發(fā)布時間：2018-05-17 16:32

  本文選題：單核苷酸多態(tài)性 + DNA酶��； 參考：《浙江大學(xué)》2017年碩士論文

【摘要】：單核苷酸多態(tài)性是人類基因組中最常見的堿基突變形式之一,研究發(fā)現(xiàn)在人類基因組的編碼區(qū)發(fā)生的單核苷酸多態(tài)性與某些疾病的發(fā)生息息相關(guān),所以建立單核苷酸多態(tài)性有效的檢測方法不僅在化學(xué)生物檢測領(lǐng)域有重要意義,而且在臨床分析方面也有潛在的應(yīng)用價值。具有辣根過氧化物酶活性的DNA酶因其合成簡單、熱穩(wěn)定性好和催化性能可調(diào)等優(yōu)勢成為生物化學(xué)分析中理想的信號報告分子,核酸信號放大技術(shù)可以實現(xiàn)一目標分子輸入,多報告分子輸出的檢測模式,大大提高檢測的靈敏度,因此將DNA酶與核苷酸信號放大技術(shù)聯(lián)用能兼具兩者的優(yōu)勢,在生化分析領(lǐng)域有廣泛的應(yīng)用前景。本論文共分為三章：第一章,作者對目前單核苷酸多態(tài)性的分析方法進行了綜述；進而簡要介紹了核酸酶的分類及作用,尤其是具有辣根過氧化物酶活性的G-四鏈體DNA酶的性質(zhì)和應(yīng)用,同時對核酸信號放大技術(shù)進行了系統(tǒng)的分類和概述。第二章,作者開展了基于DNA發(fā)夾結(jié)構(gòu)自組裝的DNA酶傳感器在單核苷酸多態(tài)性分析中的應(yīng)用研究。我們選取阿爾茲海默癥相關(guān)基因rs242557片段作為實驗?zāi)繕嘶?采用基于toehold誘導(dǎo)的、目標基因催化的發(fā)夾結(jié)構(gòu)自組裝反應(yīng)(CHA)的核酸信號放大策略,結(jié)合DNA酶催化的微流控化學(xué)發(fā)光檢測法,開展單核苷酸多態(tài)性的無酶無標記分析。實驗系統(tǒng)涉及的核苷酸鏈包括目標基因鏈和發(fā)夾DNA鏈H1和H2。首先通過NUPACK軟件模擬,對相關(guān)核苷酸鏈的二級結(jié)構(gòu)進行預(yù)測并加以比較,初步確定擬采用的核苷酸分子序列。當(dāng)系統(tǒng)中無目標基因鏈時,由于可形成G-四鏈體的富G核苷酸序列被部分包埋在H2的發(fā)夾結(jié)構(gòu)中,無法形成具有過氧化物酶活性的DNA酶；而當(dāng)系統(tǒng)中加入目標基因鏈后,則連續(xù)引發(fā)發(fā)夾H1和H2的toehold調(diào)節(jié)自組裝反應(yīng),釋放富G核苷酸序列形成DNA酶,最終催化過氧化氫和魯米諾的化學(xué)發(fā)光反應(yīng)并被檢測。該方法的絕對檢出限僅為0.3 fmol,對單核苷酸多態(tài)性檢測的區(qū)分因子可以達到20,初步展現(xiàn)了在生化分析和基因診斷等領(lǐng)域的應(yīng)用潛力。第三章,總結(jié)與展望部分。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q52;O657.3

【參考文獻】

相關(guān)期刊論文 前3條

1 鄔期望;沈宏;;基于NUPACK預(yù)測設(shè)計的Toehold誘導(dǎo)鏈置換反應(yīng)及其在DNA酶催化微流控化學(xué)發(fā)光單核苷酸多態(tài)性分析中的應(yīng)用[J];高等學(xué)校化學(xué)學(xué)報;2015年12期

2 劉其友;張云波;趙朝成;趙東風(fēng);;脫氧核酶的應(yīng)用研究進展[J];化學(xué)與生物工程;2009年08期

3 毛華偉,趙曉東,楊錫強;脫氧核酶研究進展[J];中國生物工程雜志;2003年04期

，

本文編號：1902069