Single-Base Resolution Method of 5-hmC in Biology

生物学中5-hmC的单碱基解析方法

• 批准号: 8252069
• 负责人: Shuang Chang
• 金额: $ 21.22万
• 依托单位: CINLANIAN BIOTECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8252069
• 关键词: Address; Affinity; Aging; Antibodies; Biological; Biological Markers; Biological Process; Biological Sciences; Biology; Biotin; Cells; Chemicals; Communities; Cytosine; DNA; Detection; Development; Digestion; Disease; Disease Marker; Epigenetic Process; Exodeoxyribonuclease III; Exonuclease; Future; Gene Expression; Genome; Genomic Imprinting; Genomics; Glucose; Glucosyltransferase; Human; Hypoxia; Investigation; Label; Laboratory Research; Ligation; Location; Maintenance; Malignant Neoplasms; Mammalian Cell; Medicine; Methods; Modification; Mus; Neurodegenerative Disorders; Phase; Play; Positioning Attribute; Primer Extension; Reaction; Reagent; Regulation; Research; Research Personnel; Resolution; Role; Small Business Innovation Research Grant; Technology; Testing; Tissues; Work; X Inactivation; angiogenesis; base; cancer cell; embryonic stem cell; functional group; glycosylation; human disease; leukemia; mammalian genome; new technology; next generation; sodium bisulfite; stem; success; tool; zygote

DESCRIPTION (provided by applicant): 5-Hydroxymethylcytosine (5-hmC) is a newly identified base modification in mammalian genomic DNA. Because current sequencing methods cannot differentiate 5-meC from 5-hmC, the immediate challenge is to develop robust methods to ascertain the positions of 5-hmC within the mammalian genome, a problem best addressed by adapting a new chemical labeling technology that we have developed. We show that the hydroxymethyl group of 5-hmC can be selectively labeled with chemically modified glucoses using 2-glucosyltransferase (2GT). This glycosylation offers a strategy of installing functional groups such as biotin onto 5-hmC. In this way, we can affinity capture DNA fragments containing the modified 5-hmC and develop sequencing methods to determine the precise locations of 5-hmC. Building on our early successes we propose to develop single base-resolution detection and sequencing methods to reveal the distribution of 5-hmC in mammalian genomes. We propose two different approaches, both utilizing the selective chemical labeling strategy we have developed. In one approach, we will label 5-hmC with bulky groups to hinder ligation and linear PCR reactions, thus achieving single base-resolution detection of 5-hmC. The linear PCR approach can be adapted into the next generation Solexa sequencing to perform high throughput determination of 5-hmC in mammalian genomes. In an alternative approach, we wil develop an exonuclease digestion blockage method that detects modified 5-hmC at 3' end of undigested DNA fragments using the next generation Solexa sequencing; we have already shown that the chemically modified 5-hmC blocks exonuclease III digestion at 3' end of the modified 5-hmC. The new technologies proposed in this SBIR application not only have great values as commercial research reagents, but also promise to efficiently discover and detect 5-hmC as biomarkers in various human diseases. ! PUBLIC HEALTH RELEVANCE: 5-Hydroxymethylcytosine (5-hmC) is a newly discovered base modification surprisingly abundant in the genomic DNAs of certain mammalian tissues and cells. The proposed work will develop efficient chemical labeling methods to perform single base-resolution detection and sequencing of 5-hmC in genomic DNAs. The success of the proposed work will enable the researchers to reveal the fundamental role(s) of 5-hmC in biology and develop potential disease markers.

描述（由申请人提供）：5-羟基甲基胞嘧啶（5-HMC）是哺乳动物基因组DNA的新鉴定的基础修饰。由于当前的测序方法无法将5-MEC与5-HMC区分开，因此直接挑战是开发可靠的方法，以确定哺乳动物基因组中5-HMC的位置，这是通过调整我们开发的新化学标记技术来解决这个问题的问题。我们表明，使用2-葡萄糖基转移酶（2GT），可以将5-HMC的羟基基团选择性地标记。该糖基化提供了将生物素等功能组安装到5-HMC上的策略。通过这种方式，我们可以亲和力捕获包含修改的5-HMC的DNA片段并开发测序方法以确定5-HMC的精确位置。在我们的早期成功的基础上，我们建议开发单个基础分辨率检测和测序方法，以揭示5-HMC在哺乳动物基因组中的分布。我们提出了两种不同的方法，均利用我们制定的选择性化学标记策略。在一种方法中，我们将用笨重的基团标记5-HMC，以阻止连接和线性PCR反应，从而实现5-HMC的单基分辨率检测。线性PCR方法可以改编为下一代Solexa测序，以执行哺乳动物基因组中5-HMC的高吞吐测定。在另一种方法中，我们将开发一种外核酸酶消化阻滞方法，该方法使用下一代Solexa测序在未消除的DNA片段3'末端检测5-HMC；我们已经表明，在修饰的5-HMC的3'末端，化学修饰的5-HMC块外切核酸酶III消化。该SBIR应用中提出的新技术不仅具有广泛的价值作为商业研究试剂，而且也有望有效地发现和检测5-HMC作为各种人类疾病中的生物标志物。呢 公共卫生相关性：5-羟基甲基胞嘧啶（5-HMC）在某些哺乳动物组织和细胞的基因组DNA中出人意料地发现了新发现的基础修饰。提出的工作将开发有效的化学标记方法，以在基因组DNA中对5-HMC进行单基分辨率检测和测序。拟议工作的成功将使研究人员能够揭示5-HMC在生物学中的基本作用并发展潜在的疾病标志物。