【摘要】： 核酸的復制是生命現(xiàn)象中最本質的內容之一,獲取有關核酸和聚合酶作用的實時過程信息將有助于DNA復制過程機理的研究,而且還將為藥物的篩選提供新的技術手段。傳統(tǒng)的研究DNA復制過程的分析方法往往采用放射性標記結合凝膠電泳的方法,無法獲取它們的動力學過程信息,因此發(fā)展實時的研究DNA復制過程的分析方法很有意義。 許多疾病如癌癥和遺傳病的發(fā)生都與基因的突變有關,因此對特定序列DNA的分析以及對DNA鏈中堿基突變的檢測在基因篩選、遺傳疾病的早期診斷和治療方面具有十分重要的意義。發(fā)展簡便、快速、特異的檢測方法用于基因突變研究是非常重要的。 本論文針對上述兩個問題,利用分子信標熒光探針和熒光染料探針,發(fā)展了實時監(jiān)測DNA連續(xù)復制、聚合酶活性以及基于聚合反應的單核苷酸多態(tài)性(SNP)基因分型方法。主要內容包括以下三個方面: 1、分子信標體外實時監(jiān)測DNA連續(xù)復制過程。利用分子信標實現(xiàn)了對DNA連續(xù)復制過程的實時監(jiān)測,分子信標作為DNA連續(xù)復制反應的模板和檢測分子,實時準確地獲取了DNA連續(xù)復制過程的信息。與傳統(tǒng)的研究方法相比,該方法避免了放射性同位素標記、變性凝膠電泳、放射性自顯影等復雜的操作,為DNA連續(xù)復制過程研究提供了一種新的非同位素分析方法,并在此基礎上建立了快速準確的檢測Klenow Fragment(exo-)聚合酶的分析方法。拓展了分子信標用于核酸和蛋白質相互作用研究的應用領域。 2、發(fā)展了一種簡便快速的聚合酶活性實時檢測新方法�；陔p鏈DNA結合染料能特異嵌入雙鏈DNA發(fā)出熒光的原理,發(fā)展了一種實時檢測DNA聚合酶活性的簡便方法。在檢測過程中,聚合酶的聚合反應進程被實時轉換為熒光信號,通過監(jiān)測熒光強度的變化實時檢測聚合酶的活性及藥物對聚合酶活性的影響。該方法不需要對寡核苷酸或dNTP進行放射性同位素標記和熒光標記,也不需要聚丙烯酰胺凝膠電泳和聚合酶鏈式反應,是一種簡便、快速價廉的聚合酶活性實時檢測新方法,為研究抗腫瘤藥物對聚合酶活性的影響提供了一種簡捷方法,也將為相關疾病診治和藥物篩選提供一種新的思路。3、建立了基于等位特異性延伸的SNP基因分型新方法以及用于β地中海貧血點突變的檢測。基于單堿基變化造成的堿基互補性對引物延伸反應的影響,以及染料SYBR Green I與雙鏈DNA結合發(fā)熒光的性質來識別有無聚合反應,從而達到對SNP位點的分析。該方法實驗設計簡單,無需合成標記探針,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.
【學位授予單位】：湖南大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R346

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