A real-time assay for CpG-specific cytosine-C5 methyltransferase activity.
A real-time assay for CpG-specific cytosine-C5 methyltransferase activity.
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DOI：
10.1093/nar/gkq047
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发表时间：
2010-05
影响因子：
14.9
通讯作者：
Roach PL
中科院分区：
文献类型：
--
作者：
Wood RJ;McKelvie JC;Maynard-Smith MD;Roach PL
CpG特异性胞嘧啶-C5甲基转移酶活性的实时测定已经开发。该检测试剂盒采用了一种断裂光寡核苷酸，其中未甲基化的5′-CG-3′位点的甲基化与荧光信号的产生酶促偶联。这种灵敏的测定方法可以测量低至0.34 ± 0.06 fmol/s的DNA甲基化速率。该测定具有重现性，6次独立测量的变异系数为4.5%。使用线性校准曲线根据荧光信号准确测量产物浓度，其拟合优度（R2）高于0.98。寡核苷酸底物含有三个C5甲基化胞嘧啶残基和一个未甲基化的5′-CG-3′位点。甲基化产生含有限制酶GlaI的最佳底物的寡核苷酸。完全甲基化的寡核苷酸的切割导致荧光团与猝灭剂分离，从而使荧光成比例增加。该方法已用于测定DNMT 1（人类细胞中主要的维持甲基转移酶）的活性，并用于细菌胞嘧啶-C5甲基转移酶M. SssI的动力学表征。该测定已被证明适用于以高通量形式实时监测DNMT 1活性，具有低背景信号和在长时间内获得线性甲基化速率的能力，使其成为高通量筛选抑制剂的有前途的方法。
A real-time assay for CpG-specific cytosine-C5 methyltransferase activity has been developed. The assay applies a break light oligonucleotide in which the methylation of an unmethylated 5′-CG-3′ site is enzymatically coupled to the development of a fluorescent signal. This sensitive assay can measure rates of DNA methylation down to 0.34 ± 0.06 fmol/s. The assay is reproducible, with a coefficient of variation over six independent measurements of 4.5%. Product concentration was accurately measured from fluorescence signals using a linear calibration curve, which achieved a goodness of fit (R2) above 0.98. The oligonucleotide substrate contains three C5-methylated cytosine residues and one unmethylated 5′-CG-3′ site. Methylation yields an oligonucleotide containing the optimal substrate for the restriction enzyme GlaI. Cleavage of the fully methylated oligonucleotide leads to separation of fluorophore from quencher, giving a proportional increase in fluorescence. This method has been used to assay activity of DNMT1, the principle maintenance methyltransferase in human cells, and for the kinetic characterization of the bacterial cytosine-C5 methyltransferase M.SssI. The assay has been shown to be suitable for the real-time monitoring of DNMT1 activity in a high-throughput format, with low background signal and the ability to obtain linear rates of methylation over long periods, making this a promising method of high-throughput screening for inhibitors.
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DOI：
10.1073/pnas.240460997
发表时间：
2000-12-05
影响因子：
11.1
作者：
Biggins, JB;Prudent, JR;Thorson, JS
通讯作者：
Thorson, JS
影响因子：
3.2
作者：
EHRLICH, M;WILSON, GG;GEHRKE, CW
通讯作者：
GEHRKE, CW
影响因子：
2.9
作者：
Flynn, J;Glickman, JF;Reich, NO
通讯作者：
Reich, NO
影响因子：
4.8
作者：
Hermann, A;Schmitt, S;Jeltsch, A
通讯作者：
Jeltsch, A
用于实时监测 DNA 甲基化的发夹荧光 DNA 探针
影响因子：
7.4
作者：
Li, Jun;Yan, Hongfei;Zhou, Xingwang
通讯作者：
Zhou, Xingwang