Design Of Nucleic Acids Probes And Its Significant Application In Disease Detection And Enzyme Analysis

Nowadays, the researches of life process are getting into molecular level, which is much subtle than traditional inspect level, such as individual, organism or cells. How to obtain the information of life processes at molecular level sensitively, accurately in real time has been the goal that the scientists have made great efforts to pursue all the time in biomedicine and analytical chemistry fields. Nucleic acids probe is a novel technology that nucleic acids can be designed, labeled, synthesized, and employed at molecular level. A lot of nucleic acids probes have been constructed and utilized in many fields, such as gene analysis, disease diagnosis, protein (enzyme) detection and species taxon. How to further expand the use of existing nucleic acid probes in biology and medicine, and how to develop novel nucleic acid probes to resolve new appearing problems during research process are a great challenge and also a good chance to life science and analytical chemistry researchers.In this thesis, a series of methods based on molecular beacon and dye probe have been developed for ultrasensitive detection of ribozyme and restriction enzyme cleavage products, genotyping of point mutation of gene-mutant diseases, monitoring of DNA replication process, investigation of the ligation mechanism of DNA ligase and measuring of polymerase activity. Moreover, three novel nucleic acid probes, ribozyme molecular probe, melting point difference enlarged probe and signal amplified probe have been designed and synthesized in this thesis. These new probes have been used to real time monitor ribozyme cleavage reaction and to detect point mutation inβ-thalassemia disease with high fidelity and ultra-sensitivity. The main researches included in this dissertation are presented as following:In part I, a series of methods based on molecular beacon and dye probe have been developed and used in disease detection and enzyme analysis.1. Ultrasensitive detection method for ribozyme cleavage product using molecular beacon. Ribozyme is a catalytic RNA motif that cleaves the specific viral RNA and mRNA. The property makes ribozyme become useful tools in modern medicine and biology for inactivating gene expression and as potential therapeutic agent. Traditional detection methods for ribozyme cleavage product need label ribozyme or ribozyme substrate, which result in great limitation in clinic. Using molecular beacon as the template and the signal molecule for ribozyme cleavage product, the information of cleavage product has been directly converted into fluorescent signal during RNA/DNA ligation process. This method didn't need label ribozyme and ribozyme substrate, which is fast, simple and ultrasensitive. Detection limit was 0.05nmol/L. The cleavage product of ribozyme against hepatitis C virus RNA (HCV-RNA) has been detected based on this method. Owing to its ultrasensitivity, excellent specificity, convenience and fidelity, this novel non-isotope method could provide a more convenient means for researching on enzymatic activity, offering abundant dynamic data of RNA enzyme reaction, choosing rapidly potential gene therapeutic agents. This method might hold great promise in ribozyme gene therapy.2. Ultrasensitive detection method for restriction enzyme cleavage product using molecular beacon. The molecular beacon, as the template and the signal molecule was used to monitor DNA/DNA ligation process of nucleic acids complex and to convert directly cleavage product information into fluorescence signal. In the range from 0.2 nmol/L to 30 nmol/L, the concentration of cleavage product was directly proportional to the initial ligation rate. Detection limit of the assay was 0.05 nmol/L. Moreover, the method didn't need label restriction enzymes and their substrate. The cleavage product of Bst1107 restriction enzyme has been detected based on this assay. This method will provide a platform for studying a wide variety of nucleic acids cleavage product (such as other restriction enzymes and protein enzymes).3. Study the ligation mechanism of T4 DNA ligase using molecular beacon. DNA ligase is a vital enzyme in the repair, replication and recombination of nucleic acids. The research of ligation mechanism will promote the application of ligase in disease detection, gene carrier, modification and analysis of nucleic acids. A new approach for ligation mechanism has been reported based on molecular beacon. The molecular beacon, as the template and the signal molecule for ligation reaction, was used to monitor ligation process of nucleic acids complex and to convert directly cleavage product information into fluorescence signal. Based on molecular beacon, the ligation mechanism of T4 DNA ligase, such as the ATP-depended, Mg2+-depended and template-depended has been studied conveniently. This method can be also used in studying ligation mechanism of other DNA ligases and RNA ligases.4. In vitro monitoring of polymerase process during DNA replication using molecular beacon. DNA replication plays a pivotal role in life process. The DNA replication is usually assayed by denatured gel electrophoresis and autoradiography, which are complex, time-consuming, and incapable of acquiring the dynamic data in real time. We employed molecular beacon as the probe and template for DNA replication and convert directly polymerase reaction information into fluorescence signal. The information of DNA continuous and discontinuous replication was obtained conveniently and accurately using molecular beacon. Compared with traditional methods, this method avoided radiolabeling, denatured gel electrophoresis and autoradiography. Based on the proposed principle, a convenient and accurate assay of the activity of three polymerases has been established.5. A novel simple and rapid method for real time detection of polymerase activity using dye probe. Polymerase is an important enzyme and is widely used in modern medicine and biology such as gene sequencing, gene carrier, and gene cloning. Based on the fact that the dye probe can intercalate DNA duplex to emit fluorescence, a novel method for detection of DNA polymerase has been developed. The polymerization process has been indicated by fluorescent signal in real-time. Based on this assay, the influence of anti-tumour drugs on the activity of polymerase has been investigated. This method will provide potential for diagnosis and therapy of disease and drug screening.6. A convenient method for point mutation genotyping inβ-thalassemia disease using dye probe. Thalassemia disease is inherited hemoglobin disorders characterized by reduced production of globin chain. Based on dye probe and allele specific extension, a rapid, cost effective and specific genotyping method for single nucleotide polymorphism has been proposed. Based on the proposed method, the point mutation in theβ-thalassemia has been detected accurately and rapidly.In part II, three novel nucleic acid probes have been not only designed and synthesized, but also used in the detection of diseases.7. Design of ribozyme molecular probe and monitoring in real time for HCV-RNA ribozyme cleavage process. Traditionally, the assay of ribozyme reaction processed with radial labeling, gel electrophoresis, and autoradiography. The procedures are complex and time consuming that incapable of monitoring the rapid cleavage progress and providing the dynamic data. A novel ribozyme molecular beacon has been constructed to monitor the progress of ribozyme reaction, which provided abundant dynamical data precisely, specifically and sensitively. Using the ribozyme molecular probe, the cleavage information could be provided in real time by fluorescence signal without isotope label, denaturing electrophoresis and autoradiography. Compared with previous methods, this novel non-isotope method could provide a more convenient means for studying enzymatic activity, offering abundant dynamic data of RNA enzyme reaction, choosing rapidly potential gene therapeutic agents. Furthermore, this novel approach would have potential in the research on human disease detection and diagnosis synchronously.8. Design of melting point difference enlarged probe and detection for point mutation inβ-thalassemia disease. Many genetic diseases are associated with single-base mutations of particular genes. Identification of these mutations has great applications in prevention, clinic genetic testing and molecular diagnosis of these diseases. Approaches based melting point difference analyses discriminated genotyping perfectly for investigative polymorphisms that have significant melting difference between different genotypes. However, these methods might be difficult to discriminate these point mutations that exhibited insignificant melting temperature difference. Based on fluorescence resonance energy transfer and ligase technique, a novel probe has been developed to improve melting point difference between different genotypes. This method has been validated with the genotyping of two common point mutations [CD17 (A→T) and Ivs-2-654 (C→T)] ofβ-globin gene in thalassemia disease. Owing to its simple procedures, easy interpretation and high fidelity, this method is expected to hold great promise in practical clinic diagnosis of gene-mutant diseases.9. Design of signal amplified probe and detection for point mutation inβ-thalassemia disease. Current point mutation detection methods with high sensitivity usually need expensive signal detection equipments, which cause significant limitations to their applications. Combining ligase chain reaction with microsphere-based enrichment technique, a new signal amplified probe has been constructed capable of detecting point mutations in a convenient, separation-free format. Genotypes of samples were determined through fluorescent signals of micriobeads using fluorescence microscopy. This provided a high specificity and sensitivity method to detect point mutation as little as 600 copies of target template. This approach has been demonstrated with the identification of point mutation ofβ-thalassemia disease. Owing to its high sensitivity, simple procedures, easy interpretation and high fidelity, this method is expected to hold great promise in practice clinic diagnosis of gene-mutant diseases.