基于DNA信号放大和DNA酶化学发光的单核苷酸多态性分析

发布时间：2018-05-17 16:32

本文选题：单核苷酸多态性 + DNA酶　；参考：《浙江大学》2017年硕士论文

【摘要】：单核苷酸多态性是人类基因组中最常见的碱基突变形式之一,研究发现在人类基因组的编码区发生的单核苷酸多态性与某些疾病的发生息息相关,所以建立单核苷酸多态性有效的检测方法不仅在化学生物检测领域有重要意义,而且在临床分析方面也有潜在的应用价值。具有辣根过氧化物酶活性的DNA酶因其合成简单、热稳定性好和催化性能可调等优势成为生物化学分析中理想的信号报告分子,核酸信号放大技术可以实现一目标分子输入,多报告分子输出的检测模式,大大提高检测的灵敏度,因此将DNA酶与核苷酸信号放大技术联用能兼具两者的优势,在生化分析领域有广泛的应用前景。本论文共分为三章：第一章,作者对目前单核苷酸多态性的分析方法进行了综述；进而简要介绍了核酸酶的分类及作用,尤其是具有辣根过氧化物酶活性的G-四链体DNA酶的性质和应用,同时对核酸信号放大技术进行了系统的分类和概述。第二章,作者开展了基于DNA发夹结构自组装的DNA酶传感器在单核苷酸多态性分析中的应用研究。我们选取阿尔兹海默症相关基因rs242557片段作为实验目标基因,采用基于toehold诱导的、目标基因催化的发夹结构自组装反应(CHA)的核酸信号放大策略,结合DNA酶催化的微流控化学发光检测法,开展单核苷酸多态性的无酶无标记分析。实验系统涉及的核苷酸链包括目标基因链和发夹DNA链H1和H2。首先通过NUPACK软件模拟,对相关核苷酸链的二级结构进行预测并加以比较,初步确定拟采用的核苷酸分子序列。当系统中无目标基因链时,由于可形成G-四链体的富G核苷酸序列被部分包埋在H2的发夹结构中,无法形成具有过氧化物酶活性的DNA酶；而当系统中加入目标基因链后,则连续引发发夹H1和H2的toehold调节自组装反应,释放富G核苷酸序列形成DNA酶,最终催化过氧化氢和鲁米诺的化学发光反应并被检测。该方法的绝对检出限仅为0.3 fmol,对单核苷酸多态性检测的区分因子可以达到20,初步展现了在生化分析和基因诊断等领域的应用潜力。第三章,总结与展望部分。
[Abstract]:Single nucleotide polymorphism (SNP) is one of the most common forms of base mutation in the human genome. Studies have found that single nucleotide polymorphisms in the coding region of the human genome are closely related to the occurrence of some diseases. Therefore, the establishment of an effective detection method for single nucleotide polymorphism is not only of great significance in the field of chemical and biological detection, but also of potential application value in clinical analysis. DNA enzyme with horseradish peroxidase activity is an ideal signal reporter molecule in biochemical analysis because of its advantages of simple synthesis, good thermal stability and adjustable catalytic performance. Nucleic acid signal amplification technique can realize a target molecule input. The detection mode of multi-report molecular output greatly improves the sensitivity of detection. Therefore, the combination of DNA enzyme and nucleotide signal amplification technology has the advantages of both, and has a wide application prospect in the field of biochemical analysis. This thesis is divided into three chapters: in Chapter 1, the methods of single nucleotide polymorphism analysis are reviewed, and the classification and function of nuclease are briefly introduced. In particular, the properties and applications of G-quadruplex DNA enzyme with horseradish peroxidase activity were discussed. The nucleic acid signal amplification technique was systematically classified and summarized. In chapter 2, the author studies the application of DNA enzyme sensor based on DNA hairpin structure in single nucleotide polymorphism analysis. We selected the rs242557 fragment of Alzheimer's disease related gene as the experimental target gene and adopted the nucleic acid signal amplification strategy based on toehold induced hairpin structure self-assembly reaction. DNA enzyme catalyzed microfluidic chemiluminescence assay was used to detect single nucleotide polymorphism without enzyme labeling. The nucleotide chains involved in the experimental system include target gene chains and hairpin DNA strands H _ 1 and H _ 2. The secondary structure of the related nucleotide chain was predicted and compared by NUPACK software, and the nucleotide molecular sequence was preliminarily determined. When there is no target gene chain in the system, the G-rich nucleotide sequence which can form the G-quadruplex is partially embedded in the hairpin structure of H _ 2, so the DNA enzyme with peroxidase activity can not be formed, but when the target gene chain is added to the system, the target gene chain can not be formed. Then the hairpin H1 and H2 toehold regulates the self-assembly reaction, releases the G-rich nucleotide sequence to form DNA enzyme, and finally catalyzes the chemiluminescence reaction of hydrogen peroxide and luminol and is detected. The absolute detection limit of this method is only 0.3 fmol.The discriminant factor for detection of single nucleotide polymorphism can reach 20, which shows the potential of application in biochemical analysis and gene diagnosis. The third chapter, the summary and the prospect part.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q52;O657.3

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