Profiling methylation of individual CpG sites

分析单个 CpG 位点的甲基化

• 批准号: 7478282
• 负责人: Xin Jiang
• 金额: $ 15.73万
• 依托单位: SIGNOSIS, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-24 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7478282
• 关键词: 5' Flanking Region; Biological Assay; Biological Markers; Breast; Breast Cancer Cell; Cancer cell line; Cell Line; Cells; Classification; Colon; Colon Lymphoma; CpG Islands; Cytosine Nucleotides; DNA; DNA Ligation; Diagnostic; E-Cadherin; Epigenetic Process; Event; Gene Expression; Gene Silencing; Genes; Genomics; Genus Cola; Human; Hypermethylation; Individual; Island; Ligation; Lymphoma; MGMT gene; Malignant Neoplasms; Malignant neoplasm of lung; Maps; Measures; Methods; Methylation; Microarray Analysis; Monitor; Neoplasms; Numbers; Oligonucleotides; Pathogenesis; Patients; Pattern; Phase I Clinical Trials; Polymerase Chain Reaction; Positioning Attribute; Prognostic Marker; Promoter Regions; Reporting; Series; Side; Site; Staging; Survival Rate; Technology; Testing; Uncertainty; base; bisulfite; cancer therapy; density; design; high throughput technology; innovation; malignant breast neoplasm; prognostic; promoter; tumor; tumor initiation; tumorigenesis

DESCRIPTION (provided by applicant): The CpG hypermethylation of genes are involved in tumor initiation and progression, and widely accepted as a marker and prognostic indicator of cancer. The density of methylation in a certain CpG island region has been demonstrated to be closely related to gene silencing, but not individual target CpG sites. The heterogeneous methylation patterns are associated with tumorigenesis, histological subtypes and cell origin of cancers. Most of CpG island hypermethylation is assumed based on the study of only 2 to 4 CpG sites within an island region, which may not be conclusive. We will develop a degenerate oligonucleotides ligation assay (DOLA) to map methylation status of a series of CpG sites within a promoter region upon bisulfite treatment. In the assay, we will design and synthesized two pairs of oligos to detect an individual target CpG site for the methylated (C) or unmethylated (T) states in a 40bp region, and subsequently ligate two oligonucleotides. Since the neighbor CpG sites might be very close to the target CpG site and uncertain on methylation status, we will design the degenerated R (G or A), complementary to methylated C or converted T to cover all of possibilities. We will set up the assay for E-cadherin promoter. we will design a series of unique tag sequences to each CpG site and use mircoarray analysis of the tag sequences to simultaneously measure the methylated status of individual CpG sites for five cancer-related genes, E-Cadherin, p15, p16, MGMT, and RASSF1A in five different cancer cell lines including normal and cancer breast cell lines, colon, lymphoma and lung cancer cell lines.

描述（由申请人提供）：基因的CpG超甲基化参与肿瘤的发生和进展，并被广泛接受为癌症的标志物和预后指标。已证明在某个CpG岛区域中的甲基化密度与基因沉默密切相关，而不是单个靶CpG位点。甲基化模式的异质性与肿瘤发生、组织学亚型和肿瘤的细胞起源有关。大多数CpG岛超甲基化是基于对岛区域内仅2至4个CpG位点的研究而假设的，这可能不是决定性的。我们将开发一种简并寡核苷酸连接试验（DOLA），以定位亚硫酸氢盐处理后启动子区域内一系列CpG位点的甲基化状态。在检测中，我们将设计并合成两对寡核苷酸，以检测40 bp区域中甲基化（C）或非甲基化（T）状态的单个靶CpG位点，并随后连接两个寡核苷酸。由于相邻的CpG位点可能非常接近靶CpG位点，并且甲基化状态不确定，因此我们将设计简并的R（G或A），与甲基化的C或转化的T互补，以覆盖所有的可能性。我们将建立E-cadherin启动子的检测方法。我们将为每个CpG位点设计一系列独特的标记序列，并使用标记序列的微阵列分析来同时测量五种不同癌细胞系（包括正常和癌乳腺细胞系、结肠癌、淋巴瘤和肺癌细胞系）中五种癌症相关基因（E-钙粘蛋白、p15、p16、MGMT和RASSF 1A）的单个CpG位点的甲基化状态。