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姓名	瑞吉雅(Guia Raymundo)  查詢紙本館藏	畢業系所	生物物理研究所
論文名稱	光學奈米流道應用於至十萬鹼基對之DNA極速尺寸分析 (Ultrafast size profiling of 100 kilo-base paired DNA using optonanofluidic device)
檔案	[Endnote RIS 格式]    [Bibtex 格式]    [相關文章]   [文章引用]   [完整記錄]   [館藏目錄]   至系統瀏覽論文 (2025-1-1以後開放)
摘要(中)	DNA 長度的量測在生物領域中是項非常重要的技術。例如DNA 指紋鑑定(DNA fingerprinting)、 限制酶定位法(restriction mapping)、流行病學基因分型 (epidemiological genotyping)、次世代定序(next-generation sequencing)等等的生物 技術中，都需要量測DNA 之長度。最傳統的DNA 長度量測方式為凝膠電泳法(Gel electrophoresis)。然而此方法只適用於量測長度50 kbps 以下之DNA。對於50 kbps 以上之DNA，則需要藉由週期性改變電場方向的方式，也就是脈衝場凝膠電泳法(Pulsed Field Gel Electrophoresis)，來完成DNA 長度的量測。然而脈衝場凝膠電泳的量測時間 需要數小時至數天。此篇論文將展示一種新的DNA 長度量測技術。此技術結合奈米流道生物晶片以及單分子數位影像分析，目前已經可以於10~60 分鐘，量測長度最長 100 kbps左右之DNA。未來此技術將有潛力達到於30 分鐘內，量測1000 kbps 長度以上之DNA。
摘要(英)	DNA sizing is one of the most crucial processes in molecular biology. It is important for processes in DNA fingerprinting, restriction mapping , epidemiological genotyping, and the growing utility of next-generation sequencing. In the past decades, DNA gel electrophporesis has been the main tool at lab-bench to separateDNA fragments; however, challenges persist when sizing DNA molecules up to 50 kbp. Although pulse-field gel electrophoresis (PFGE) can separate long DNA fragments up to mega-base pairs by the periodic change of the electric field direction, PFGE usually lasts from hours to days. Here, we provide a simple single-molecule based DNA profiling device and methodology with designated algorithm to achieve an ultrafast size profiling. Samples up to 100 kbp DNA molecules were efficiently sized into bands from 10 to 60 minutes. Our results establish the ability, far beyond the conventional gel electrophoresis, for easy and quick DNA sizing up to 100-base pairs in complex DNA samples. We expect our method can size DNA molecules up to mega-base pairs for less than 30 minutes.
關鍵字(中)	★ 指紋鑑定 ★ 限制酶定位法 ★ 流行病學基因分型 ★ 次世代定序	關鍵字(英)	★ DNA fingerprinting ★ restriction mapping ★ epidemiological genotyping ★ next-generation sequencing
論文目次	Abstract vii Contents xi List of Figures xiii List of Tables xvii 1 Introduction 1 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Outline of the thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 DNA polyelectrolyte in external field 3 3 Methods for DNA sizing 7 3.1 Gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3.2 Pulsed field gel electrophoresis . . . . . . . . . . . . . . . . . . . . 11 3.3 Capillary electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . 14 3.4 Micro-/nanofluidic separation . . . . . . . . . . . . . . . . . . . . 15 3.4.1 Rectified diffusion . . . . . . . . . . . . . . . . . . . . . . . 16 3.4.2 Entropic traps . . . . . . . . . . . . . . . . . . . . . . . . . . 18 3.4.3 Nanochannels . . . . . . . . . . . . . . . . . . . . . . . . . . 20 3.4.4 Pressure driven . . . . . . . . . . . . . . . . . . . . . . . . . 21 3.5 Molecular combing . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.6 Flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3.7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 4 Materials and methods 31 4.1 Device fabrication . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 4.1.1 Fabrication of micro-/nanofludic channels . . . . . . . . . 31 4.2 Loading hole drilling . . . . . . . . . . . . . . . . . . . . . . . . . . 34 4.2.1 PSQ bonding . . . . . . . . . . . . . . . . . . . . . . . . . . 35 4.3 Fluorescently labeled dsDNA and buffer solution . . . . . . . . . 36 4.4 Data acquisition and analysis . . . . . . . . . . . . . . . . . . . . . 38 4.4.1 Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 4.4.2 Image Processing . . . . . . . . . . . . . . . . . . . . . . . . 38 Noise reduction . . . . . . . . . . . . . . . . . . . . . . . . . 39 Segmentation . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Excluding non-uniform illumination . . . . . . . . . . . . . 41 4.4.3 Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Pixel area vs. mean intensity . . . . . . . . . . . . . . . . . 42 Effective size distribution histogram . . . . . . . . . . . . . 42 5 Results and discussions 45 5.1   DNA-HindIII Digest . . . . . . . . . . . . . . . . . . . . . . . . . 45 5.2   DNA-Mono Cut Mix . . . . . . . . . . . . . . . . . . . . . . . . . 50 5.3 MidRange PFG Marker and   DNA-HindIII Digest . . . . . . . . 55 5.4 MidRange PFG Marker . . . . . . . . . . . . . . . . . . . . . . . . . 62 5.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 6 Conclusion and future prospects 71 Bibliography 73
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指導教授	周家復 陳志強(Chia-Fu Chou Chi-Keung Chen)	審核日期	2022-9-28
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