A novel methyl-binding domain protein enrichment method for identifying genome-wide tissue-specific DNA methylation from nanogram DNA samples.
A novel methyl-binding domain protein enrichment method for identifying genome-wide tissue-specific DNA methylation from nanogram DNA samples.
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DOI：
10.1186/1756-8935-6-17
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发表时间：
2013-06-07
影响因子：
3.9
通讯作者：
Merbs SL
中科院分区：
文献类型：
--
作者：
Oliver VF;Wan J;Agarwal S;Zack DJ;Qian J;Merbs SL
越来越多的证据表明，DNA甲基化在组织特异性分化中发挥了作用。目前利用甲基结合结构域蛋白(MBD)进行甲基组分析的方法仅限于大的(≥1μg)DNA样本，限制了对小组织样本的分析。在这里，我们提出了一种技术，它能够从纳克量的DNA中表征全基因组组织特定的甲基化模式。我们开发了一种方法，利用MBD2b/MBD3L1富集甲基化DNA，经激酶处理的连接介导的PCR扩增(MeKL)和杂交，以获得全面的高通量相对甲基化(CHAME)定制平铺阵列，我们称之为MeKL-CHIP。与添加聚乙二醇组分相结合的激酶修饰使连接介导的聚合酶链式反应扩增倍数增加到20倍以上，使起始DNA扩增倍数达到400倍。我们已经证明，MeKL-ChIP可以应用于小至20 ng的DNA，从而能够对小DNA样本进行全面分析。将MeKL芯片应用于小鼠视网膜(一个有限的组织来源)和脑，共鉴定了2498个组织特异性差异甲基化区域(T-DMRS)。通过焦磷酸测序验证了前5个T-DMRS(Rgs20、Hes2、NFIC、Cockbr和Six3os1)。由于MBD2b/MBD3L1蛋白复合体的结合特性，MeKL-CHIP能够对纳克量的DNA进行全基因组甲基化分析，并具有广泛的观察到与预期的CpG比率。这一方法使首次对小鼠视网膜全基因组甲基化进行了分析，从而表征了新的T-DMR。
Growing evidence suggests that DNA methylation plays a role in tissue-specific differentiation. Current approaches to methylome analysis using enrichment with the methyl-binding domain protein (MBD) are restricted to large (≥1 μg) DNA samples, limiting the analysis of small tissue samples. Here we present a technique that enables characterization of genome-wide tissue-specific methylation patterns from nanogram quantities of DNA. We have developed a methodology utilizing MBD2b/MBD3L1 enrichment for methylated DNA, kinase pre-treated ligation-mediated PCR amplification (MeKL) and hybridization to the comprehensive high-throughput array for relative methylation (CHARM) customized tiling arrays, which we termed MeKL-chip. Kinase modification in combination with the addition of PEG has increased ligation-mediated PCR amplification over 20-fold, enabling >400-fold amplification of starting DNA. We have shown that MeKL-chip can be applied to as little as 20 ng of DNA, enabling comprehensive analysis of small DNA samples. Applying MeKL-chip to the mouse retina (a limited tissue source) and brain, 2,498 tissue-specific differentially methylated regions (T-DMRs) were characterized. The top five T-DMRs (Rgs20, Hes2, Nfic, Cckbr and Six3os1) were validated by pyrosequencing. MeKL-chip enables genome-wide methylation analysis of nanogram quantities of DNA with a wide range of observed-to-expected CpG ratios due to the binding properties of the MBD2b/MBD3L1 protein complex. This methodology enabled the first analysis of genome-wide methylation in the mouse retina, characterizing novel T-DMRs.
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影响因子：
3.7
作者：
Merbs SL;Khan MA;Hackler L Jr;Oliver VF;Wan J;Qian J;Zack DJ
通讯作者：
Zack DJ
影响因子：
3.8
作者：
Dedeurwaerder, Sarah;Defrance, Matthieu;Fuks, Francois
通讯作者：
Fuks, Francois
影响因子：
14.9
作者：
PHEIFFER, BH;ZIMMERMAN, SB
通讯作者：
ZIMMERMAN, SB
影响因子：
5
作者：
Rauch, T;Pfeifer, GP
通讯作者：
Pfeifer, GP
影响因子：
46.9
作者：
通讯作者：
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