EVALUATION OF SEQUENCE SPECIFIC CHANGES IN GENOMIC DNA

我们希望建立一个技术基础设施， DNA甲基化的病理生理学研究的基础 癌已经表明，DNA的表观遗传修饰形式是 CpG甲基化可导致突变，癌症，并可能老化。为 例如，已经建立了一个非常明确的机制，将5 MeC与 通过5 MeC到 胸苷此外，CpG甲基化的变化与 基因沉默、功能性杂合性缺失（洛）和缺失 表观遗传印记目前没有任何景观允许研究 单细胞/单染色体水平的甲基化变化。 该建议的核心重点是建立一个强大的原地 用于检测CpG甲基化中序列特异性变化的技术 在哺乳动物染色体上。这项技术将是序列特异性的， 能够同时测量多个位点， 中期染色体的三维组织，并能够 确定细胞分裂中期染色体上胞嘧啶甲基化的变化， 单细胞将其打造成一项实用且可转移的技术 可以定义某些特定的技术成就。这些 增量步骤将得到满足， 在模型系统中进行评估，以建立性能基线。 这种显微镜方法将允许研究人员区分 在单个细胞中存在或不存在特异性和/或整体甲基化， 在肿瘤发展的特定阶段。这项技术还将 允许研究者了解正常的 在分离的单细胞中甲基化基因组的发育。

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.