Global DNA Methylation Analysis Using Methylcytosine Dioxygenase
Global DNA Methylation Analysis Using Methylcytosine Dioxygenase
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DOI：
10.1007/978-1-0716-1229-3_9
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Natsumi Taka;W. Yoshida
中科院分区：
文献类型：
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作者：
Natsumi Taka;W. Yoshida
由于在各种癌细胞中检测到整体DNA甲基化水平的改变，因此整体DNA甲基化水平有望作为癌症诊断的生物标志物。基于亚硫酸氢盐的测定已被广泛用作用于定量总体DNA甲基化水平的传统方法。也使用了基于高效液相色谱法（HPLC）的测定和酶联免疫吸附测定（ELISA）;然而，这些方法需要耗时的DNA处理，如亚硫酸氢盐处理、消化或在分析前固定基因组DNA。为了以简单的步骤分析整体DNA甲基化水平，开发了使用十-十一易位（泰特）的酶测定，其氧化基因组DNA上的5-甲基胞嘧啶（5 mC）。琥珀酸在5 mC氧化期间产生;因此，由TET介导的氧化产生的琥珀酸的量与基因组DNA的整体DNA甲基化水平相关。在这一章中，我们详细描述了泰特在大肠杆菌中的表达，并测量了由TET介导的反应产生的琥珀酸的量。基于TET的测定可检测至少100 ng基因组DNA中的整体DNA甲基化水平。
Because the alteration of global DNA methylation levels is detected in various cancer cells, the global DNA methylation level is expected as a biomarker for cancer diagnostics. Bisulfite-based assays have been widely used as a traditional method for the quantification of global DNA methylation levels. High-performance liquid chromatography (HPLC)-based assay and enzyme-linked immunosorbent assay (ELISA) have also been used; however, these methods require time-consuming DNA treatments, such as bisulfite treatment, digestion, or immobilization of genomic DNA before analysis. To analyze global DNA methylation levels in simple steps, an enzymatic assay using Ten-eleven translocation (TET), which oxidizes 5-methylcytosine (5mC) on genomic DNA, was developed. Succinate is produced during 5mC oxidation; thus, the amount of succinate produced by TET-mediated oxidation is correlated with the global DNA methylation level of genomic DNA. In this chapter, we describe the details of the expression of TET inEscherichia coliand the measurement of the amount of succinate produced from the TET-mediated reaction. The TET-based assay detects global DNA methylation levels in at least 100 ng genomic DNA.