Methylome Profiling via DNA Methyltransferase-directed Labeling

通过 DNA 甲基转移酶定向标记进行甲基化分析

• 批准号: 7361511
• 负责人: Art Petronis
• 金额: $ 11.87万
• 依托单位: CENTRE FOR ADDICTION AND MENTAL HEALTH
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-20 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7361511
• 关键词: Age; Aging; Asthma; Base Sequence; Biological; Biological Sciences; Biopolymers; Biotin; Cells; Childhood; Chromosomes; Clinical; Clinical Medicine; Complex; Coupling; Cytosine; DNA; DNA Methylation; DNA Methyltransferase; DNA Modification Methylases; DNA Modification Process; DNA Sequence; Deposition; Development; Diabetes Mellitus; Diagnostic; Diet; Disease; Elements; Engineering; Enzymes; Epigenetic Process; Exhibits; Gene Expression Regulation; Genes; Genome; Genomics; Glass; Goals; Histones; Human; Human Biology; Human Genome; Human Genome Project; Individual; Label; Laboratories; M.SssI; Malignant Neoplasms; Maps; Methods; Methyltransferase; Microarray Analysis; Modification; Molecular; Muscle; Neurons; Nucleotides; Organism; Outcome; Positioning Attribute; Procedures; Process; Reporter; Research; Role; S-Adenosylmethionine; Scanning; Schizophrenia; Sensitivity and Specificity; Series; Side; Site; Skin; Streptavidin; Syndrome; Techniques; Technology; Tissue Differentiation; Tissues; Variant; amino group; analog; base; biological research; bisulfite; brain cell; chemical group; cofactor; cost; cyanine; design; dopamine system; epigenomics; genetic element; genome sequencing; innovation; interest; methyl group; mutant; new technology; novel strategies; plasmid DNA; research study; sex; technological innovation

DESCRIPTION (provided by applicant): The ultimate goal of the Human Genome Project was to determine the sequence of the 3 billion "genetic elements", or nucleotides, that make up the human genome. The human sequence of the human genome is of enormous value and promises to revolutionize biological research and clinical medicine. It also comes with the realization that the complete genome sequence is only a beginning in our understanding of human biology. One of the greatest mysteries is the regulation of genes present within each cell. Despite being genetically identical, cells from different tissues look differently and perform very different functions. For example, although dopamine system genes are present in the cells of brain, muscle, and skin, such genes are active in neurons but not in muscle or skin cells. It is now believed that tissue-specific expression is achieved by the epigenetic regulation of genes via processes such as DNA methylation. More specifically, one of the nucleotides, namely cytosine, can be present in two functional states methylated or unmethylated. Methylated cytosines are sometimes referred as the 5th base of human DNA. DNA methylation profiles are highly variable across different cells, even in the same organism, and such variation depends on tissue, age, sex, diet, and numerous other factors. Over the last decade a series of new methods have been developed to investigate DNA methylation profiles across large DNA regions - chromosomes and even entire genomes. Unfortunately, all these methods exhibit significant limitations as they require large amounts of DNA, interrogate only a small fraction of methylatable nucleotides or are able to scan only short DNA fragments. This project is dedicated to development of a new technology for genome wide DNA methylation, or methylome, analysis. The key innovation consists of a combination of two powerful technologies: targeted deposition of extended groups of biopolymers on DNA, and the application of microarrays. Engineered DNA methyltrasferases, enzymes that methylate DNA, will be used to attach fluorescent labels on unmethylated cytosines, and these labeled DNA fragments will be interrogated on tiling microarrays. Microarrays are small pieces of glass containing millions of short DNA sequences that will hybridize to and highlight the unmethylated DNA fragments. This new approach exhibits numerous advantages over the existing methods for DNA methylation profiling in terms of simplicity, sensitivity, informativeness, and robustness. This technology may significantly contribute to our understanding of development, tissue differentiation, aging, and the molecular basis of complex disease, among numerous other fundamental questions of the life sciences.

描述（由申请人提供）：人类基因组项目的最终目标是确定构成人类基因组的30亿个“遗传元素”或核苷酸的序列。人类基因组的人类序列具有巨大的价值，并有望彻底改变生物学研究和临床医学。它还认识到，完整的基因组序列只是我们对人类生物学的理解的开始。最大的奥秘之一是调节每个细胞中存在的基因。尽管在遗传上相同，但来自不同组织的细胞看起来有所不同，并且表现非常不同的功能。例如，尽管多巴胺系统基因存在于脑，肌肉和皮肤的细胞中，但这种基因在神经元中活跃，但在肌肉或皮肤细胞中不具有活性。现在，人们认为组织特异性表达是通过基因通过DNA甲基化的过程表观遗传调节来实现的。更具体地说，其中一种核苷酸，即胞嘧啶，可以存在于两个功能状态甲基化或未甲基化的功能状态。甲基化的胞嘧啶有时被称为人DNA的第五个碱。即使在同一生物体中，DNA甲基化谱在不同细胞之间也有高度变化，并且这种变化取决于组织，年龄，性别，饮食和许多其他因素。在过去的十年中，已经开发了一系列新方法来研究大型DNA区域的DNA甲基化谱 - 染色体甚至整个基因组。不幸的是，所有这些方法都表现出显着的局限性，因为它们需要大量的DNA，仅询问一小部分甲基化核苷酸或仅能扫描短的DNA片段。 该项目致力于开发一种用于基因组宽DNA甲基化或甲基化分析的新技术。关键创新包括两种强大的技术的组合：针对DNA的扩展生物聚合物组的靶向沉积以及微阵列的应用。工程的DNA甲基转移酶，甲基酸DNA的酶将用于在未甲基化的细胞氨酸上附着荧光标签，这些标记的DNA片段将在平铺微阵列上进行询问。微阵列是一小块玻璃，其中包含数百万个短DNA序列，可以杂交并突出未甲基化的DNA片段。这种新方法比现有的DNA甲基化分析的方法具有许多优势，从简单，灵敏度，信息性和鲁棒性方面。这项技术可能会大大有助于我们对生命科学的许多其他基本问题以及复杂疾病的发展，组织分化，衰老和分子基础的理解。