Detecting cytosine methylation at the single DNA molecule level

在单个 DNA 分子水平检测胞嘧啶甲基化

• 批准号: BB/I022791/1
• 负责人: Tracy Melvin
• 金额: $ 109.87万
• 依托单位: University of Southampton
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI022791%2F1
• 关键词: Detecting; cytosine; methylation; single; DNA

Although the genomes of many organisms (humans, plants, invertebrates and vertebrates) have been sequenced and many of the genes identified, our understanding of the regulation of the genes is limited due to lack of analysis technology. DNA is composed of four nucleic acid bases, adenine, guanine, cytosine and thymine. Some of these nucleic acid bases can be modified by enzymes and as a result have an additional methyl group; here we will investigate new technologies for the detection of methylcytosine and unmethylated cytosine within single DNA molecules. The methylation of cytosine nucleic acid bases is associated with gene silencing. In humans DNA methylation is considered to play a critical role in development and is aberrant in many diseases, but as yet the complete role remains unclear. There are numerous techniques for the detection of methylated cytosine in DNA, but the current methodologies do not yet provide a simple, fast, reliable cheap approach. A major problem is the need to evaluate DNA from cell samples that will contain the same DNA sequence but which are heterogeneous with respect to the cytosine residues that are methylated. So an average is often obtained. Those techniques that do allow single DNA strands to be evaluated are highly laborious and limited. Here we will develop a new approach for detecting sequences containing methylated cytosines at the single molecule level. There are currently other groups working in the field of DNA sequencing of single molecules, but these methods are slow and the DNA is investigated as a single strand. We will interrogate double-stranded DNA and this will allow us to detect methylated or unmethylated cytosine molecules on each strand, called hemi-methylation. Our approach is to create an artificial form of DNA, an oligonucleotide, that associates and wraps within the major groove of double-stranded DNA molecule at specific sequences. This artificial form of DNA when associated is called a triplex and the molecules synthesised will also contain a fluorophore. When the DNA sample has been treated with these triplex forming oligonucleotides the helix will contain fluorophores at different points along it. We will inject the DNA sample into a small channel that will result in unravelling and straightening of the strand so that it is then threaded into an optical interrogation channel. The fluorophores will be excited with light, which in the presence of nanostructures within the nanochannel will result in fluorescence intensity changes. The change in intensity will provide a code that indicates the methylation status of the different cytosine containing sequences (unmethylated, hemi-methylated, doubly methylated). A simple technique to detect the methylated and unmethylated cytosines within DNA sequences will be important for a wide academic, clinical and industrial research community, since this will allow a greater understanding of gene regulation. There are many research areas where cytosine methylation is considered to play a significant role in humans, such as diet related disease, inflammatory diseases, embryonic development to name a few, or in plants for understanding the effect of environmental stress. But as noted above, cytosine methylation is important for many organisms, and a technique that allows for the analysis of the patterns of methylation within genes has the potential to be commercially valuable in the longer-term. First a better understanding of DNA methylation is required, but it is possible that a form of the approach proposed here will yield a diagnostic tool.

尽管许多生物体（人类、植物、无脊椎动物和脊椎动物）的基因组已被测序，并且许多基因已被鉴定，但由于缺乏分析技术，我们对基因调控的了解有限。 DNA由四种核酸碱基组成：腺嘌呤、鸟嘌呤、胞嘧啶和胸腺嘧啶。其中一些核酸碱基可以被酶修饰，从而具有额外的甲基；在这里，我们将研究检测单个 DNA 分子内甲基胞嘧啶和非甲基化胞嘧啶的新技术。胞嘧啶核酸碱基的甲基化与基因沉默有关。在人类中，DNA 甲基化被认为在发育中发挥着关键作用，并且在许多疾病中出现异常，但其完整作用仍不清楚。有多种技术可用于检测 DNA 中的甲基化胞嘧啶，但目前的方法尚未提供一种简单、快速、可靠、廉价的方法。一个主要问题是需要评估来自细胞样品的 DNA，这些样品将包含相同的 DNA 序列，但在甲基化的胞嘧啶残基方面是异质的。因此通常会得到平均值。那些能够评估单链 DNA 的技术非常费力且受到限制。在这里，我们将开发一种在单分子水平上检测含有甲基化胞嘧啶的序列的新方法。目前还有其他小组致力于单分子 DNA 测序领域，但这些方法速度缓慢，而且 DNA 是作为单链进行研究的。我们将询问双链 DNA，这将使我们能够检测每条链上甲基化或未甲基化的胞嘧啶分子，称为半甲基化。我们的方法是创造一种人工形式的 DNA，即一种寡核苷酸，它以特定序列结合并包裹在双链 DNA 分子的主沟内。这种人造形式的 DNA 结合后称为三链体，合成的分子也将含有荧光团。当 DNA 样品用这些形成三链体的寡核苷酸处理时，螺旋将在沿其不同点处包含荧光团。我们将 DNA 样本注入一个小通道，这将导致 DNA 链解开并拉直，然后将其穿入光学询问通道。荧光团将被光激发，在纳米通道内存在纳米结构的情况下将导致荧光强度变化。强度的变化将提供指示不同胞嘧啶含有序列的甲基化状态（未甲基化、半甲基化、双甲基化）的代码。检测 DNA 序列中甲基化和非甲基化胞嘧啶的简单技术对于广泛的学术、临床和工业研究界来说非常重要，因为这将使人们更好地了解基因调控。在许多研究领域，胞嘧啶甲基化被认为在人类中发挥着重要作用，例如饮食相关疾病、炎症性疾病、胚胎发育等等，或者在植物中了解环境压力的影响。但如上所述，胞嘧啶甲基化对于许多生物体都很重要，并且能够分析基因内甲基化模式的技术从长远来看具有潜在的商业价值。首先需要更好地了解 DNA 甲基化，但这里提出的一种方法可能会产生一种诊断工具。

项目成果

Stretching of single DNA molecules under pressure-driven flow in straight and curved microfluidic channels

直线和弯曲微流体通道中压力驱动流下单个 DNA 分子的拉伸

• 发表时间: 2015
• 作者: Horak P

Triplex-mediated analysis of cytosine methylation at CpA sites in DNA.

• DOI: 10.1039/c3cc45917a
• 发表时间: 2014-01
• 期刊: Chemical communications
• 影响因子: 4.9
• 作者: Marie W Johannsen;S. Gerrard;Tracy Melvin;Tom Brown-

A nanoporous gold membrane for sensing applications.

• DOI: 10.1016/j.sbsr.2016.01.001
• 发表时间: 2016-03
• 期刊: Sensing and Bio-Sensing Research
• 影响因子: 5.3
• 作者: Oo SZ;Silva G;Carpignano F;Noual A;Pechstedt K;Mateos L;Grant-Jacob JA;Brocklesby B;Horak P;Charlton M;Boden SA;Melvin T
• 通讯作者: Melvin T

Study of single lambda-DNA molecule stretching based on microfluidic devices

基于微流控装置的单λ-DNA分子拉伸研究

• 发表时间: 2013
• 作者: Noual A

Nanopores within 3D-structured gold film for sensing applications

用于传感应用的 3D 结构金膜内的纳米孔

• DOI: 10.1049/cp.2016.0933
• 发表时间: 2016
• 作者: Carpignano F