EVALUATION OF SEQUENCE SPECIFIC CHANGES IN GENOMIC DNA

我们希望创建一个技术基础设施，作为 DNA CpG甲基化的病理生理学研究基础 癌症。已经证明，DNA的表观遗传修饰形式是 CpG甲基化可能导致突变、癌症，甚至可能导致衰老。为 例如，已经建立了一个非常明确的机制，将5个MEC与 通过5 MeC到5 MeC的转变增加突变负荷 胸苷。此外，CpG甲基化的变化也与 基因沉默、功能性杂合性丢失(LOH)和丢失 表观遗传印记。目前没有一种景观允许研究 单细胞/单染色体水平的甲基化变化。 这项提议的中心焦点是建立一个强大的现场 检测CpG甲基化中序列特异性变化的技术 在哺乳动物的染色体上。这种技术将是特定于序列的， 可同时测量多个轨迹，保存三个 中期染色体的空间组织，并能够 慢性粒细胞白血病中期染色体胞嘧啶甲基化变化的研究 单细胞。将其作为一种实用的、可转让的技术 某些特定的技术成就是可以定义的。这些 增量步骤将得到满足，由此产生的技术 在模型系统中进行评估，以建立绩效的基线。 这种微观方法将使调查者能够区分 在单个基因中存在或不存在特定和/或全局甲基化 肿瘤发展中特定时间点的细胞。这项技术还将 允许调查员发展对正常情况的理解 在分离的单细胞中甲基化基因组的发展。

We wish to create a technical infrastructure that would serve as a foundation for the study of pathophysiology of DNA CpG methylation in cancer. It has been shown that epigenetic modification of DNA in form of CpG methylation can result in mutations, cancer, and possibly aging. For example, a very clear mechanism has been established relating 5 MeC to an increase in mutational load through the transition of 5MeC to thymidine. In addition, changes in CpG methylation have been correlated with gene silencing, functional Loss Of Heterozygosity (LOH), and loss of epigenetic imprint. No current landscape allows for the study of changes in methylation at the single cell/single chromosome level. The central focus of this proposal is to establish a robust in situ technology for detection of sequence specific changes in CpG methylation on mammalian chromosomes. This technology would be sequence specific, capable of surveying multiple loci simultaneously, preserve the three dimensional organization of the metaphase chromosome, and be capable of ascertaining changes in cytosine methylation on metaphase chromosomes in single cells. To create this as a practical and transferable technology certain specific technical achievements can be defined. These incremental steps will be satisfied and the resulting technology evaluated in model systems to establish a base line of performance. This microscopic method will permit the investigator to distinguish the presence or absence of specific and/or global methylation in single cells at specific points in tumor development. This technology will also permit the investigator to develop an understanding of the normal development of the methylated genome in isolated single cells.