Investigation of DNA Restriction Mapping by Capillary ectrophoresis

• Main Authors: Chang, Gau-Shuoh, 張高碩
• Other Authors: You-Zung,Hsieh
• Format: Others
• Language: zh-TW
• Published: 1995
• Online Access: http://ndltd.ncl.edu.tw/handle/65056040432781602065

碩士 === 國立交通大學 === 應用化學系 === 83 === Separation and detection of DNA restriction fragments are the essential and major steps in gene analysis or physical mapping of genes. Slab gel electrophoresis is the trational way to separate DNA fragments,however, the method is time consuming ,labor intensive, hard to quantitate, and large sample quantity is required for analysis. To detect DNA fragment in a slab gel either ethidium bromide or radioisotope is emploted and these chemicals can pose futher health hazardous problem. Capillary electrophoresis is an automated separation technique offering the benefits of rapid and high resolution separation of DNA molecules. Capillary electrophoresis has been a maturenique for separating proteins, peptide, organic chemicals, and small fragments separations, several hurdles imposed by the nature of large DNA molecules needed to be overcome before the technique can be realized. In this thesis, I explore several capillary electrophoresis conditions to separate large DNA restriction fragments and apply the technique to DNA restriction mapping which is an essential tool to many molecular biology labs. Laser induced fluorescence detection and partial digest mapping method are adopted and modified to map DNA fragments by capillarytrophoresis. The pros and cons of capillary electrophoresis in DNA mapping as compared to the tradtional slab gel electrophoresis are discussed by using pBluescript SK(+) phagemid DNA as a model. A restriction map of human interleukine 5 receptor gene is constructed using capillary electrophoresis and the technique developed in this thesis to demonstrate and conclude the feasibility of this thesis project.