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HIGH-THROUGHPUT RETRIEVAL OF MOLECULAR CLONES FOR POLYNUCLEOTIDE SYNTHESIS : 폴리뉴클레오타이드 합성을 위한 초고속 분자 클론 추출 기술 개발
DC Field | Value | Language |
---|---|---|
dc.contributor.advisor | 권성훈 | - |
dc.contributor.author | 김효기 | - |
dc.date.accessioned | 2017-07-13T06:58:06Z | - |
dc.date.available | 2017-07-13T06:58:06Z | - |
dc.date.issued | 2013-02 | - |
dc.identifier.other | 000000010421 | - |
dc.identifier.uri | https://hdl.handle.net/10371/118911 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 전기·컴퓨터공학부, 2013. 2. 권성훈. | - |
dc.description.abstract | Molecular clone is a large population of DNA replicated from a single DNA molecule. Production, analysis and retrieval of the molecular clones are the key techniques in current biotechnology. Here, high-throughput method for the manipulation of molecular clones is presented using highly parallel sequencing and radiation-based non-contact molecular clone retrieval system.
Specifically, production and analysis methods of the molecular clones are investigated using a highly parallel sequencing platform. In emulsion polymerase chain reaction (PCR) at the sample preparation of parallel pyrosequencing, large amount of molecular clones are created. After the production of large amount of molecular clones, individual molecular clone is sequenced so that sequence and position information of each molecular clone are identified. Molecular clone retrieval system based on non-contact energy transmission is developed for rapid and precise retrieval of each molecular clone. Pulse laser, optical component, vision module, and motorized stage are integrated in the system. Also, system control software is developed combining functions such as: (i) extraction of sequence and positional information of each molecular clone from output data of highly parallel sequencing, (ii) scanning sequencing plate and high-resolution image reconstruction, (iii) algorithmic identification of molecular clone in the reconstructed image, (iv) synchronization of sequencing plate movement, pulse-laser radiation, and carrier plate movement. Furthermore, retrieval method of molecular clones produced by bacterial cell is presented. Mixture of genetically diverse bacterial cell stock is spread and grown on a solid growth medium. After the short period of growth, position of each cell clone is identified using microscope vision scanning system, and then each cell clone is retrieved using focused pulse laser. Retrieved individual cell clone is contained in carrier micro well plate. DNA of retrieved individual cell clone is tagged with barcode DNA, and analyzed and identified by highly parallel sequencing. Method and system developed here may be applicable in a wide range of biotechnology area such as synthetic DNA production, antibody drug discovery, and functional genome study with exceptional cost reduction and increased throughput due to highly parallel nature of the method. | - |
dc.description.tableofcontents | Abstract i
Contents iii List of Figures vi Chapter 1 Introduction 1 Chapter 2 Fundamentals 7 2.1 Conventional Molecular Cloning 7 2.1.1 Definition of Molecular Clone 8 2.1.2 Polymerase Chain Reaction 9 2.1.3 Vector Cloning 10 2.1.4 Retrieval of Molecular Clones 12 2.1.5 Discussion of the Conventional Molecular Cloning 13 2.2 Column-based Chemical DNA Synthesis 13 2.2.1 Structure of DNA 13 2.2.2 Phosphoramidite DNA Synthesis Chemistry 14 2.2.3 Commercial Column-based DNA Synthesizer 17 2.3 DNA Microarray 19 2.3.1 Fabrication Method for DNA Microarray 19 2.3.2 Discussion on the Coupling Efficiency 21 2.4 Chain-termination Sequencing 23 2.5 Highly Parallel Sequencing 24 2.5.1 Sequencing-by-Ligation 25 2.5.2 Sequencing-by-Synthesis 26 2.6 Comparison of Sequencing Platforms 27 Chapter 3 System Overview 28 Chapter 4 Sample Preparation 31 4.1 Sequence Design for Microarray DNA synthesis 31 4.2 Emulsion PCR for Reduced Amplification Bias 34 4.3 Optimization of Emulsion PCR Condition 34 4.4 Adapter Ligation for Next Generation Sequencing 37 4.5 Comparison of the Emulsion PCR and Normal PCR 39 4.6 shRNA Producing Oligo Design 43 4.7 Protocol for Emulsion PCR 45 Chapter 5 Non-contact Retrieval of Molecular Clones 48 5.1 Pulse Laser based Molecular Clone Retrieval System 48 Chapter 6 Microfluidic DNA Preconcentrator 60 6.1 Introduction 60 6.2 Materials and Methods 62 6.3 Results and Discussion 67 Chapter 7 Conclusion 72 Appendix 73 Bibliography 106 Abstract in Korean 1 | - |
dc.format | application/pdf | - |
dc.format.extent | 3910792 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject.ddc | 621 | - |
dc.title | HIGH-THROUGHPUT RETRIEVAL OF MOLECULAR CLONES FOR POLYNUCLEOTIDE SYNTHESIS | - |
dc.title.alternative | 폴리뉴클레오타이드 합성을 위한 초고속 분자 클론 추출 기술 개발 | - |
dc.type | Thesis | - |
dc.description.degree | Doctor | - |
dc.citation.pages | 124 | - |
dc.contributor.affiliation | 공과대학 전기·컴퓨터공학부 | - |
dc.date.awarded | 2013-02 | - |
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