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論文中文名稱:利用單股核苷酸修飾之金奈米粒子偵測單核苷酸多型性之研究 [以論文名稱查詢館藏系統]
論文英文名稱:Detection of single nucleotide polymorphisms using ssDNA-Au nanoparticles [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:徐巧齡
英文姓名:Chiao-Ling Hsu
研究生學號:94688003
學位類別:碩士
語文別:中文
口試日期:2007-07-23
論文頁數:77
指導教授中文名:侯劭毅
口試委員中文名:王勝仕;黃志宏
中文關鍵詞:單核苷酸多型性(SNP)金奈米粒子單股特異性核酸酶
英文關鍵詞:Single nucleotide polymorphismsgold nanoparticlessingle-strand specific
論文中文摘要:單核苷酸多型性(SNP)是最普遍發生的一種遺傳變異,在基因組DNA中每隔幾百到幾千個鹼基就會發生。個人化醫療的概念是根源於這些獨特多型性的鑑定,在疾病診斷和治療上提供了一個機會。到目前為止,許多技術已經被發展出來作為SNP的偵測方法。但是,這些傳統的方法相對耗時且成本較高。在這個研究中,我們希望能以單股特異性核酸酶和金奈米粒子為基礎的呈色分析系統,發展出一套簡單、低成本、且不需要精確溫控就能偵測完全互補的目標DNA分子與單一個鹼基配對錯誤的目標DNA分子間的差異。
這個SNP的偵測方法是經由三個步驟完成的,首先將目標DNA分子與assay probe雜交後的雙股DNA,利用mung bean nuclease在雜交雙股DNA中錯誤配對的位置處進行斷裂反應,而對於完全配對的雜交雙股DNA則不會有斷裂反應發生。反應後隨即加入capture Au-probe及anti-assay probe與上述酵素作用之產物反應,用離心的方式將互補於capture Au-probe的目標DNA回收下來。最後加入detector Au-probe和互補在capture Au-probe上的目標DNA分子進行反應,藉由全波長的掃瞄來分析目標DNA中是否有單一個配對錯誤的鹼基。
本研究所用的呈色分析系統可以偵測出樣本中在某個鹼基範圍內有SNP存在,但是至目前為止,還無法偵測到SNP確切的位置。
論文英文摘要:Single nucleotide polymorphisms (SNP) are the most abundant form of genetic variation and occur as often as every few hundred to few thousand base pairs in genomic DNA. The concept of personalized medicine is rooted in the identification of these unique polymorphisms, providing opportunities in both the diagnosis and treatment of diseases. Up to now, many techniques have been developed for SNP detection. However, conventional methods are of relatively time-consuming and high cost. In this study, we develop a novel colorimetric detection system based on single-strand specific nucleases and gold nanoparticles, the assay provided a convenient yet powerful colorimetric detection that enabled a straightforward single-base discrimination without the need of precise temperature control.
The assay coluld be implemented via three steps: a hybridization reaction that allowed assay probe to hybrid with the target DNA strand, a cleavage reaction that generates between mismatched target and assay probe, but no cleavage reaction that generates between perfectly matched target and assay probe.When the reaction mixture was added capture gold-probe and anti-assay probe. After reaction, the solution by centrifugation to retrieve target DNA. Finally, the redispersed solution by adding a detector gold-probe.After reaction, the solution using a UV-vis spectrophotometer analysis was performed.
To our knowledge, this is the first report concerning SNP detection based on the single-strand specific nucleases and the gold nanoparticles assembly.
論文目次:中文摘要 i
英文摘要 ii
誌謝 iv
目錄 v
表目錄 vii
圖目錄 viii
第一章 緒論 1
1.1 前言 1
1.2 研究動機及目的 4
第二章 研究背景與原理介紹 5
2.1 單核苷酸多型性(SNPs) 5
2.2 現行SNP檢測方法及原理 5
2.2.1 DNA定序法 (DNA sequencing) 6
2.2.2 等位基因特異性雜交螢光測定法 6
2.2.3 DNA微陣列法(DNA Microarray) 9
2.2.4 以金奈米粒子偵測SNP 9
2.3 奈米金粒子檢測技術介紹 10
2.3.1 膠體金結構 10
2.3.2 膠體金特性 10
2.3.3 膠體金粒子吸收光譜 11
2.3.4 膠體金製備方法 11
2.3.5 膠體金粒子於DNA檢測上之原理及應用 12
2.3.5.1 使用於溶液中的檢測法 12
2.3.5.2 使用於固態載體表面上的檢測法 18
2.4 綠豆核酸酶(Mung Bean nuclease)的特性介紹 29
2.5 S1核酸酶(S1 nuclease)的特性介紹 30
2.6 奈米金粒子檢測相關文獻比較 30
第三章 實驗方法與步驟 34
3.1 實驗架構 34
3.2 寡核苷酸之設計 37
3.3 以硫醇基將DNA固定在金奈米粒子表面 38
3.4 金奈米粒子上的硫醇-寡核苷酸的定量 38
3.5 SNP的偵測 39
3.6 聚丙乙烯膠體電泳分析 40
3.6.1 膠體製備 40
3.6.2 樣本製備 42
3.6.3 凝膠電泳分離 42
3.6.4 影像分析 42
3.7 實驗藥品與設備 43
3.7.1 實驗藥品 43
3.7.2 實驗設備 44
第四章 結果與討論 46
4.1 SNP的偵測實驗 46
4.2 金奈米粒子-硫醇-寡核苷酸探針的製備及特性 46
4.3 金奈米粒子上的硫醇-寡核苷酸的定量 47
4.4 金奈米粒子探針與目標DNA分子的結合反應 49
4.5 綠豆核酸酶對於單一鹼基錯誤配對的斷裂實驗 50
4.6 T42mer Perfect match DNA與T32-42mer mutant DNA以綠豆核酸酶
酶切及回收後之全波長比較 53
4.7 不同長度DNA分子之Tm值 55
4.8 A700/A400之Tm比值對不同濃度的T42與C32 (Control 32mer DNA)之比較 60
4.9 綠豆核酸酶酶切與回收實驗之探討 61
第五章 結論 63
參考文獻 64
附錄 69
附錄一 Sigama網站提供的20 nm金奈米粒子的資料 70
附錄二 金奈米粒子上的硫醇-寡核苷酸的定量之參考圖表
72
附錄三 Promega網站提供的Mung Bean nuclease的資料 73
附錄四 口試後實驗 76
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