以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：22

、訪客IP：18.219.22.169

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
參考文獻	[1] Katie M Horsman et al. “Forensic DNA analysis on microfluidic devices: a review”. In: Journal of forensic sciences 52.4 (2007), pp. 784–799. [2] Stephen C Jacobson and J Michael Ramsey. “Integrated microdevice for DNA restriction fragment analysis”. In: Analytical chemistry 68.5 (1996), pp. 720–723. [3] Louis Dubeau et al. “Southern blot analysis of DNA extracted from formalin-fixed pathology specimens”. In: Cancer Research 46.6 (1986), pp. 2964–2969. [4] Kit-SumWong and Jolita Uthe. “New Advances in NGS Library Prep QC: gDNA Metric, Larger Fragments, Higher Sample Sensitivity”. In: Genetic Engineering & Biotechnology News 35.5 (2015), pp. 22–23. [5] David C Schwartz and Charles R Cantor. “Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis”. In: cell 37.1 (1984), pp. 67–75. [6] Bnice W Birren et al. “Optimized conditions for pulsed field gel electrophoretic separations of DNA”. In: Nucleic acids research 16.15 (1988), pp. 7563–7582. [7] Georges F Carle, Mark Frank, and Maynard V Olson. “Electrophoretic separations of large DNA molecules by periodic inversion of the electric field”. In: Science 232.4746 (1986), pp. 65–68. [8] Kevin D Dorfman et al. “Beyond gel electrophoresis: Microfluidic separations, fluorescence burst analysis, and DNA stretching”. In: Chemical reviews 113.4 (2013), pp. 2584–2667. [9] Pier Giorgio Righetti. Capillary electrophoresis in analytical biotechnology: a balance of theory and practice. Vol. 4. CRC press, 1995. [10] Herb Schwartz and Andras Guttman. Separation of DNA by capillary electrophoresis. Citeseer, 1995. [11] Chia-Fu Chou et al. “Sorting by diffusion: An asymmetric obstacle course for continuous molecular separation”. In: Proceedings of the National Academy of Sciences 96.24 (1999), pp. 13762–13765. [12] Jongyoon Han and Harold G Craighead. “Separation of long DNA molecules in a microfabricated entropic trap array”. In: Science 288.5468 (2000), pp. 1026–1029. [13] Joshua David Cross, Elizabeth A Strychalski, and Harold G Craighead. “Size-dependent DNA mobility in nanochannels”. In: Journal of Applied Physics 102.2 (2007), p. 024701. [14] Derek Stein et al. “Pressure-driven transport of confinedDNApolymers in fluidic channels”. In: Proceedings of the National Academy of Sciences 103.43 (2006), pp. 15853–15858. [15] Chia-Fu Chou et al. “Electrodeless dielectrophoresis of single-and doublestranded DNA”. In: Biophysical journal 83.4 (2002), pp. 2170–2179. [16] Kuo-Tang Liao and Chia-Fu Chou. “Nanoscale molecular traps and dams for ultrafast protein enrichment in high-conductivity buffers”. In: Journal of the American Chemical Society 134.21 (2012), pp. 8742–8745. [17] Eileen T Dimalanta et al. “A microfluidic system for large DNA molecule arrays”. In: Analytical chemistry 76.18 (2004), pp. 5293–5301. [18] Dmitry Torchinsky and Yuval Ebenstein. “Sizing femtogram amounts of dsDNA by single-molecule counting”. In: Nucleic acids research 44.2 (2016), e17–e17. [19] WPatrick Ambrose et al. “Flow cytometric sizing of DNA fragments”. In: Topics in fluorescence spectroscopy. Springer, 2003, pp. 239–270. [20] Alonso Castro, Frederic R Fairfield, and E Brooks Shera. “Fluorescence detection and size measurement of single DNA molecules”. In: Analytical Chemistry 65.7 (1993), pp. 849–852. [21] Jean-Louis Viovy. “Electrophoresis of DNA and other polyelectrolytes: Physical mechanisms”. In: Reviews of Modern Physics 72.3 (2000), p. 813. [22] What is gel electrophoresis? 2021. URL: https://www.yourgenome.org/ facts/what-is-gel-electrophoresis. [23] KA Ferguson. “Starch-gel electrophoresis—application to the classification of pituitary proteins and polypeptides”. In: Metabolism 13.10 (1964), pp. 985–1002. [24] Philippe Cluzel et al. “DNA: an extensible molecule”. In: Science 271.5250 (1996), pp. 792–794. [25] Leonard S Lerman and Harry L Frisch. “Why does the electrophoretic mobility of DNA in gels vary with the length of the molecule?” In: Biopolymers: Original Research on Biomolecules 21.5 (1982), pp. 995–997. [26] Gary W Slater et al. “Quantitative analysis of the three regimes of DNA electrophoresis in agarose gels”. In: Biopolymers: Original Research on Biomolecules 27.3 (1988), pp. 509–524. [27] Gary W Slater. “Theory of band broadening for DNA gel electrophoresis and sequencing”. In: Electrophoresis 14.1 (1993), pp. 1–7. [28] Tom Duke. “The Physics of DNA Electrophoresis”. In: Biologically Inspired Physics. Springer, 1991, pp. 71–80. [29] Mary Elizabeth Kaufmann. “Pulsed-field gel electrophoresis”. In: Molecular bacteriology. Springer, 1998, pp. 33–50. [30] Katheleen Gardiner. “Pulsed field gel electrophoresis”. In: Analytical chemistry 63.7 (1991), pp. 658–665. [31] Cassandra L Smith and Charles R Cantor. “[28] Purification, specific fragmentation, and separation of large DNA molecules”. In: Methods in enzymology 155 (1987), pp. 449–467. [32] TAJ Duke and RH Austin. “Microfabricated sieve for the continuous sorting of macromolecules”. In: Physical review letters 80.7 (1998), p. 1552. [33] Jongyoon Han, SW Turner, and Harold G Craighead. “Entropic trapping and escape of long DNA molecules at submicron size constriction”. In: Physical review letters 83.8 (1999), p. 1688. [34] J Calvin Giddings et al. Unified separation science. Wiley, 1991. [35] Thomas T Perkins, Douglas E Smith, and Steven Chu. “Single polymer dynamics in an elongational flow”. In: Science 276.5321 (1997), pp. 2016– 2021. [36] F Brochard and PG De Gennes. “Dynamical scaling for polymers in theta solvents”. In: Macromolecules 10.5 (1977), pp. 1157–1161. [37] KK Sriram et al. “DNA combing on low-pressure oxygen plasma modified polysilsesquioxane substrates for single-molecule studies”. In: Biomicrofluidics 8.5 (2014), p. 052102. [38] A Bensimon et al. “Alignment and sensitive detection of DNA by a moving interface”. In: Science 265.5181 (1994), pp. 2096–2098. [39] Zhengping Huang, James H Jett, and Richard A Keller. “Bacteria genome fingerprinting by flow cytometry”. In: Cytometry: The Journal of the International Society for Analytical Cytology 35.2 (1999), pp. 169–175. [40] Jeffrey T Petty et al. “Characterization of DNA size determination of small fragments by flow cytometry”. In: Analytical Chemistry 67.10 (1995), pp. 1755–1761. [41] Zhengping Huang et al. “Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones”. In: Nucleic acids research 24.21 (1996), pp. 4202–4209. [42] Robert C Habbersett and James H Jett. “An analytical system based on a compact flow cytometer for DNA fragment sizing and single-molecule detection”. In: Cytometry Part A: The Journal of the International Society for Analytical Cytology 60.2 (2004), pp. 125–134. [43] MatthewMFerris et al. “Performance assessment of DNA fragment sizing by high-sensitivity flow cytometry and pulsed-field gel electrophoresis”. In: Journal of clinical microbiology 42.5 (2004), pp. 1965–1976. [44] George B Arfken and Hans J Weber. Mathematical methods for physicists. 1999. [45] Ronald Newbold Bracewell and Ronald N Bracewell. The Fourier transform and its applications. Vol. 31999. McGraw-Hill New York, 1986. [46] Tadeusz Guszczynski et al. “Capillary zone electrophoresis of large DNA”. In: Electrophoresis 14.1 (1993), pp. 523–530. [47] Alexandra Agronskaia et al. “Two-color fluorescence in flow cytometry DNA sizing: Identification of single-molecule fluorescent probes”. In: Analytical chemistry 71.20 (1999), pp. 4684–4689.
指導教授	周家復 陳志強(Chia-Fu Chou Chi-Keung Chen)	審核日期	2022-9-28
推文	facebook   plurk   twitter   funp   google   live   udn   HD   myshare   reddit   netvibes   friend   youpush   delicious   baidu
網路書籤	Google bookmarks   del.icio.us   hemidemi   myshare