Technology to Detect Genome wide DNA Methylation Changes

描述：(申请人提供) DNA甲基化的体细胞表观遗传改变与 发育、细胞分化和肿瘤转化。例如, 启动子区域的CpG岛的超甲基化已日益增多 与肿瘤抑制基因转录失活有关 致癌。虽然确定甲基化程度的技术 已经有了特定的DNA片段或总DNA，但很少有 高效扫描和识别基因甲基化变化的技术 整个基因组。 我们开发了一种名为甲基化敏感扩增片段的方法 长度多态(MS-AFLP)。基于聚合酶链式反应的无偏DNA指纹分析 这项技术允许鉴定显示DNA的切割位点 甲基化改变，随后允许分离DNA片段 在这些地点的尽头。高甲基化/低甲基化很容易 分别以谱带强度的降低/增加来区分。 MS-AFLP需要少量的模板DNA和多个 并行采样可轻松识别一致的常见 不同之处。利用匹配的正常/肿瘤DNA进行NOTL-MSEL MS-AFLP实验 显示出高度可重复性的带型差异，其中一些是 与肿瘤表型有明确关联。对其中一些条带进行测序 已经确定了多个同源异型基因以及参与 同源异型基因表达的调控。这些结果证明了 MS-AFLP在鉴定与脑脊髓炎相关的表观遗传学改变方面的潜力 细胞分化与癌症。 我们将通过转化凝胶进一步发展这种强大的MS-AFLP方法 基于DNA微阵列的电泳法指纹分析技术 杂交技术在甲基化变异分析中的应用 几个生物学问题。在R21阶段，我们将构建一个试点DNA 微阵列面板，考察几种方法的可行性和敏感性 利用先导DNA的MS-AFLP和非PCR杂交方法 基因芯片，并确定最佳方法(S)进行进一步发展。在 R33期，我们将寻找前列腺癌和乳腺癌的特异性DNA 甲基化改变，分析基因表达，重新检查一些 钠对DNA甲基化和基因表达的影响 亚硫酸氢盐修饰法和多重RT-PCR。我们还将构建一个 用于临床检测DNA甲基化的肿瘤特异性DNA微阵列 改装。

DESCRIPTION: (Provided by applicant) Somatic epigenetic alterations in DNA methylation are tightly linked to development, cell differentiation and neoplastic transformation. For instance, hypermethylation of CpG islands in promoter regions has been increasingly associated with transcriptional inactivation of tumor suppressor genes in carcinogenesis. Although techniques to determine the degree of methylation in specific DNA segments or in total DNA have been available, there are few techniques to efficiently scan and identify changes in methylation in the entire genome. We have developed a method called Methylation Sensitive-Amplified Fragment Length Polymorphism (MS-AFLP). This PCR-based unbiased DNA fingerprinting technique permits the identification of the cleavage sites that exhibit DNA methylation alterations and subsequently allows the isolation of DNA fragments with these sites at their ends. Hyper/hypomethylation can easily be differentiated by the decrease/increase of band intensity, respectively. MS-AFLP requires low amounts of template DNA and electrophoresis of multiple samples in parallel enables easy identification of consistent common differences. Notl-Msel MS-AFLP experiments using matched normal/tumor DNA have shown highly reproducible differences in banding patterns some of which were specifically linked with the tumor phenotype. Sequencing some of these bands has identified multiple numbers of homeotic genes and the genes involved in the regulation of homeotic gene expression. These results demonstrate the potential of MS-AFLP in identifying epigenetic alterations associated with cell differentiation and cancer. We will further develop this powerful MS-AFLP method by transforming the gel electrophoresis-based fingerprinting technique into a DNA microarray-based hybridization technique for general use of methylation alteration analysis of several biological problems. In the R21 phase, we will construct a pilot DNA microarray panel, examine the feasibility and sensitivity of several hybridization-based MS-AFLP and non-PCR methods using the pilot DNA microarray, and determine the best method(s) for further development. In the R33 phase, we will search for the prostate and breast cancer-specific DNA methylation alterations, analyze the gene expression, re-examine some of the identified alterations in DNA methylation and gene expression by the sodium bisulfite modification method and multiplex RT -PCR. We will also construct a cancer-specific DNA microarray for the clinical detection of DNA methylation alterations.