The Study Of DNA Methylases Based On Hairpin Fluorescence Molecular Probes

DNA methylation,caused by the interaction between DNA and methylase, is one of important modifications of DNA bases. It effects the structure of nucleic acids and the interaction between DNA and protein, then results in changing of gene expression, tumor occur and tumor growth. Therefore, DNA methylation has aroused an extensive interest. In this thesis, three parts of work were finished to investigate the interaction between DNA and methylase using fluorescence molecular probes. The designs of the probes are easy and flexible.1. An assay for the activity of DNA methylase based on a hairpin fluorescence molecular probe and its application in drug screeningA hairpin molecular probe with a longer stem was designed, which was more stable than the traditional molecular beacons. The recognition sites of DNA methylase and corresponding restriction endonuclease were designed in the stem part of the probe. The fluorophore attached at the 5' terminus can be quenched by the quencher linked at the 3' terminus. The unmethylated probe can be cut in the recognition site by restriction endonuclease, causing the restoration of the fluorescence of fluorophore. Hence, the activity of methylase can be detected according to the degree of fluorescence restoration. Based on this assay, the influence of anti-tumor drugs on the activity of methylase was investigated by adding drugs in the methylation reaction. This method provides potential for drug screening for tumors caused by altered gene methylation pattern.2.Real time monitoring of DNA methylation catalyzed by Taq I methylase based on a hairpin fluorescence molecular probeA molecular probe with two bordered recognition sequences of Taq I methylase were designed and synthesized. When the stem was methylated by Taq I methylase, probe could be cleaved by Dpn I, causing the fluorescence restoration. The DNA methylation process catalyzed by Taq I methylase was monitored in real-time based on the restoration of fluorescence. The activity of Taq I methylase was detected simply and rapidly, according to the initial velocity of the reaction.3.The influence of DNA methylation outside the enzyme recognition sequence on the interaction between enzymes and DNADifferent molecular probes were designed and synthesized, which contain methylated cytosines and adesines outside the recognition sequence of the restriction...