Preparation of immobilized polynucleotides on non-porous polymer microbeads and their applications for DNA hybridization and recombination
| Autor: | Hui-Jung Lin, 林慧蓉 |
|---|---|
| Rok vydání: | 2002 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 90 Immobilized oligonucleotides have been widely used in the studies of molecular biology and clinical analysis, including affinity chromatography, peptide and oligonucleotide synthesis, biosensor and DNA-microchip technology. In this study, non-porous polymer beads with a particle diameter of ca. 2.0 m were prepared by the dispersion polymerization of glycidyl methacrylate (GMA) and styrene. For the applications in recombinant DNA and DNA hybridization, the copolymerized beads were chemically modified to introduce aldehyde and amino groups, and then covalently linked with protein and oligonucleotide, respectively. To reduce the purification steps in the conventional recombinant DNA method and improve the efficiency by preventing lose of plasmid and cloned DNA fregment, a novel method of DNA recombination on the non-porous polymer beads was proposed. The separation of DNA could be easily done by centrifugation, according to this solid phase recombinant DNA method. The oligonucleotide-immobilized non-porous particles were also used for DNA hybridization. These beads were proved to be stable and reusable by the study of repeated hybridization. After repeated hybridization/denaturation for five times, the efficiency of hybridization could retain at its initial value. For the application of solid phase PCR, non-porous polystyrene beads were also prepared by the method dispersion polymerization, and then treated with nitration and hydrogenation to introduce the amino groups on the surface. After covalent coupling of the primer (oligonucleotide) on their surface via amino group, these heatproof polystyrene beads were able to go through PCR. After the solid phase PCR, non-porous beads immobilizing with a specific double-stranded DNA were obtained and can be reusable for any biological or clinical purpose. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
| Externí odkaz: |
|