| Summary: | 碩士 === 國立嘉義大學 === 應用化學系研究所 === 99 === In the molecular biology, restriction endonucleases used as a molecular scissors plays an very important role. It has been widely employed in molecular cloning gene transfection and RFLP etc. The efficiencies of restriction endonucleases could be influenced by the incubation temperature, DNA fringe end, and DNA sequences, etc. Agarose gel electrophoresis can provide a convenient and efficient way to detect the result of the enzyme digestion. However, one of the limitation is that the method can’t be detected simultaneously.
Fluorescence spectroscopy can offer the advantages of sensitity, simplicility, and wealth of molecular information. One of important features of fluorescence is that there is a direct connection the spectral observation and molecular features of the sample. It is very easy to visualize how the spectral properties are affected by the local environment or the present of nearby acceptors.
In this study, we use the fluorescence spectrophotometer to detect the fluorescence intensity change upon the environment change. This result could lead to calculation of the affinity between two molecules. It is also employed to measure the distance between two sites by the fluorescence rescence energy transfer (FRET).
We design the DNA substrates containing 17 nucleotides which have two restriction endonuclease restriction sites, BamH1(GGATCC) and Xho1(CTCGAG). Two different fluorescence probes FAM and CY3 were covalenly attached to 5’end of each DNA strand. Therefore we can use fluorescence spectrophotometer to simultaneously detect how the resriction endonucleases work on DNA substrates.
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