羟甲基化DNA和胸腺嘧啶DNA糖苷酶电化学发光传感研究

发布时间：2018-02-26 11:08

本文关键词： 电化学发光羟甲基化DNA 胸腺嘧啶DNA糖苷酶纳米材料核酸信号放大　出处：《西北大学》2017年硕士论文　论文类型：学位论文

【摘要】：在哺乳动物基因组DNA中,最新发现的5-羟甲基胞嘧啶(5-hydroxymethylcytosine,5-hmC)被称为继5-甲基胞嘧啶(5-methylcytosine,5-mC)之后的“第六种碱基”,是一种新的表观遗传修饰物。5-hmC存在于人和小鼠脑组织以及小鼠的胚胎干细胞中,通过TET(ten-eleven translocation)酶氧化 5-mC 产生。研究表明,5-hmC 不仅参与了 DNA 主动去甲基化、体细胞重新编程和胚胎发育等重要的生物过程,也与多种疾病有关,如癌症,它在多种癌细胞中含量的降低使其有可能成为癌症早期诊断的标志物。胸腺嘧啶DNA糖苷酶(TDG)是一种碱基切除修复酶,它可特异性识别并移除5-mC、5-hmC去氨基后的产物以及5-hmC连续氧化的产物,引发碱基切除修复通路(BER)完成碱基的正常配对,在DNA去甲基化过程中也扮演了重要的表观遗传角色。因此,建立高灵敏检测羟甲基化DNA(5-hmC-DNA)和TDG活性的方法对于DNA去甲基化机理的研究和癌症早期的诊断具有重要的意义。相比已报道的检测羟甲基化DNA和TDG活性方法,电化学发光法因具有电化学方法和化学发光法的双重优势,如灵敏度高、反应可控性好、响应速度快、易于微型化等优点而备受关注。基于此,本文建立了两种高灵敏检测羟甲基化DNA和TDG活性的电化学发光生物传感新方法。全文共分为三章,主要内容如下:第一章,绪论。首先,概述了电化学发光的定义和特点;其次,综述了核酸信号放大和纳米材料信号放大策略与应用;最后,提出了本论文的研究内容和意义。第二章,基于T4噬菌体β-葡萄糖转移酶(T4β-glycosyltransferase,β-GT)在葡萄糖供体尿核苷二磷酸葡萄糖(UridineDiphosphoglucose,UDP-Glu)存在的情况下,特异性的糖基化 5-hmC 形成 β-葡萄糖基-5-羟甲基胞嘧啶(β-glucosyl-5-hydroxyme-thylcytosine,5-ghmC),利用氧化石墨烯(GO)负载多个信号分子进行信号放大,构建了一种高选择性、高灵敏的新型电化学发光生物传感器,用于羟甲基化DNA的检测。结果表明,在优化条件下,该传感器检测羟甲基化DNA的线性范围为0.01 pM～1.00 nM,检出限为3.84 fM。第三章,基于生物条形码(bio-barcode)引发杂交链式反应(HCR)进行无酶双重信号放大,结合电化学发光信号物质邻菲Up啉钌(Ru(phen)32+)能高效、稳定地嵌入DNA双螺旋结构的特点,构建了一种高灵敏的新型电化学发光生物传感器,用于TDG活性的分析。结果表明,在优化条件下,该传感器检测TDG的线性范围为5×10-5U/μL～1×10-2U/μL,检出限为 1.1×10-5U/μL(0.0028ng/mL)。
[Abstract]:In mammalian genomic DNA, The newly discovered 5-hydroxymethylcytosine 5-hydroxycytosine (5-HMC) is known as "6th bases" after 5-methylcytosine (5-methylcytosine). It is a new epigenetic modifier. 5-hmC is present in human and mouse brain tissues and embryonic stem cells in mice. TET(ten-eleven translocation enzymes oxidize 5-mC production. Studies have shown that 5-HMC is involved not only in the active demethylation of DNA, somatic cell reprogramming and embryo development, but also in many diseases, such as cancer. Its reduced levels in a variety of cancer cells make it possible to become a marker for early cancer diagnosis. Thymine DNA glycosidase is a base excision repair enzyme. It can specifically recognize and remove the product of 5-hmC deaminination and 5-hmC continuous oxidation, and trigger the base excision repair pathway (ber) to complete the normal pairing of the base, which also plays an important epigenetic role in the process of DNA demethylation. The establishment of a highly sensitive method for the detection of hydroxymethylated DNA-5-hmC-DNA and TDG activity is of great significance for the study of the mechanism of DNA demethylation and the early diagnosis of cancer, compared with the reported methods for the detection of hydroxymethylated DNA and TDG activities. Electrochemiluminescence (ECL) has attracted much attention for its advantages of both electrochemical and chemiluminescence methods, such as high sensitivity, good controllability, fast response and easy miniaturization. In this paper, two novel electrochemiluminescence biosensors with high sensitivity for the detection of hydroxymethylated DNA and TDG activities have been developed. The main contents are as follows: chapter 1, introduction. Firstly, the definition and characteristics of ECL are summarized. Secondly, the strategies and applications of nucleic acid signal amplification and nanomaterial signal amplification are reviewed. Based on T4 phage 尾 -glycosyltransferase (尾 -GT) in the presence of glucose donor Uridine Diphosphoglucose (UDP-Glu), A novel electrochemiluminescence biosensor with high selectivity and high sensitivity was constructed by the specific glycosylation of 5-hmC to form 尾 -glucosyl-5-hydroxythylcytosine (5-hydroxythylcytosine) -loaded with several signal molecules for signal amplification. The results show that under the optimized conditions, the linear range of the sensor for detection of hydroxymethylated DNA is 0. 01 pM~1.00 / nM. the detection limit is 3. 84 fM. Chapter 3, Based on the bio-barcode-based hybrid chain reaction (DNA), the enzyme free double signal amplification combined with the electrochemical luminescent signal substance, o-phenanthroline ruthenium Rufen 32) can efficiently and stably embed the DNA double helix structure. A novel electrochemiluminescence biosensor with high sensitivity was constructed for the analysis of TDG activity. The results show that the linear range of the biosensor for detecting TDG is 5 脳 10 ~ (-5) U / 渭 L ~ (-1) 脳 10 ~ (-2) U / 渭 L, and the detection limit is 1.1 脳 10 ~ (-5) U / 渭 L ~ (0.0028) ng / m ~ (-1).
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q52;O657.1

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