High-Sensitivity Mutation Scanning of Genes Novel Detection Platform and Conceptual module Design
| Autor: | Shih-Hsuan Kao, 高士軒 |
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| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 103 Target drugs are commonly used to treat tumors, which based on blocking the target genes’ signaling pathway; however, once mutation occurred in target genes could effect tumor cells insensitive to target drugs. Herein, to rapidly detect the certain mutations in the cells, high-sensitivity mutation detection platform (HMDP) has been developed, aim to rapidly screening of certain mutations, and to replace the conventional technics, for example: sequencing, enzyme mismatch cleavage etc. The features of HMDP could accurately discriminate small sequence or even single base differences between mutated sequences from wild type sequence of cells. Yb2Ti2O7 sensing films was used on the HMDP. Total 167 bp of sample sequence was hybridized with certain mutated sequences probes by mixing with DNA hybridized solution in 1% agarose gel, and fix on the sensor film for further detection. The C−V curves for different probes were measured through an Ag/AgCl reference electrode (commercial liquid-junction electrode) using a Hewlett-Packard (HP) 4284A precision LCR meter. Our results showed that the resolution of HMDP could identify single nucleotide mutation by our designed probes. Moreover, the stability of HMDP could reach to 50% in a hundred tests so far and identification pattern of positive was tested for verification. However, to goal the highest resolution, some issues we still struggled with: (1) the point mutation occurred on the same locus of gene could result in different identification rates,; (2) the mutation spots located at the c.35 has higher detection rate than at c.38 in 27 bp of probes; (3) the influence resulted from wafer, environment and artificial factor; for example, the amount of agarose could be altered by temperature changes, the problem of air bubble in/on the agarose gel, the reusing problems by status of wafer. Therefore, an automatic module design was proposed for effectively solve and monitored factors in this research. The second law of chemical thermodynamics was used to explain how the identification reasons is. For identification of a single point mutation with C-V figure, we speculate that because of the number of hydrogen bonds and stability of hydrogen bonds was measured. Our HMDP could replace the traditional technics not only shorten the exam time and also reduce lot of cost. Hopefully, it will benefit cancer patients in the near future. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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