Monitoring Nucleic Acids Replication And Polymerase Activity And Detecting Point Mutation Based On Nucleic Acid Probes

DNA replication is one of the most essential contents in life. Acquiring real time process information of interactions between nucleic acids and polymerases would not only benefit to understand mechanism of DNA replication, but also provide a new research tool for drug screening. In traditional assay on DNA replication, the isotope labeling, gel electrophoresis and autoradiography were usually used, which are complicated, laborious and incapable of acquiring the dynamic data in real time. Therefore it is significant to develop a real time method to investigate of DNA replication process. Many diseases, such as cancer and genetic diseases, are accompanied by gene mutation. Therefore, mutation detection of DNA sequence is of great importance for gene screening, early diagnosis and therapy of genetic diseases. It is necessary to develop technology for convenient, rapid and specific assay on gene mutation.In this thesis, new methods using molecular beacons and fluorescent dye probes were developed for real time monitoring of DNA continuous replication, detection of polymerase activity and genotyping. This thesis is composed of the following there parts:1. In vitro real time monitoring of DNA continuous replication process based on molecular beacons.DNA continuous replication process was investigated in real time. Molecular beacons were not only the probe for monitoring replication, but also the template for DNA substrate formation, Real time process information of DNA continuous replication was obtained accurately. Compared with traditional methods, this method avoided radio-labeling, denatured gel electrophoresis and autoradiography and provided a new non-isotope method for investigation of DNA replication. Based on the same principle, a convenient and accurate assay for monitoring the activity of Klenow Fragment(exo-) was established. This novel approach will extend the application fields of investigation of interaction and relationship between proteins and nucleic acids based on molecular beacons.2. A novel simple and rapid method for real time detection of polymerase activity.Based on the fact that the fluorescent dye SYBR Green I can intercalate double-stranded DNA to emit fluorescence, a novel fluorescence method for...