博碩士論文 91324015 詳細資訊




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:41 、訪客IP:18.118.137.243
姓名 高立安(Li-An Kao)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 以表面電漿共振儀研究單股去氧核醣核酸之二級結構於去氧核糖核酸雜交在動力學與反應機制的效應
(Studies of the Effect of Secondary Structure of single-stranded DNA on the Kinetics and Mechanism of Hybridizationby Surface Plasmon Resonance)
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摘要(中) 表面電漿共振(Surface Plasmon Resonance,SPR)為可利用檢測光學直接偵測晶片表面上因生物反應造成折射率微量變化來達到免標定之生醫光電技術。SPR 技術具有靈敏度遠高於其他檢測方法且可排除標定的繁複過程,並且能即時地分析生物分子之間的交互作用(biomolecular interaction analysis,BIA),而大大地縮短檢測的時間並提供了更多且精確的生物資訊。近年來,已被廣泛地應用在很多生物分子診斷的領域上,諸如receptor-ligand 或抗原(antigen)與抗體(antibody)之交互作用、蛋白質分子的非特定吸附、薄膜與蛋白質間交互反應或去氧核醣核酸分子雜交(DNAhybridization)等。
本實驗室在本校機械系陳顯禎教授之協助下,自行研發的表面電漿共振系統已成功應用於各式生物分子診斷等領域,如建立完整的核酸雜交動力學研究程序,並實際檢測寡去氧核糖核酸序列雜交試驗。使用此系統於檢測、研究分子間的交互反應動力學機制可提供一套非常準確、即時的資訊。
本研究是利用表面電漿共振儀,配合DNA 分子結構預測軟體(Oligo)所計算之DNA 二級結構,來量測不同二級結構之單股DNA 於不同溫度下雜交的行為。藉此得知處於不同雜交環境以及不同的DNA 分子結構下雜交反應之動力數據,再配合質傳限制模式(Mass transport limitation model)以動力學的角度進行數據分析,並使用Van’t Hoff 方程式在DNA 雜交反應之熱力學數據對DNA 雜交反應的機制作較詳細的討論。
摘要(英) This study utilizes a surface plasmon resonance (SPR) biosensor and a theoretical secondary structure calculation program(Oligo) to investigate the influence of secondary structures of ssDNA on the DNA hybridization. It is found that the SPR angular shifts associated with the
three pairs of 60mer oligo-nucleotides with prominent secondary structures are lower than those observed for the two pairs of oligonucleotides with no obvious secondary structures. It is also determined that increasing the DNA hybridization temperature from 35 oC to 45 oC reduces secondary structure effects on hybridization. On the hybridization with mixture target oligo-nucleotides, the SPR results demonstrate that presence of oligo-nucleotides mixture due to non-specific interactions
between non-complementary probes and targets interfere the extent of kinetics of hybridization significantly.
In this investigation, thermodynamics information was obtained by Van’t Hoff equation and combining with the kinetics data from SPR, the
hybridization mechanism were proposed and discussed.
關鍵字(中) ★ 表面電漿共振光譜儀
★ 二級結構
★ 寡核苷酸雜交
關鍵字(英) ★ surface plasmon resonance
★ oligo-nucleotide hybridization
★ secondary structure
論文目次 第一章前言...................................................................1
第二章文獻回顧...............................................................3
2.1 表面電漿共振(Surface Plasmon Resonance,SPR) ....................3
2.1.1 表面電漿現象原理.........................................................3
2.1.2 光學激發表面電漿之方式..................................................4
2.1.3 SPR 檢測生物反應.........................................................7
2.1.4 表面電漿共振光譜儀於分子交互作用上可提供之資訊.................8
2.1.5 其他類型之表面電漿共振..................................................9
2.2 SPR 於生物檢測上之應用領域................................................15
2.2.1 蛋白質變(復)性研究......................................................15
2.2.2 DNA 與Protein 之間的交互作用.....................................16
2.2.3 Epitop Mapping..........................................................18
2.2.4 抗原(Antigen)與抗體(Antibody)與之交互作用..............19
2.2.5 生物膜(membrane)與酯雙層(lipid bilayer)......................20
2.2.6 阿茲海默症致病研究(Alzheimer) ....................................22
2.2.7 高分子膜(polymer films)研究..........................................23
2.2.8 非特異吸附生物分子交互作用-生物相容性(biocompatibility)...............24
2.2.9 組織工程...............................................................25
2.3 去氧核醣核酸及核酸雜交反應................................................28
2.3.1 去氧核醣核酸分子簡介....................................................28
2.3.2 核酸雜交反應...........................................................30
2.3.3 單股核酸之二級結構.....................................................36
第三章實驗藥品與儀器設備.....................................................38
3.1 實驗藥品.................................................................38
3.2 實驗儀器設備..............................................................40
3.2.1 表面電漿共振儀(Surface Plasmon Resonance) ...............40
3.2.2 Oligo 序列模擬軟體.....................................................44
3.3 實驗目的..............................................................46
3.4 實驗方法..................................................................46
3.4.1 實驗溶液配製..........................................................46
3.4.2 實驗步驟............................................................47
第四章結果與討論..........................................................51
4.1 Oligo 模擬核酸序列之結果..............................................51
4.2 二級結構對核酸雜交的影響.............................................59
4.2.1 具明顯二級結構雜交實驗之結果....................................59
4.2.2 非明顯二級結構雜交實驗之結果....................................61
4.3 溫度效應對二級結構核酸雜交之影響.....................................62
4.3.1 溫度效應對具明顯二級結構於核酸雜交反應之影響...62
4.3.2 溫度效應對非明顯二級結構於核酸雜交反應之影響...65
4.4 雜交反應動力學分析........................................................67
4.4.1 反應模式的選擇........................................................67
4.4.2 動力曲線擬合............................................................70
4.4.3 擬合動力學參數之合理評估............................................75
4.4.4 動力學分析...........................................................77
4.5 混合樣品之雜交反應.......................................................80
4.5.1 實驗設計................................................................80
4.5.2 混合樣品之雜交反應................................................80
4.6 雜交反應熱力學參數分析..................................................85
第五章結論...............................................................87
Reference ..................................................................90
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指導教授 陳文逸(Wen-Yih Chen) 審核日期 2004-6-17
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