Study Of The Mechanism Of DNA Polymerase Reaction And Detection Of Single-base DNA Mutation With Fluorescence Resonance Energy Transfer Technique

DNA polymerase is playing the vital role in the DNA replication, the reorganization and in the basic group excision repair process. The deoxyribonucleic acid is the main genetic material and playing the vital role in organism's vital activity. Therefore, the enzyme mechanism research as well as the DNA detection is especially important. The fluorescence resonance energy transfer analysis method has advantages as follows:the simple instrument, the high sensitivity, rapid procedure and so on. So it has been widely applied in the research of biological macromolecule.In the second chapter of this thesis, we use the cation conjugate polymer(PFP) and fluorescein as fluorescence probes to research the action mechanism of DNA polymerase. In the presence of DNA polymerase fluorescein-labeled deoxyguanosine triphosphate(dGTP) interacts with DNA and forms E-DNA-dGTP complex. Then cationic conjugate polymer was added, the conjugate polymer and DNA bonded by a strong electrostatic force, so the distance between fluorescein and cationic conjugate polymer became shoter. Therefor fluorescence resonance energy transfer occurs from cation conjugate polymer to fluorescein. We use different DNA to interact with DNA polymerase, then detect the fluorescent intensity of fluorescein. We found that Klenow I DNA polymerase combine with double-strand part of DNA, and Taq DNA polymerase combine with the gap of DNA.In the third chapter of this thesis, we use the cationic conjugate polymer(PFP) and Au nanoparticles as fluorescence probes to detect single-base DNA mutation. Firstly Au nanoparticles were coated by chitosan, then guanosine 5'-monophosphate(GMP) were attached onto the chitosan-coated Au nanoparticles through their 5'phosphate group via the formation of a phosphoramidate bond with the free amino groups of the immobilized chitosan. If the mutation site has a base complementary to the GMP immobilized on Au nanoparticles, in the presence of Klenow I DNA polymerase, AuNPs-dsDNA complex were formed via base-paring. Then cationic conjugate polymer was added, the conjugate polymer and DNA bonded by a strong electrostatic force, so the distance between Au nanoparticles and cationic conjugate polymer became shoter. Therefor, fluorescence resonance energy transfer occurs from cation conjugate polymer to Au nanoparticles and the fluorescence is quenched. So we can detect single-base DNA mutation. This method does not need fluorescence labeling, and Au nanoparticles modification is simple. So we established one kind rapid, convenient and low-cost method for detection of DNA mutation.