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論文名稱 Title |
核酸及鎖核酸錯配敏感雜化之原子力顯微鏡研究 Study on Mismatch-Sensitive Hybridization of DNA-DNA and LNA-DNA by Atomic Force Microscopy |
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系所名稱 Department |
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畢業學年期 Year, semester |
語文別 Language |
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學位類別 Degree |
頁數 Number of pages |
76 |
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研究生 Author |
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指導教授 Advisor |
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召集委員 Convenor |
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口試委員 Advisory Committee |
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口試日期 Date of Exam |
2008-07-21 |
繳交日期 Date of Submission |
2008-07-25 |
關鍵字 Keywords |
錯配雜化反應、核酸雜化反應、鎖核酸、核酸自組裝單層膜、原子力顯微鏡 DNA hybridization, mismatched hybridization, locked nucleic acid, DNA self-assembled monolayers, atomic force microscopy |
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統計 Statistics |
本論文已被瀏覽 5643 次,被下載 10 次 The thesis/dissertation has been browsed 5643 times, has been downloaded 10 times. |
中文摘要 |
本研究將原子力顯微鏡(AFM)應用於奈米顯影,觀察核酸自組裝薄膜因雜化反應所導致之膜厚變化。首先在探針去氧核糖核酸(DNA)及探針鎖核酸(LNA)自組裝薄膜上刻畫奈米尺度凹型圖案,觀察奈米圖案深度因雜化反應形成豎直之雙股鏈導致的高度變化,調控探針濃度探討探針密度對於雜化反應之影響,尋求最佳辨識標靶DNA探針密度,測得當探針DNA濃度為2μM及探針LNA濃度為1μM所得到之雜化反應高度變化最顯著。其次為得知雜化結合反應之熱力學資訊,在最佳雜化探針密度條件下研究與不同標靶DNA濃度由AFM觀測的高度變化與雜化時間關係,將實驗數據以Langmuir model擬合得到解離平衡常數(Kd)及最大雜化效率。更進一步將核酸雜化實驗延伸至具錯配雜化情況予以偵測,在DNA:DNA及LNA:DNA錯配雜化反應所量測到之高度變化僅約1nm且約數十分鐘的反應時間高度變化即達到飽和,與完全配對雜化實驗所量測之結果具有明顯之差異,亦證實DNA與LNA在完全互補與錯配雜化可藉由AFM奈米顯影術表現出辨認之差異性。 |
Abstract |
In this study we use AFM-based nanolithography technique to produce nanofeatures of the single strand DNA and LNA probe molecules which are prepared via thiolated nucleic acid self-assembled monolayers (SAMs) on gold substrates. The goal is to observe the topographic changes of the DNA film structures resulting from the formation of rigid double strand DNA when the target and probe DNAs bind together. The so-called hybridization depends strongly on the probe density on the substrate surface. To find the proper probe density for hybridization, we vary the concentration of the probe DNA and search for the optimal conditions for measuring the height changes of the nanofeatures. We also monitor the topographic changes of the DNA nanofeatures in the different target DNA concentrations as a function of time, and the binding isotherms are fitted with the Langmuir adsorption model to derive the equilibrium dissociation constant and maximum hybridization efficiency. In addition, we extend the nanoscale hybridization reaction detection to mismatched DNA:DNA and LNA:DNA hybridization, and observe that topographic change of mismatched hybridization is inconspicuous and rapidly reach equilibrium. The results reveal the apparent difference between the perfect match and mismatch conditions, and validate that this approach can be applied to differentiate the situations for both perfect match and mismatch cases, demonstrating its potentials in the gene chip technology. |
目次 Table of Contents |
中文摘要 i 英文摘要 ii 目錄 iii 圖表目錄 v 第壹章 緒論 1 1-1 前言 1 1-2 研究背景 2 1-2.1 核酸雜化反應及鹼基錯配反應 2 1-2.2 鎖核酸雜化反應 4 1-3 研究動機 8 第貳章 儀器與實驗 9 2-1 實驗儀器 9 2-2 原子力顯微鏡原理 9 2-3 Contact Mode AFM氣、液相掃描 10 2-4 實驗材料 14 2-5 實驗操作方法 16 2-5.1 清洗容器 16 2-5.2 製備gold substrate/Au(111) 16 2-5.3 生物樣品配製 18 2-5.4 製備核酸自組裝單層膜 22 2-5.5 核酸雜化反應 22 2-6 原子力顯微鏡奈米顯影術操作步驟 22 2-7 實驗分析量測 24 第參章 核酸雜化反應觀測 27 3-1 DNA自組裝單層膜 27 3-2 AFM觀測DNA雜化反應 30 3-3 探針密度(濃度)對於雜化反應之影響 36 3-4 雜化反應之熱力學資訊-解離平衡常數及最 大雜化效率 39 3-5 AFM觀測鎖核酸(LNA)雜化反應 45 第肆章 DNA:DNA及LNA:DNA錯配靈敏度之偵測 51 4-1 核酸鹼基錯配雜化相關研究 51 4-1.1 DNA鹼基錯配雜化 51 4-1.2 LNA鹼基錯配雜化 53 4-2 AFM觀測DNA:DNA及LNA:DNA鹼基錯配雜 化 55 第伍章 結論 58 參考文獻 60 |
參考文獻 References |
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