Technology to Detect Genome wide DNA Methylation Changes

检测全基因组 DNA 甲基化变化的技术

• 批准号: 6335456
• 负责人: FUMIICHIRO YAMAMOTO
• 金额: $ 19.5万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-11 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6335456
• 关键词: DNA methylation; breast neoplasms; fluorescent dye /probe; genome; human genetic material tag; human tissue; method development; microarray technology; neoplasm /cancer genetics; nucleic acid hybridization; prostate neoplasms

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.

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。比如说， 启动子区域中CpG岛的超甲基化已经越来越多地被 与肿瘤抑制基因的转录失活相关， 致癌作用尽管测定细胞中甲基化程度的技术 尽管已经有了特定的DNA片段或总DNA， 技术，以有效地扫描和鉴定甲基化的变化， 整个基因组 我们开发了一种叫做甲基化敏感扩增片段的方法 长度多态性（MS-AFLP）。这种基于PCR的无偏DNA指纹图谱 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 这些地方在他们的尽头。高/低甲基化可以很容易地 分别通过谱带强度的降低/增加来区分。 MS-AFLP需要少量的模板DNA和电泳的多个 平行采样可轻松识别一致的常见 差异使用匹配的正常/肿瘤DNA的Notl-Msel MS-AFLP实验具有以下优点： 显示了高度可重复的带型差异，其中一些是 与肿瘤表型特异性相关。对其中一些条带进行测序 已经确定了多个同源异型基因， 同源异型基因表达的调控。这些结果证明 MS-AFLP在鉴定与以下疾病相关的表观遗传学改变中的潜力 细胞分化和癌症。 我们将进一步发展这种强大的MS-AFLP方法， 基于DNA微阵列的指纹技术 通用甲基化改变分析杂交技术 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，检查几个的可行性和灵敏度 基于杂交的MS-AFLP和使用先导DNA的非PCR方法 微阵列，并确定进一步发展的最佳方法。在 R33期，我们将寻找前列腺癌和乳腺癌特异性DNA 甲基化改变，分析基因表达，重新检查一些 在DNA甲基化和基因表达中发现了钠的改变， 亚硫酸氢盐修饰法和多重RT-PCR。我们还将建立一个 癌症特异性DNA微阵列用于DNA甲基化的临床检测 改变。