The application of methylation specific electrophoresis (MSE) to DNA methylation analysis of the 5' CpG island of mucin in cancer cells.
The application of methylation specific electrophoresis (MSE) to DNA methylation analysis of the 5' CpG island of mucin in cancer cells.
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DOI：
10.1186/1471-2407-12-67
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发表时间：
2012-02-14
期刊：
影响因子：
3.8
通讯作者：
Yonezawa S
中科院分区：
文献类型：
--
作者：
Yokoyama S;Kitamoto S;Yamada N;Houjou I;Sugai T;Nakamura S;Arisaka Y;Takaori K;Higashi M;Yonezawa S
基因组 DNA 中 CpG 位点的甲基化在基因调控尤其是基因沉默中发挥着重要作用。我们报道了粘蛋白表达的表观遗传调控机制，粘蛋白是潜在恶性肿瘤和人类肿瘤早期检测的标志物。启动子区域的表观遗传变化似乎是粘蛋白表达的第一步。因此，检测启动子甲基化状态对于癌症的早期诊断、监测肿瘤行为以及评估肿瘤对靶向治疗的反应具有重要意义。然而，传统的DNA甲基化分析方法需要大量DNA且灵敏度较低。在这里，我们报告了使用巢式 PCR 方法的亚硫酸氢盐-DGGE（变性梯度凝胶电泳）的改进版本。我们将该方法命名为甲基化特异性电泳（MSE）。 MSE 方法由以下步骤组成：(a) 基因组 DNA 的亚硫酸氢盐处理，(b) 通过巢式 PCR 方法扩增目标 DNA，以及 (c) 应用于 DGGE。为了检验 MSE 方法是否能够分析各种样品中粘蛋白基因的 DNA 甲基化，我们将其应用于从状态细胞系、乙醇固定的结肠隐窝和人胰液中获得的 DNA。 MSE方法大大减少了DNA输入量。区分不同甲基化状态的检测下限为< 0.1%，可检测的最小DNA量为20 pg，只需从少数细胞中即可获得。我们还表明，MSE 可用于分析具有挑战性的样本，例如人类分离的结肠隐窝或人类胰液，从中只能提取少量 DNA。 MSE方法可以提供甲基化序列谱的定性信息。 MSE 方法可以对几乎任何多个 CpG 位点块的 DNA 甲基化模式进行灵敏且特异的分析。 MSE方法可应用于分析许多不同临床样本中的DNA甲基化状态，这可能有助于识别新的风险标记。
Methylation of CpG sites in genomic DNA plays an important role in gene regulation and especially in gene silencing. We have reported mechanisms of epigenetic regulation for expression of mucins, which are markers of malignancy potential and early detection of human neoplasms. Epigenetic changes in promoter regions appear to be the first step in expression of mucins. Thus, detection of promoter methylation status is important for early diagnosis of cancer, monitoring of tumor behavior, and evaluating the response of tumors to targeted therapy. However, conventional analytical methods for DNA methylation require a large amount of DNA and have low sensitivity. Here, we report a modified version of the bisulfite-DGGE (denaturing gradient gel electrophoresis) using a nested PCR approach. We designated this method as methylation specific electrophoresis (MSE). The MSE method is comprised of the following steps: (a) bisulfite treatment of genomic DNA, (b) amplification of the target DNA by a nested PCR approach and (c) applying to DGGE. To examine whether the MSE method is able to analyze DNA methylation of mucin genes in various samples, we apply it to DNA obtained from state cell lines, ethanol-fixed colonic crypts and human pancreatic juices. The MSE method greatly decreases the amount of input DNA. The lower detection limit for distinguishing different methylation status is < 0.1% and the detectable minimum amount of DNA is 20 pg, which can be obtained from only a few cells. We also show that MSE can be used for analysis of challenging samples such as human isolated colonic crypts or human pancreatic juices, from which only a small amount of DNA can be extracted. The MSE method can provide a qualitative information of methylated sequence profile. The MSE method allows sensitive and specific analysis of the DNA methylation pattern of almost any block of multiple CpG sites. The MSE method can be applied to analysis of DNA methylation status in many different clinical samples, and this may facilitate identification of new risk markers.
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影响因子：
7.4
作者：
Yonezawa, Suguru;Kitajima, Shinichi;Higashi, Michiyo;Osako, Masahiko;Horinouchi, Michiko;Yokoyama, Seiya;Kitamoto, Sho;Yamada, Norishige;Tamura, Yukihiro;Shimizu, Takeshi;Tabata, Mineo;Goto, Masamichi
通讯作者：
Goto, Masamichi
影响因子：
78.5
作者：
Ushijima, T
通讯作者：
Ushijima, T
影响因子：
82.9
作者：
Kononen, J;Bubendorf, L;Kallioniemi, OP
通讯作者：
Kallioniemi, OP
影响因子：
3
作者：
Yamada, Norishige;Nishida, Yukari;Yonezawa, Suguru
通讯作者：
Yonezawa, Suguru
DOI：
10.1007/s00534-006-1203-4
发表时间：
2007-11-01
期刊：
JOURNAL OF HEPATO-BILIARY-PANCREATIC SURGERY
影响因子：
--
作者：
Shinozuka, Nozomi;Okada, Katsuya;Koyama, Isamu
通讯作者：
Koyama, Isamu