Direct DNA methylation profiling using methyl binding domain proteins.
Direct DNA methylation profiling using methyl binding domain proteins.
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DOI：
10.1021/ac1010316
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发表时间：
2010-06-15
影响因子：
7.4
通讯作者：
Chagovetz, Alexander
中科院分区：
文献类型：
--
作者：
Yu, Yinni;Blair, Steve;Gillespie, David;Jensen, Randy;Myszka, David;Badran, Ahmed H.;Ghosh, Indraneel;Chagovetz, Alexander
DNA甲基化通过建立抑制转录的异染色质结构导致基因沉默，研究表明启动子区域CpG岛的胞嘧啶甲基化是早期癌症发展的前兆。已知哺乳动物天然存在的甲基结合域（MBD）蛋白与甲基化CpG二核苷酸（mCpG）结合，随后招募其他染色质修饰蛋白抑制转录。检测甲基化DNA的常规方法包括在进行测定之前进行亚硫酸氢盐处理或免疫沉淀。我们专注于使用表面结合甲基化探针的直接微阵列分析的概念验证研究。重组蛋白1xMBD-GFP识别杂交dsDNA的CpG序列的半甲基化和对称甲基化，同时对对称甲基化基序表现出更大的亲和力。从这些研究中，对于对称mCpG， 1xMBD-GFP的KD范围为106 nM至870 nM，这取决于甲基化位点与传感器表面的接近程度。非对称甲基化基序的KD值始终较高（> 2µM），但对称和半甲基化基序之间的结合选择性在4到30之间，对靠近表面的位点或邻近的多个位点的选择性降低，我们将其归因于空间效应。将偏态正态概率密度函数拟合到我们的数据中，我们估计我们的方法识别甲基化CpG位点的准确率为97.5%，可以通过优化探针设计和表面密度来提高这一精度。
Methylation of DNA is responsible for gene silencing by establishing heterochromatin structure that represses transcription, and studies have shown that cytosine methylation of CpG islands in promoter regions acts as a precursor to early cancer development. The naturally occurring methyl binding domain (MBD) proteins from mammals are known to bind to the methylated CpG dinucleotide (mCpG), and subsequently recruit other chromatin-modifying proteins to suppress transcription. Conventional methods of detection for methylated DNA involve bisulfite treatment or immunoprecipitation prior to performing an assay. We focus on proof-of-concept studies for a direct microarray-based assay using surface-bound methylated probes. The recombinant protein 1xMBD-GFP recognizes hemi-methylation and symmetric methylation of the CpG sequence of hybridized dsDNA, while displaying greater affinity for the symmetric methylation motif, as evaluated by SPR. From these studies, for symmetric mCpG, the KD for 1xMBD-GFP ranged from 106 nM to 870 nM, depending upon the proximity of the methylation site to the sensor surface. The KD values for non-symmetrical methylation motifs were consistently greater (> 2 µM), but the binding selectivity between symmetric and hemi-methylation motifs ranged from 4 to 30, with reduced selectivity for sites close to the surface or multiple sites in proximity, which we attribute to steric effects. Fitting skew normal probability density functions to our data, we estimate an accuracy of 97.5% for our method in identifying methylated CpG loci, which can be improved through optimization of probe design and surface density.
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DOI：
10.1073/pnas.93.18.9821
发表时间：
1996-09-03
影响因子：
11.1
作者：
Herman, JG;Graff, JR;Baylin, SB
通讯作者：
Baylin, SB
影响因子：
14.9
作者：
Jørgensen HF;Adie K;Chaubert P;Bird AP
通讯作者：
Bird AP
影响因子：
14.9
作者：
Gonzalgo, ML;Jones, PA
通讯作者：
Jones, PA
影响因子：
4.7
作者：
Hermisson, M;Klumpp, A;Weller, M
通讯作者：
Weller, M
影响因子：
158.5
作者：
Hegi, ME;Diserens, A;Stupp, R
通讯作者：
Stupp, R