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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 盧彥文(Yen-Wen Lu) | |
| dc.contributor.author | Pei-Chun Kao | en |
| dc.contributor.author | 高培鈞 | zh_TW |
| dc.date.accessioned | 2021-06-16T17:23:09Z | - |
| dc.date.available | 2015-09-01 | |
| dc.date.copyright | 2012-08-28 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-16 | |
| dc.identifier.citation | Alom Ruiz, S. and C. S. Chen. 2007. Microcontact printing: A tool to pattern. Soft Matter, 3:168-177.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/63921 | - |
| dc.description.abstract | 單核苷酸多型性 (Single Nucleotide Polymorphism)，為最常見之基因變異，這種變異亦為一重要的生物分子遺傳標記，其不但影響了物種個體間之差異，亦可能造成個體具有過敏體質、遺傳性疾病或是對於藥物之抗藥性等。目前多數的單核苷酸多型性檢測技術仍存在缺點，如須要耗費大量的試劑以及繁複之處理過程。因此，本研究致力於發展微小化之動態等位基因特異性雜交法技術，並整合微珠微流道技術以減少試劑使用量。本系統採用微珠作為固態載體，將待測的去氧核醣核苷酸固定於微珠表面，而後利用流體將微珠限制於流道固定位置，以進行熔解曲線分析達成單核苷酸多型性之基因分型。本研究中以ataxia-telangiectasia突變 (ATM) 基因為例，成功以合成DNA以及由藍瑞斯母豬取得之基因體DNA完成位點ATM-A之基因分型。此微珠微流道系統成功地減少試劑使用量，並簡化了聚合酶鏈鎖反應之程序，同時保有了動態等位基因特異性雜交法之靈活性及準確度。 | zh_TW |
| dc.description.abstract | Single-nucleotide polymorphism (SNP) not only is one of the most common genetic variances in human genome, but also serves a crucial bio-marker greatly affecting phenotypes of individuals. Most of today’s SNP detection techniques utilize enzymes or modification of DNA, leading to the requirement of high reagent cost or complex procedures. Thus, a SNP genotyping system is developed by integrating a miniaturized Dynamic Allele-Specific Hybridization (DASH) technique with a bead-based microfluidic device. The microbeads function as a solid vehicle for immobilizing DNA duplexes, confined by the fluidic traps in the device. Meanwhile, the Melting Curve analysis of the DASH technique was applied for SNP detection. Our proposed device was successfully tested, detecting the SNP of ataxia-telangiectasia-mutated (ATM-A) from both synthetic DNA and genomic DNA of Landrace sows. This bead-based device required a minimal reagent amount and simplified Polymerase Chain Reaction (PCR) procedures in preparations, while preserved as a flexible and accurate SNP detection scheme by integrating with DASH technique. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T17:23:09Z (GMT). No. of bitstreams: 1 ntu-101-R99631010-1.pdf: 7167693 bytes, checksum: 60569390a8949aac4751cddd58f0a93a (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 致謝 i
中文摘要 ii Abstract iii Table of Contents iv List of Figures viii List of Tables xiii Chapter 1 Introduction 1 1.1 Single Nucleotide Polymorphism and Ataxia-Telangiectasia Mutated Gene 1 1.2 Bead-Based Microfluidic Platform 2 1.3 Overall Structure of Thesis 3 Chapter 2 Literature Review 4 2.1 Bead-Based Microfluidic Platforms and Trapping Mechanisms 4 2.2 SNP Genotyping Mechanisms 6 2.3 Primer Extension Method 6 2.4 Dynamic Allele-Specific Hybridization (DASH) Method 7 2.5 Ligation Method 9 2.6 Enzymatic Cleavage Method 10 2.7 Asymmetric Polymerase Chain Reaction (PCR) 11 2.8 SNP Genotyping System 13 Chapter 3 Materials and Methods 18 3.1 SNP Target Selection and DNA Sample Preparation 19 3.1.1 Ataxia-Telangiectasia Mutated (ATM) Gene 19 3.1.2 DNA Extraction and SNP Discovery 19 3.1.3 Symmetric Polymerase Chain Reaction 21 3.1.4 Asymmetric Polymerase Chain Reaction 24 3.2 Validation of Dynamic Allele-Specific Hybridization Method 26 3.3 Bead-Based Microfluidic 27 3.3.1 Design and Principle 27 3.3.2 Fabrication 28 3.4 Development of Melting Curve Analysis System 32 3.4.1 Overall System 32 3.4.2 Temperature controller Unit 34 3.4.3 Optical Unit 34 3.5 Data Processing and Human-Machine Interface 36 3.6 Genotyping with Melting Curve Analysis System 37 3.6.1 Sample Preparation for Genotyping on Developed System 37 3.6.2 Genotyping with Melting Curve Analysis 38 3.6.3 Fluorescent Signal Quantification 38 Chapter 4 Results and Discussion 40 4.1 Validation of Dynamic Allele-Specific Method 40 4.1.1 Measurement of Optical Density Value 40 4.1.2 Electrophoresis Experiment for PCR Products 41 4.1.3 Electrophoresis Experiment for Fluorescence Samples 42 4.1.4 Validation of DASH method 43 4.2 Bead-Based Microfluidic Device 45 4.3 Melting Curve Analysis System 49 4.3.1 Temperature controller Unit 49 4.3.2 Optical Unit 50 4.4 Genotyping with the Developed System 53 4.4.1 Genotyping of Synthetic DNA 53 4.4.2 Genotyping of Genomic DNA from Landrace sow 54 4.5 Comparison between DASH and Golden Gate Genotyping Method 58 Chapter 5 Conclusion 59 5.1 Conclusion 59 5.2 Prospective 60 Appendix 1 61 Appendix 2 62 Appendix 3 63 Reference 66 | |
| dc.language.iso | en | |
| dc.title | 應用微珠微流道及等位基因特異性雜交技術於單核苷酸多型性檢測 | zh_TW |
| dc.title | Single Nucleotide Polymorphism Detection Using DASH Technology in Bead-Based Microfluidics | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 丁詩同(Shih-Torng Ding),王倫(Lon (Alex),林恩仲(En-Chung Lin) | |
| dc.subject.keyword | 微珠微流道,單核苷,酸多型性,熔解曲線分析,動態等位基因特異性雜交法,ataxia-telangiectasia突變基因, | zh_TW |
| dc.subject.keyword | bead-based microfluidc,single nucleotide polymorphism,dynamic allele-specific hybridization,melting curve analysis,ataxia-telangiectasia mutated gene, | en |
| dc.relation.page | 68 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-16 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 生物產業機電工程學研究所 | zh_TW |
| 顯示於系所單位： | 生物機電工程學系 | |
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