Shiratori Hiromi, Feinweber Carmen, Knothe Claudia, Lötsch Jörn, Thomas Dominique, Geisslinger Gerd, Parnham Michael J, Resch Eduard
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.
PLoS One. 2016 Oct 17;11(10):e0163184. doi: 10.1371/journal.pone.0163184. eCollection 2016.
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.
DNA甲基化是基因转录的主要调控过程，DNA甲基化异常与包括癌症在内的多种疾病相关。据报道，许多化合物可改变DNA甲基化状态。尽管对具有表观遗传效应的药物的临床应用以及癌症中全基因组低甲基化诊断标志物的使用兴趣日益增加，但用于测量药物对DNA甲基化影响的大规模筛选系统却很有限。在本研究中，我们改进了先前建立的基于荧光偏振的全基因组DNA甲基化检测方法，使其更适用于人类基因组DNA。我们的甲基敏感荧光偏振（MSFP）检测方法具有高度可重复性（批间变异系数 = 1.5%）且准确（r2 = 0.99）。根据信号线性，对于384孔板形式，每个反应仅需50 - 80 ng人类基因组DNA。MSFP是一种简单、快速的方法，因为所有生化反应和最终检测都可在384孔板的一个孔中进行，无需纯化步骤，耗时不到3.5小时。此外，我们证明了MSFP与LINE - 1焦磷酸测序检测方法（一种广泛使用的全基因组DNA甲基化检测方法）之间存在显著相关性。MSFP可用于影响全基因组DNA甲基化状态的化合物的预筛选以及某些类型癌症的诊断。
