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標題: | 量子點螢光標記於DNA生物晶片之應用 Application of Quantum Dot Labeling to DNA Microarray |
作者: | Zhen-Zhi Jian 簡禎志 |
指導教授: | 莊曜宇(Yao-Yu Chuang) |
關鍵字: | 量子點,微陣列晶片,雜合反應, Quantum dots,DNA microarray,Hybridization, |
出版年 : | 2008 |
學位: | 碩士 |
摘要: | DNA 微陣列晶片技術為現今廣泛應用大規模偵測基因表現的生物技術,
其正確性仰賴cDNA與cRNA的螢光標記偵測。然而,目前廣泛使用的有機螢光染劑之光學特性均易受強光破壞。目前亟需一可取代的螢光染劑以作為DNA微陣列晶片使用。 本論文目的為建立與優化ㄧ利用量子點標記的DNA微陣列晶片系統。本論文中,我以生物素標記cDNA與微陣列晶片上的互補核酸序列進行雜合反應,再以表面修飾鏈黴素之量子點進行反應。並以雷射掃瞄器偵測其營光強度。實驗中發現鏈黴素修飾之量子點於空氣中較常用的尿嘧啶修飾之Cy5具有較高的光學穩定性;且鏈黴素修飾之量子點之藍移現象於空氣中一小時內並不顯著。本論文中測試四種反應條件、兩種生物素修飾方法與兩種玻面表面材質以優化系統。並以人類肺腺癌細胞株CL1-5作為RNA之來源與甘油醛-3-磷酸脫氫酶基因的60個寡核甘酸序列作為檢測之序列作為模式的微陣列晶片系統。其所建立的系統其線性區間約為兩個數量級。 DNA Microarray technology is extremely powerful for detecting gene expression in large scale. The accuracy and precision of microarray depends on the fluorescent signal of fluorescent labeled cDNA or cRNA probe. Netherless, conventional organic fluorophores are easily photobleaching. The most used dyes, cyanine 3 and cyanine 5, are affected by ozone, especially cyanine 5. An alternative fluorophore for labeling are required for DNA microarray analysis. The aim of this thesis was to establish and optimize a quantum dot labeling system for DNA microarray. In this thesis, I present a microarray system based on quantum dot labeling method, in which cDNA were labeled with biotin and hybridized with 60 mer oligo probe on the glass slide. After incubation with quantum dot streptavidin conjugated, the fluorescent signal intensity of the quantum dot streptavidin conjugated bound to the biotin labeled cDNA detected by a laser scanner. I found that quantum dots are more photostable than Cy5-dUTP at ambient air. The bluing effect of streptavidin conjugated Qdot®655 was not obvious at ambient air within one hour. Four incubation conditions, two kinds of biotin labeling methods and two kinds of coated slides were testified to optimize the system. Besides, I used a lung cancer cell line-CL1-5 total RNA and a housekeeping gene, Glyceraldehyde-3- phosphate dehydrogenase, 60mer probe as RNA and probe in model microarrays. The detection dynamic range are about 2 orders of magnitude. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/40286 |
全文授權: | 有償授權 |
顯示於系所單位: | 電機工程學系 |
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