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論文中文名稱:直接量測於設計之長片段DNA內之電子傳遞 [以論文名稱查詢館藏系統]
論文英文名稱:Direct Measurement of Electrical Transport Through Long DNA Fragment with Designed Sequence [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
中文姓名:李漢軒
英文姓名:Han-Hsuan Lee
研究生學號:93688006
學位類別:碩士
語文別:中文
口試日期:2006-07-28
論文頁數:54
指導教授中文名:侯劭毅
指導教授英文名:Shao-Yi Hou
口試委員中文名:黃志宏;黃榮堂;王勝仕
中文關鍵詞:DNA導電度微電極原子力顯微鏡
英文關鍵詞:DNAconductivitymicroelectrodeAtomic Force Microscopy (AFM)
論文中文摘要:在先前許多團隊的研究下,DNA是否具有導電能力的疑問已經慢慢有了一些答案,在他們的努力下,我們知道了DNA可能具有導電能力的關鍵可能在於鳥糞嘌呤 (guanine,G)和鳥糞嘌呤間的距離。因為Marcus的理論以及鳥糞嘌呤是較有機會提供電子轉移的鹼基,所以每個GC鹼基對間要維持在10到15埃(Å)之內,以B-form DNA分子兩個鹼基對間的垂直距離約是3.4埃(Å)來計算,則相當於每個GC鹼基對間最多僅能容納2到3個AT鹼基對。
本研究撰寫FORTRAN程式進行序列的比對,藉以找到完全符合條件之DNA片段來進行研究。藍色鏈黴菌(Streptomyces coelicolor A3(2))之染色體DNA序列可以找出GC鹼基對間最多僅能容納2個AT鹼基對之最長片段5413bps約1.8μm。隨後,以聚合酶連鎖反應將目標DNA片段之量放大,並在5’端修飾上硫醇基,用以接於金微電極間。最後將目標DNA片段固定在微電極之兩端藉以量測電流,再以原子力顯微鏡觀測電極間DNA數量,藉此研究單一DNA片段之導電度。
從實驗結果中我們觀察到,在控制之相對溼度(75%及95%)下,無論是僅有單一DNA分子連接於電極之間,或因數條DNA分子糾結而連接電極之兩端,得到之電流值變化結果皆沒有顯著變化。而離子對於DNA導電特性之影響,測試結果也顯示了電流值的變化並不明顯。
論文英文摘要:The conductivity of DNA has been surveyed by several research groups, however, the result is still controversy. Based on Giese assumptions, the conductivity of DNA maybe depend on the distance between Guanines. Giese assumes that guanine is the most potential candidate in the four nucleotides for electron transfer. Electrons can take individual "hops" from guanine to guanine, allowing long-distance travel. In Marcus theory, the electrons can only travel 10 to 15 angstroms (Å). The vertical distance between two bases is 3.4 Å in B-form DNA, hence, two to three adenine-thymine (AT) base pairs (bps) are allowed between two guanine-cytosine (GC) bps.
A Fortran program which was written by us for this study was used to find out the suitable DNA sequences. On the chromosome of Streptomyces coelicolor A3(2), a 5413 bps or 1.8μm DNA fragment with at most two AT bps between two GC bps was served as template for polymerase chain reaction (PCR). The modified PCR primers contain thiol group at 5’ end to bind golden microelectrodes. The current of target DNA fragments were measured. The number of DNA fragments connected between microelectrodes was observed by Atomic Force Microscopy.
In this study, we found that the electric current along either single molecule or bundles of DNA with designed sequence is very low in air no matter that relative humidity is 75% or 95%. Our conclusion is that DNA is insulator in air.
論文目次:中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
表目錄 vi
圖目錄 vii
第一章 簡介 1
1.1 前言 1
1.2 研究動機及目的 3
第二章 研究背景與原理介紹 4
2.1 DNA之基本特性 4
2.2 相互矛盾的研究結果 6
2.3 電荷轉移理論 10
2.4 藍色鏈黴菌(Streptomyces coelicolor A3(2)) 12
2.5 Fortran電腦程式語言 12
2.6 原子力顯微鏡之簡介 13
2.4.1 原子力顯微鏡之原理 13
2.4.2 原子力顯微鏡之操作模式 17
第三章 實驗方法與步驟 19
3.1 實驗架構 19
3.2 Fortran電腦程式撰寫 19
3.3 菌種選擇與目標片段搜尋 21
3.4 DNA之製備步驟 21
3.4.1 染色體純化 21
3.4.2 DNA的酶切與接合反應 22
3.4.3 瓊脂糖膠電泳分離及萃取 24
3.4.3.1 瓊脂糖膠電泳分離 24
3.4.3.2 凝膠萃取 24
3.4.4 引子之設計 25
3.4.5 聚合酶連鎖反應之原理與參數設定 25
3.6 電極之製作流程簡介 29
3.6.1 繪製光罩圖案 26
3.6.2 微影製程及金屬薄膜沉積 27
3.7 以硫醇基將DNA固定及連接於微電極 30
3.8 電流量測方法 31
3.9 AFM之操作條件與流程 31
3.10 實驗藥品與設備 34
3.10.1 實驗藥品 34
3.10.1 實驗設備 35
第四章 結果與討論 38
4.1 利用電腦程式搜尋目標片段 38
4.2 DNA樣品之製備 41
4.3 微電極製作結果 42
4.4 將DNA連接於微電極偵測電流並以AFM觀測 43
4.4.1 電極間僅一條DNA分子連接 43
4.4.2 電極間DNA為束狀結構 44
4.4.3 電極間DNA為連接不完全 46
第五章 結論 50
參考文獻 51
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論文全文使用權限:同意授權於2007-08-24起公開