Profiling methylation of individual CpG sites

描述（由申请人提供）：基因CpG高甲基化参与肿瘤的发生和发展，被广泛接受为癌症的标志物和预后指标。特定CpG岛区域的甲基化密度已被证明与基因沉默密切相关，但与单个目标CpG位点无关。异质甲基化模式与肿瘤发生、组织学亚型和癌症的细胞起源有关。大多数CpG岛超甲基化的假设是基于对岛屿区域内仅2至4个CpG位点的研究，这可能不是决定性的。我们将开发一种退化寡核苷酸连接试验（DOLA）来绘制亚硫酸盐处理后启动子区域内一系列CpG位点的甲基化状态。在实验中，我们将设计和合成两对寡核苷酸来检测40bp区域中甲基化(C)或非甲基化(T)状态的单个目标CpG位点，并随后连接两个寡核苷酸。由于邻近的CpG位点可能非常接近目标CpG位点，并且甲基化状态不确定，我们将设计退化的R (G或A)，与甲基化的C或转化的T互补，以涵盖所有可能性。我们将建立e -钙粘蛋白启动子的检测方法。我们将为每个CpG位点设计一系列独特的标签序列，并对标签序列进行微阵列分析，同时测量五种不同癌细胞系（包括正常和癌乳腺癌细胞系、结肠癌、淋巴瘤和肺癌细胞系）中E-Cadherin、p15、p16、MGMT和RASSF1A五种癌症相关基因的CpG位点的甲基化状态。

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.