Biology and life sciences
                Genetics
                DNA
                DNA modification
                DNA methylation
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA modification
                DNA methylation
                Biology and life sciences
                Genetics
                Epigenetics
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                DNA methylation
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                Genetics
                Gene expression
                DNA modification
                DNA methylation
                Biology and Life Sciences
                Genetics
                Genomics
                Human Genomics
                Research and Analysis Methods
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                Spectrophotometry
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                Cell Signaling
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                Electrophoretic Techniques
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                Nucleic acids
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                Bioassays and Physiological Analysis
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                Biology and life sciences
                Molecular biology
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                Research and analysis methods
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