莊曜宇Chuang, Yao-Yu臺灣大學:電機工程學研究所簡禎志Jian, Zhen-ZhiZhen-ZhiJian2010-07-012018-07-062010-07-012018-07-062008U0001-3007200812523200http://ntur.lib.ntu.edu.tw//handle/246246/187987DNA 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，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.he 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. 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.Table of Contentsbstract………………………………………………………………………….……1要…………………………………………………………….. ……………………2hapter 1 Introduction……………………………………………………………..31. 1 Motivations………………………………………………………………...3. 2 DNA microarray…………………………….……………………………..3. 3 Fluorescent dyes for DNA microarray labeling………………………..…4. 4 Direct and indirect labeling……………………………………………....10. 5 Target-labeling strategies of different DNA microarray platforms….12. 6 Overview of Quantum dots………………………….……………………14. 7 Quantum dots for DNA microarray……………………………………..16hapter 2 Materials and methods………………………………………………....20. 1 Photostability comparison of streptavidin conjugated Qdot®655 and Cy5-dUTP…………………………………….…………………………..20. 2 The emission spectrum measurement of streptavidin conjugated Qdot®655 ………….………………………….…………………………..20. 3 Optimization of photomultiplier gain setting for streptavidin conjugated Qdot® 655 ………….………...……………….…………………………..22. 4 Cell culture……………..………………………………………………….25. 5 Total RNA extraction……………………………………………………..25. 6 Microarray fabrication for poly-L-lysine slides…………………..…….25. 7 Microarray fabrication for Corning® epoxide coated slides……………26. 8 Biotin labeling……………………………………………………………..26. 9 Prehybridization, hybridization and post-hybridization washes……... 28. 10 Incubation with streptavidin conjugated Qdot®655, post-incubation washes and scans……………………………………………………...….29. 11 Assessment of the steric hindrance of streptavidin conjugated Qdot®655 in microarray system………………………………………....29. 12 Optimization of probe concentration…………………………………..31hapter 3 Results…….…………………………………………..…………………34. 1 Photostability comparison of streptavidin conjugated Qdot®655 with Cy5-dUTP………………………………………………………………...34. 2 The emission spectrum of streptavidin conjugated Qdot®655…………34. 3 Optimization of photomultiplier gain setting…………………………...34. 4 Comparison of two kinds of biotin labeling methods…………………...36. 5 Spotting buffer selection for epoxide coated slides……………………...38. 6 Comparison of the incubation systems in epoxide coated slides……….38. 7 Evaluation of detection limit and dynamic range in streptavidin conjugated Qdot®655 and biotin-labeled probe system………………..39. 8 Probe concentration condition…………………………………………...39hapter 4 Discussion………………………………………………………………..48. 1 Photostability of Cy5-dUTP and streptavidin conjugated Qdot®655….48. 2 The emission spectrum of streptavidin conjugated Qdot®655…………48. 3 Calibration curves for streptavidin conjugated Qdot®655……………..49. 4 Biotin labeled method…………………………………………………….49. 5 Comparison of poly-L-lysine and epoxide coated slides………………..50. 6 Spotting buffer selection………………………………………………….50. 7 Detect limit and dynamic range in streptavidin conjugated Qdot®655 and biotin-labeled probe system………………………………………...51. 8 Future works for Quantum dot labeling……………………………...…51hapter 5 Conclusion……………………………………………………………….53ist of Figuresig. 1-1 Scheme of the DNA microarray experiment……………..……….……….5ig. 1-2 The chemical structures of fluorescent dyes derived from Alexa Flour® dyes………………………………………………………………………..…7ig. 1-3 The chemical structures of cyanine dye-labeled dNTP…………………...9ig. 1-4 The structures of streptavidin and biotin………………………………...11ig. 1-5 The processes of excitation, relaxation and emission in bulk semi-conductor…………………………………………………………….15ig. 1-6 The energy landscapes of bulk semiconductors and quantum dots….…15ig. 1-7 The emission wavelength of different Quantum dot core materials……17ig. 2-1 Experimental setups of streptavidin conjugated Qdot®655 emission spectrum measurement…………………………..………………………..21ig. 2-2 Layout of the calibration slide for streptavidin conjugated Qdot®655...22ig. 2-3 Layout of two kinds of biotinylated probe chips for assessing the steric hindrance of streptavidin conjugated Qdot®655 in the microarray system………………………………………………………………………32ig. 2-4 Layout of a slide for optimizing probe concentration condition………..33ig. 3-1 Photostability comparison of streptavidin conjugated Qdot®655 and Cy5-dUTP………………………………………………………………….35ig. 3-2 The emission spectrum of streptavidin conjugated Qdot®655………….35ig. 3-3 Verification of biotin labeling for cDNA by adding streptavidin conjugated Qdot® 655 in 1% agarose gel without dye…………………..37ig. 3-4 The calibration curves under different PMT gains……………...………42ig. 3-5 Biotin-14-dCTP for biotin labeling in different incubation systems (poly-L-lysine slides)………………...…………………………………….43ig. 3-6 Comparison of spotting buffers for Corning epoxide coated slides….....45ig. 3-7 Comparison of different incubation systems for Corning epoxide coated slides……………………………………………………………………...…45ig. 3-8 Evaluation of detection limit and dynamic range in biotin- streptavidin conjugated Qdot® 655 system……………………………………………..46ig. 3-9 Probe concentration condition………………………………………........47 ist of Tablesable 1-1 Spectroscopic characteristics of fluorescent dyes used in microarray...6able 1-2 Target labeling strategies of different DNA microarray platforms…...13able 1-3 Comparison of the properties of organic fluorophores and quantum dots……………………………………………………………………….18able 2-1 The original concentration of streptavidin conjugated Qdot®655 in calibration slide……………...………………………………………….23able 2-2 Nucleotide sequence of the probes used for the thesis…………………27able 2-3 Different incubation conditions for streptavidin conjugated Qdot®655 ……………………………………………………………………………30able 3-1 Incubation system comparison for poly-L-lysine slides…………….…40able 3-2 Spotting buffer comparison……………………………………………40able 3-3 Incubation system comparison for poly-L-lysine slides……………….411001922 bytesapplication/pdfen-US量子點微陣列晶片雜合反應Quantum dotsDNA microarrayHybridization[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/187987/1/ntu-97-R95921115-1.pdf