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研究生:邱偉倉
研究生(外文):Wei-tsang Chiou
論文名稱:奈米金粒子與緩衝液對於自然對流式聚合酵素連鎖反應影響之研究
論文名稱(外文):Study of the effect of nano-gold and buffer on natural-convection polymerase chain reaction
指導教授:陳明志陳明志引用關係
指導教授(外文):Ming-Jyh Chern
學位類別:碩士
校院名稱:國立臺灣科技大學
系所名稱:機械工程系
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:100
中文關鍵詞:聚合酵素連鎖反應效率自然對流基因複製奈米金粒子粒子影像速度儀
外文關鍵詞:Polymerase chain reactionnatural convectionDNA replicationgold nanoparticlesParticle image velocimetry
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我們觀察不同的聚合酵素連鎖反應(PCR) 的緩衝液, 如去離子水、TBE 緩衝液及膠體金溶液對於其反應之影響。並使用傳統熱循環式與自然對流式兩種不同的PCR 進行實驗。實驗得到的產物則應用電泳技術, 來觀察隨著反應循環數增加而產生的產量與特異性變化。結果發現TBE 緩衝液在超過標準的PCR 循環數(35個循環) 後, 仍可以保持PCR 中複製DNA 片段的特異性。接著並發現膠體金溶液及其中分離出來的金保存液, 兩者皆會對PCR 產生抑制力, 使得產量降低,但仍可以保持其特異性。而若是使用去離子水稀釋兩者, 再進行PCR 仍可以得到足夠的DNA 片段產量。我們使用粒子影像速度儀(Particle Image Velocimetry,PIV) 技術, 觀察自然對流式PCR 的流場結構, 了解以自然對流為主要機制的PCR, 是如何進行反應。並探討是什麼原因可以讓自然對流PCR 在較短的時間內, 達到與傳統PCR 同樣的複製DNA 效果。同時探討膠體金溶液對自然對流式PCR 流場結構的改變。
We investigate the effects of PCR (Polymerase Chain Reaction) buffer, such
as Deionized water (DI water), TBE buffer and nano gold colloid, on PCR. A conventional PCR and natural-convection PCR are considered to explore the effects.
Electrophoresis was conducted to analyze the PCR product. We found that the
amount of PCR product without any buffer and specificity were changed when the
number of cycle of PCR increased. In addition, we found that adding the TBE
buffer can keep the specificity even the number of PCR cycle exceeds 35 cycles. On the other hand, nano gold colloid and its buffer restrained PCR, but they still kept the specificity. It is, however, revealed that the enough PCR yield was obtained by diluted nano gold colloid. Subsequently, we utilized a PIV (Particle image velocimetry) technique to observe the flow structure of natural-convection PCR.
The mechanism why natural-convection PCR is more efficient than conventional
PCR was studied. The variation of flow structure of natural-convection PCR due
to nano gold colloids was found in PIV results.
中文摘要. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i
英文摘要. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii
致謝. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
目錄. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
符號索引. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii
圖目錄. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
表目錄. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv
1 導論1
1.1 研究動機. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1.1 DNA的歷史. . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.1.2 PCR的原理. . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.1.3 對流(Convectional) PCR 的原理. . . . . . . . . . . . . . . 8
1.1.4 奈米金粒子. . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.2 文獻回顧. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.3 論文架構. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2 實驗材料與方法17
2.1 PCR步驟與藥品製備. . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.1.1 特異性(Specificity) . . . . . . . . . . . . . . . . . . . . . . . 19
2.1.2 TBE緩衝液. . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.1.3 實驗試劑配方. . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2 C-PCR步驟. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.3 RB-PCR步驟. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.3.1 PID溫度控制器. . . . . . . . . . . . . . . . . . . . . . . . . 24
2.3.2 RB-PCR的流場可視化. . . . . . . . . . . . . . . . . . . . . 27
2.4 粒子影像速度儀(Particle image velocimetry, PIV) . . . . . . . . . 29
2.4.1 灰階圖片的表示. . . . . . . . . . . . . . . . . . . . . . . . . 30
2.4.2 PIV運算及分析. . . . . . . . . . . . . . . . . . . . . . . . . 31
2.4.3 PIV二維校正. . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.4.4 PIV動態校正. . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.4.5 影格數的獨立性. . . . . . . . . . . . . . . . . . . . . . . . . 33
2.4.6 次視窗大小的選用. . . . . . . . . . . . . . . . . . . . . . . 35
2.5 PCR產物分析: 膠體電泳. . . . . . . . . . . . . . . . . . . . . . . . 37
2.6 膠體金溶液配製步驟. . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.6.1 光譜儀量測. . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.6.2 奈米金粒子顆粒大小及濃度計算. . . . . . . . . . . . . . . 43
2.7 小結. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3 奈米金粒子及緩衝液對於PCR 的影響47
3.1 PCR緩衝液濃度的定義. . . . . . . . . . . . . . . . . . . . . . . . . 47
3.2 不同的PCR 緩衝液加入C-PCR 和RB-PCR 的影響. . . . . . . . 48
3.3 用金保存液稀釋不同濃度的膠體金溶液加入C-PCR和RB-PCR的
影響. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3.4 以去離子水稀釋奈米金粒子,觀察有無奈米金粒子對C-PCR和RBPCR
的影響. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.5 小結. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.5.1 PCR緩衝液的影響. . . . . . . . . . . . . . . . . . . . . . . 60
3.5.2 奈米金粒子與金保存液的交互作用. . . . . . . . . . . . . . 60
4 RB-PCR的流場型態觀測與分析63
4.1 RB-PCR反應腔中自然對流的流場結構. . . . . . . . . . . . . . . . 63
4.2 不同膠體金溶液濃度對RB-PCR 自然對流流場的影響. . . . . . . . 65
4.3 PCR試劑對RB-PCR 自然對流流場的影響. . . . . . . . . . . . . . 66
4.4 不同PCR 緩衝液對RB-PCR 自然對流流場的影響. . . . . . . . . 68
4.5 以去離子水稀釋膠體金溶液及金保存液為0.072x 對RB-PCR 流場
的影響. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
4.6 小結. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
4.6.1 RB-PCR的反應機制. . . . . . . . . . . . . . . . . . . . . . 71
4.6.2 緩衝液及奈米金粒子對RB-PCR 的影響. . . . . . . . . . . 72
5 結論與建議73
5.1 結論. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
5.2 建議. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
參考文獻. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
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