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.
Highlights
DNA cytosine methylation at position 5 in the pyrimidine ring (5mC) is predominantly observed in the context of CpG dinucleotides in human [1]
All the restriction enzymes we used generate a CpG overhang at the 5'-terminus on both complementary strands, serving as templates during the sequential terminal extension step with TAMRA-dCTP, a fluorescent labelled cytosine (Step 2)
Fluorescence polarization (FP) detects the binding of small fluorescent molecules to larger objects because fluorescent labelled molecules excited by polarized light emit light with a degree of polarization that is inversely proportional to the rate of molecular rotation [40]
Summary
DNA cytosine methylation at position 5 in the pyrimidine ring (5mC) is predominantly observed in the context of CpG dinucleotides in human [1]. It is a hallmark of transcriptional gene silencing and heterochromatin formation in conjunction with chromatin remodelling factors [2,3,4,5]. It plays crucial roles in key physiological processes, including differentiation and chromosome stability [6,7,8]. 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. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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