Single-Base Resolution Method of 5-hmC in Biology

生物学中5-hmC的单碱基解析方法

基本信息

  • 批准号:
    8252069
  • 负责人:
  • 金额:
    $ 21.22万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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(由申请方提供):5-羟甲基胞嘧啶(5-hmC)是哺乳动物基因组DNA中新发现的碱基修饰。由于目前的测序方法不能区分5-meC从5-hmC,直接的挑战是开发强大的方法,以确定5-hmC在哺乳动物基因组中的位置,最好通过适应我们已经开发的一种新的化学标记技术来解决这个问题。我们表明,5-hmC的羟甲基基团可以选择性地标记与化学修饰的葡萄糖使用2-葡萄糖基转移酶(2GT)。这种糖基化提供了一种将生物素等功能基团安装到5-hmC上的策略。通过这种方式,我们可以亲和捕获含有修饰的5-hmC的DNA片段,并开发测序方法来确定5-hmC的精确位置。在我们早期成功的基础上,我们建议开发单碱基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,都利用我们已经开发的选择性化学标记策略。在一种方法中,我们将用大体积基团标记5-hmC以阻碍连接和线性PCR反应,从而实现5-hmC的单碱基分辨率检测。线性PCR方法可适用于下一代Solexa测序,以进行哺乳动物基因组中5-hmC的高通量测定。在替代方法中,我们将开发外切核酸酶消化阻断方法,其使用下一代Solexa测序在未消化的DNA片段的3'末端检测修饰的5-hmC;我们已经表明化学修饰的5-hmC阻断外切核酸酶III在修饰的5-hmC的3'末端的消化。SBIR应用中提出的新技术不仅具有商业研究试剂的巨大价值,而且有望有效地发现和检测5-hmC作为各种人类疾病的生物标志物。! 公共卫生关系:5-羟甲基胞嘧啶(5-hmC)是一种新发现的碱基修饰,在某些哺乳动物组织和细胞的基因组DNA中含量惊人地丰富。这项工作将开发有效的化学标记方法,以进行单碱基分辨率检测和测序的基因组DNA中的5-hmC。这项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,并开发潜在的疾病标志物。

项目成果

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