原子力顯微鏡(AFM)的解析度雖仍不及電子顯微鏡，但液相AFM配合輕敲掃描卻是最合適在接近生理環境下觀測生物分子之技術。然其執行上必須克服一些問題方足以應用於生物體系之研究。本論文發現在溶液中使用含二價陽離子之緩衝液能有效固定DNA分子，另外針對懸臂驅動頻率、輕敲時振幅之設定、回饋控制參數及掃描速度尋求最佳工作條件。在探討Ssp I-linearized pUC19 DNA/EcoR I限制酶交互作用體系，可明顯觀察複合體某些特殊結構。例如：在限制酶切割DNA鏈時，捕捉到EcoR I在特定識別位置上使DNA呈銳角彎曲之中間態影像，並引申出作用過程中酶運動之滑行、跳躍或在迴圈構型上互相換位等模式。AFM即時偵測酶/核酸反應過程研究中則已有初步結果，但欲取得真正動態資訊，仍有待努力。

The image resolution of atomic force microscopy (AFM) is still less superior to that of the electron microscopy (EM). However AFM operated in liquids complemented by Tapping-mode (TM) detection proves to be more suitable for imaging biomolecules in physiological-like environments. Nevertheless, manipulation of AFM in solution turned out to be non-trivial, several technical difficulties were encountered. In the thesis, I report using divalent cation-containing buffer as a feasible method to immobilize DNA molecules effectively for imaging in liquid media. AFM operating conditions, such as cantilever oscillating drive frequency, setpoint amplitude, feedback control parameters and scan rates were studied to obtain the optimized function. Various AFM images of Ssp I-linearized pUC19 DNA/EcoR I restriction enzyme complexes were captured, revealing the molecular details of their complex machineries. For example, the intermediate stage of the enzyme cleavage action was displayed by images showing that DNA was bent by an acute angle at the active site in the presence of one single EcoR I molecule. Some evidence for a jumping, sliding or intersegmental transfer mechanism is achieved. To trace the enzyme-DNA interaction dynamic in real time, preliminary results were obtained, but further improvements are required.

中文摘要………….……………………………………………………i
英文摘要………………………………………………………………ii
目錄………………………………………………………………….iii
圖表目錄………………………………………………………………vi

第壹章 緒論………………………………………………………1
1-1 前言…………………………………………………………1
1-2 研究背景………………………………………………....2
1-2.1 AFM研究液相DNA分子………………………...…….……3
1-2.2 DNA與蛋白質的交互作用………………………...……..5
RNA polymerase complexes……..….….…….....…5
Enzymatic degradation…………………………………6
1-3 研究動機……………………………………………………8
第貳章 儀器與實驗介紹…………………………………………9
2-1 實驗儀器…………………………………………………………9
2-2 原子力顯微鏡原理………………………………………………9
2-3 TM-AFM氣、液相之掃描………………………………………..10
氣相操作步驟…………………………………………………..12
液相操作步驟…………………………………………………..13
2-4 實驗材料…………………………………………………………15
基板………………………………………………………………15
樣品………………………………………………………………15
緩衝液……………………………………………………………15
成像環境…………………………………………………………15
2-5 實驗操作方法………………………………………………18
2-5.2 製備修飾基板………………………………………………18
精胺(spermine)-雲母片的製備………………………….18
2-5.2 生物樣品配置………………………………………………18
DNA溶液…………………………………………………..18
Plasmid DNA之限制酶切割……………………………..19
DNA fragments extraction kit……………………….20
2-5.3 製作生物樣品基板………………………………………..21
DNA分子…………………………………………………..21
稀薄濃度的酶/核酸複合體………………………………22
液相槽即時動態追蹤之酶/核酸反應……………………23
2-6 實驗分析量測………………………………………………23
第參章 掌握原子力顯微鏡之偵測效能…………………………25
3-1 基板…………………………………………………………25
3-2 液相下輕敲式原子力顯微鏡操作變因研究………………28
3-2.1 固定DNA…………………………………………………..28
3-2.2 懸臂驅動頻率對成像的影響…………………………….30
3-2.3 選擇Setpoint amplitude……………………………….32
3-2.4 修正回饋訊號…………………………………………….33
第肆章 酶與核酸之交互作用……………………………………...35
4-1 限制酶與限制酶/核酸複合體…………………………….35
4-2 限制酶切割雙股DNA in vitro之AFM成像研究………….38
4-2.1 Ssp I切割環型plasmid pUC19 DNA之成像…………….39
4-2.2 Ssp I及EcoR I先後切割環型plasmid pUC19 DNA之成像.42
4-2.3 Ssp I-linerized pUC19 DNA/EcoR I複合體之成像….44
4-2.4 Ssp I-linerized pUC19 DNA/EcoR V複合體之成像….51
4-3 AFM即時動態觀測限制酶與雙股DNA作用…………………54
第伍章 結論………………………………………………………….60
參考文獻……………………………………………………………….62

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