臺灣博碩士論文加值系統

本研究利用兩種形態微陣列探針（Probe）與多樣的探針濃度，進行去氧核醣核苷酸（DNA）標的物（Target）預雜交反應（Pre-hybridization），以探討第二標的物樣品的雜交效率。使用三組經微陣列點樣之DNA探針和與其自身序列互補的標的物，每組包含一對DNA探針和一對與其自身序列互補並標定兩種易區分的螢光染色分子，經過特別設計的每個探針序列使標的物能分別互補於探針兩相鄰區域，分子空間效應對於雜交效率也透過比較單一及兩個標的物之間螢光訊號強度來探討。結果顯示在低探針濃度時有較好的雜交效率，且利用短序列DNA標的物進行第一次雜交反應可改善第二標的物鍵結至相同探針的雜交效率。然而第一標的物也發現因為第二標的物的鍵結而犧牲其自身訊號強度。

The effect of pre-hybridizing genetic DNA target on the hybridization efficiency of the second target samples is studied on two probe configurations and various probe concentrations. Three sets of microarrayed DNA probes and their complementary samples are utilized. Each set contains a pair of DNA probes and a pair of their complementary samples labeled with two distinct fluorescent dyes. The sequence of each probe is especially designed so that two targets are simultaneously complementary to two adjacent sections of the probe. The molecular steric effect on the hybridization efficiency is investigated by comparing the dye signals between configurations of one-target and two-target hybridization scenarios. The results show that a low probe concentration gives better hybridization efficiency and the first-hybridization conducted by a shorter-size DNA target improves the hybridization efficiency of the second target coupling onto the same probe. The first DNA target is also found to sacrifice signal intensity for the second one.

中文摘要…………….……………………………………………………… I
英文摘要…………….……………………………………………………... II
誌謝辭………………………...…………………………………………… III
總目錄……………………………………………………………………… V
圖目錄……………………………………………………………………… X
表目錄……………………….…………………………………………... XIII
第一章 緒論
1-1 生物晶片的發展背景………………………………………………….. 1
1-2 生物晶片的類型……………………………………………………….. 2
1-2.1 微陣列晶片（Microarray-chip）………………………………… 2
1-2.2 微流體晶片（Microfluidic-chip）………………………………… 3
1-3 晶片的應用…………………………………………………………….. 4
1-4 表面改質……………………………………………………………….. 5
1-5 晶片點陣固定方式…………………………………………………….. 7
1-5.1 光引導原位合成法（Light-directed synthesis, Photolithography）… 7
1-5.2 接觸式點樣法（Pin-touch, Mechanical micro-spotting）……… 8
1-5.3 噴墨法（Inkjet printer, Ink jetting）………………………………. 9
1-6 聚合酵素鏈鎖反應（Polymerase Chain Reaction）………………….. 11
1-7 DNA熔解溫度（Tm值）……………………………………………… 12
1-7.1 Wallace公式……………………………………………………… 12
1-7.2 Marmur公式…………………………………………………….. 12
1-7.3 Howley公式…………………………………………………….. 13
1-7.4 Nearest-neighbor公式…………………………………………... 13
1-8 膠體電泳（Gel electrophoresis）………………………………………… 14
1-8.1 電泳的演進背景…………………………………………………… 14
1-8.2 影響電泳速度的外界因素………………………………………… 15
1-9 標的物基因背景…………………………………………………….... 16
1-10 訊號標記螢光………………………………………………………… 17
1-11 探針（Probe）與雜交（Hybridization）的名詞解釋…….......18
1-12 晶片掃瞄儀器………………………………………………………… 18
1-13 影響生物晶片偵測效率的因素……………………………………... 19
1-14 實驗動機……………………………………………………………... 21
第二章 實驗原理
2-1 預雜交（Pre-hybridization）…………………………………………… 23
2-2 晶片表面化學塗佈改質……………………………………………… 27
2-3 DNA微陣列…………………………………………………………... 28
2-4 PCR原理…………………………………………………………… 30
2-5 膠體電泳原理……………………………………………………… 31
2-6 雜交溫度設定………………………………………………………… 33
2-7 吸光度量測…………………………………………………………… 34
2-8 DNA雜交原理………………………………………………………... 36
2-9 螢光顯色……………………………………………………………… 39
第三章 實驗步驟
3-1 實驗材料……………………………………………………………… 41
3-1.1 螢光標的物（Targets）…………………………………………….. 41
3-1.2 探針（Probes）…………………………………………………….. 43
3-1.3 正控點（Immobilization Control or Positive control ）………… 45
3-1.4 化學詴劑………………………………………………………... 46
3-2 實驗儀器……………………………………………………………… 48
3-3 微陣列晶片製作……………………………………………………… 55
3-3.1 核酸矩陣晶片點樣機打點前準備……………………………... 55
3-3.2 基因點陣和固定化……………………………………………... 60
3-3.3 固定化後清洗…………………………………………………... 62
3-4 晶片雜交程序………………………………………………………… 63
3-4.1 標的物配製……………………………………………………... 63
3-4.2 專一性測詴 …………………………………………………. 65
3-4.3 60mer標的物單獨雜交測詴…………………………...…... 67
3-4.4 17mer標的物單獨雜交測詴………………………………... 67
3-4.5 先17mer標的物後60mer標的物堆疊雜交測詴………………. 68
3-4.6 先60mer標的物後17mer標的物堆疊雜交測詴………………. 69
3-5 清洗步驟……………………………………………………………… 69
3-6 螢光掃瞄訊號………………………………………………………… 70
3-7 數據整理……………………………………………………………… 71
第四章 結果與討論
4-1 專一性測詴………………………………………………….………… 72
4-2 標定Cy3之60mer標的物單獨雜交測詴……………….……... 73
4-3 標定Cy5之17mer標的物單獨雜交測詴….…….…………..… 76
4-4 先pda06175-Cy5後ATP5O-Cy3堆疊雜交測詴………………….. 78
4-5 先ATP5O-Cy3後pda06175-Cy5堆疊雜交測詴………………….. 80
4-6 先pda06122-Cy5後PSMA5-Cy3堆疊雜交測詴…………...………. 82
4-7 先PSMA5-Cy3後pda06122-Cy5堆疊雜交測詴……..…….………. 84
4-8 先pda13015-Cy5後CANX-Cy3堆疊雜交測詴……..……………. 86
4-9 先CANX-Cy3後pda13015-Cy5堆疊雜交測詴……..……………. 88
第五章 結論………………………………………………………………. 92
第六章 參考文獻 ……………………………………………………. 93
第七章 附錄
7-1 來源基因全序列……………………………………………………… 100
7-1.1 阿拉伯芥基因全序列……………………………………………. 100
7-1.2 人類基因全序列………………………………………………… 103
7-2 晶片掃描原始分析數據…………………………………………….... 108



圖目錄
圖1-1： 微陣列技術………………………………………………………... 10
圖2-1：DNA探針與互補標的物實驗設計圖…………………………….. 26
圖2-2：酸酐改質與DNA固定化…………………………………………. 27
圖2-3：接觸角偵測圖……………………………….…………………… 28
圖2-4：晶片製作程序示意圖……………………………………………... 29
圖2-5：PCR複製機制示意圖……………………………………………… 31
圖2-6：吸光度量測示意圖………………………………………………... 36
圖2-7：DNA鹼基配對…………………………………………………… 38
圖2-8：晶片雜交測詴示意圖…………………………………………….. 39
圖2-9：螢光分子結構式…………………………………………………... 40
圖3-1：低速離心機（Centrifuge CN-6000, Hsiangtai Machinery, Taiwan）…………………………………………………………………… 49
圖3-2：核酸矩陣晶片點樣機（Cartesian PixSys7500, Genomic Solutions, Michigan, USA）…………………………………………………………… 50
圖3-3 ：紫外光可見光分光光譜儀（DU® 800 UV/Visible Spectrophotometer, Beckman Coulter, California, USA …………………………… 51
圖3-4：迴轉式震盪機（S101D, Firstek Scientific, Taipei, Taiwan）……………………………………………………………………. 53
圖3-5：共軛焦距螢光掃瞄儀（4000B, Molecular Devices, California, USA）………………………………………………………………………. 54
圖3-6：核酸矩陣晶片點樣機打點前準備圖示…………………………... 56
圖3-7：DNA微陣列晶片配置圖………………………………………….. 61
圖3-8：PCR反應所用藥品……………………………………………… 64
圖3-9：晶片雜交程序示意圖……………………………………………... 66
圖3-10：基因標的物各雜交區域位置圖………………………………… 68
圖3-11：晶片雜交後清洗程序示意圖……………………………………. 70
圖4-1：螢光標的物於3對探針進行專一性測詴………………………… 72
圖4-2：標定Cy3螢光標的物於兩探針進行單獨雜交……………………..75
圖4-3：標定Cy5螢光標的物於兩探針進行單獨雜交………..………….. 77
圖4-4：螢光標的物—先pda06175-Cy5 後ATP5O-Cy3進行堆疊式雜交…………………………………………………………….……………... 79
圖4-5：螢光標的物—先ATP5O-Cy3後pda06175-Cy5進行堆疊式雜交…………………………………………………………….……………... 81
圖4-6：螢光標的物—先pda06122-Cy5 後PSMA5-Cy3進行堆疊式雜交…………………………………………………………….……………... 83
圖4-7：螢光標的物—先PSMA5-Cy3 後pda06122-Cy5進行堆疊式雜交…………………………………………………………….……………... 85
圖4-8：螢光標的物—先pda13015-Cy5 後CANX-Cy3進行堆疊式雜交…………………………………………………………….……………... 87
圖4-9：螢光標的物—先CANX-Cy3後pda13015-Cy5進行堆疊式雜交…………………………………………………………….……………... 89



表目錄
表2-1：本研究所使用DNA標的物資訊………………………………25
表2-6：各基因片段熔解溫度………………………………………………34
表3-1：點樣機參數設定…………………………………………………... 57
表7-1：60 mer-Cy3標的物單獨雜交測詴晶片原始分析數據…………108
表7-2：17 mer-Cy5標的物單獨雜交測詴晶片原始分析數據…………109
表7-3：各組基因堆疊雜交測詴晶片分析數據（先17 mer-Cy5 後 60 mer-Cy3）………………………………………………………………..111
表7-4：各組基因堆疊雜交測詴晶片分析數據（先60 mer-Cy3 後 17 mer-Cy5）………………………………………………………………..114

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