核酸探针用于核酸复制与酶活性的实时监测及点突变分析

发布时间：2018-07-29 19:56

【摘要】： 核酸的复制是生命现象中最本质的内容之一,获取有关核酸和聚合酶作用的实时过程信息将有助于DNA复制过程机理的研究,而且还将为药物的筛选提供新的技术手段。传统的研究DNA复制过程的分析方法往往采用放射性标记结合凝胶电泳的方法,无法获取它们的动力学过程信息,因此发展实时的研究DNA复制过程的分析方法很有意义。许多疾病如癌症和遗传病的发生都与基因的突变有关,因此对特定序列DNA的分析以及对DNA链中碱基突变的检测在基因筛选、遗传疾病的早期诊断和治疗方面具有十分重要的意义。发展简便、快速、特异的检测方法用于基因突变研究是非常重要的。本论文针对上述两个问题,利用分子信标荧光探针和荧光染料探针,发展了实时监测DNA连续复制、聚合酶活性以及基于聚合反应的单核苷酸多态性(SNP)基因分型方法。主要内容包括以下三个方面: 1、分子信标体外实时监测DNA连续复制过程。利用分子信标实现了对DNA连续复制过程的实时监测,分子信标作为DNA连续复制反应的模板和检测分子,实时准确地获取了DNA连续复制过程的信息。与传统的研究方法相比,该方法避免了放射性同位素标记、变性凝胶电泳、放射性自显影等复杂的操作,为DNA连续复制过程研究提供了一种新的非同位素分析方法,并在此基础上建立了快速准确的检测Klenow Fragment(exo-)聚合酶的分析方法。拓展了分子信标用于核酸和蛋白质相互作用研究的应用领域。 2、发展了一种简便快速的聚合酶活性实时检测新方法。基于双链DNA结合染料能特异嵌入双链DNA发出荧光的原理,发展了一种实时检测DNA聚合酶活性的简便方法。在检测过程中,聚合酶的聚合反应进程被实时转换为荧光信号,通过监测荧光强度的变化实时检测聚合酶的活性及药物对聚合酶活性的影响。该方法不需要对寡核苷酸或dNTP进行放射性同位素标记和荧光标记,也不需要聚丙烯酰胺凝胶电泳和聚合酶链式反应,是一种简便、快速价廉的聚合酶活性实时检测新方法,为研究抗肿瘤药物对聚合酶活性的影响提供了一种简捷方法,也将为相关疾病诊治和药物筛选提供一种新的思路。3、建立了基于等位特异性延伸的SNP基因分型新方法以及用于β地中海贫血点突变的检测。基于单碱基变化造成的碱基互补性对引物延伸反应的影响,以及染料SYBR Green I与双链DNA结合发荧光的性质来识别有无聚合反应,从而达到对SNP位点的分析。该方法实验设计简单,无需合成标记探针,不需要昂
[Abstract]:Nucleic acid replication is one of the most essential contents in life phenomena. Obtaining real-time process information about nucleic acid and polymerase action will be helpful to study the mechanism of DNA replication process and will also provide a new technical means for drug screening. The traditional analysis methods of DNA replication process often use the method of radioactive labeling and gel electrophoresis, so it is very important to develop real-time analysis methods of DNA replication process because they can not get the information of their kinetic processes. Many diseases, such as cancer and genetic diseases, are associated with mutations in genes, so analysis of specific sequences of DNA and detection of base mutations in DNA strands are in gene screening. The early diagnosis and treatment of genetic diseases is of great significance. It is very important to develop a simple, rapid and specific detection method for gene mutation. In order to solve the above two problems, a real-time method for monitoring DNA continuous replication, polymerase activity and (SNP) genotyping based on single nucleotide polymorphism (SNP) was developed by using molecular beacon fluorescent probes and fluorescent dye probes. The main contents are as follows: 1. In vitro, molecular beacons monitor the continuous replication of DNA. Molecular beacons are used to monitor the continuous replication of DNA in real time. Molecular beacons are used as templates and detection molecules for continuous replication of DNA. The information of continuous replication of DNA is obtained in real time and accurately. Compared with traditional methods, this method avoids complex operations such as radioisotope labeling, denaturing gel electrophoresis and radioautography, and provides a new non-isotope analysis method for the study of continuous replication of DNA. On this basis, a rapid and accurate method for the detection of Klenow Fragment (exo-) polymerase was established. The application of molecular beacons in the study of nucleic acid / protein interaction is expanded. 2. A simple and fast method for real-time detection of polymerase activity is developed. Based on the principle that double-stranded DNA binding dyes can specifically embed double-stranded DNA to emit fluorescence, a simple method for real-time detection of DNA polymerase activity was developed. In the process of detection, the polymerization process of polymerase was converted into fluorescence signal in real time. The activity of polymerase and the effect of drugs on the activity of polymerase were detected by monitoring the change of fluorescence intensity. This method does not require radioisotope labeling or fluorescence labeling of oligonucleotides or dNTP, nor does it require polyacrylamide gel electrophoresis and polymerase chain reaction. It is a simple, rapid and inexpensive method for real-time detection of polymerase activity. It provides a simple and convenient method to study the effect of antitumor drugs on polymerase activity. It will also provide a new idea for the diagnosis and treatment of related diseases and drug screening. A new SNP genotyping method based on allele-specific extension and the detection of 尾 -thalassemia point mutation have been established. Based on the effect of base complementarity caused by single base variation on primer extension reaction and the fluorescence properties of dye SYBR Green I combined with double-stranded DNA, the polymerization reaction was identified and the SNP site was analyzed. The experimental design of this method is simple, no need for synthetic labeling probe and no need for high power.
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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