全內反射螢光顯微系統於DNA探針動態行為之研究

The Dynamic behavior study of DNA probe by TIRF

許哲嘉(Che-Chia Hsu)

指導教授 ： 許志楧 ； 吳見明

國立清華大學/原子科學院/生醫工程與環境科學系/碩士(2008年)

摘要

中文摘要
固化在基因晶片上的DNA探針之動態行為是科學界感興趣的問題。固化方式一般分為兩種：(1)藉由鋪在玻片上的poly-L-lysine以正負電荷吸引的方式來固定DNA；(2)以共價鍵結的方式將DNA的一端固定在玻片上。我們希望可以鑑別兩種固化方式之DNA在溶液中的形態，究竟DNA是像海草般在溶液中搖擺(海草模式)；或是像根繩索般平躺在玻片表面(平躺模式)。這兩種模式是影響DNA雜合效率的重要因素。
由於TIRFM產生之漸逝波具有隨高度指數衰減的特性，漸逝波的強度將隨z軸改變而產生敏感變化。被漸逝波激發的螢光所產生的訊號強度也會隨著螢光在z軸的位移而改變，所以TIRFM系統對螢光z軸位置變化有敏銳的偵測能力。我們以TIRFM偵測一條20base pairs長且標有Cy3的DNA，DNA以biotin-streptavidin 的方式固定在玻片表面，並判斷DNA之行為屬於上述何種模式。即時偵測螢光訊號強度隨時間的變化來估計螢光在Z軸上的位移，並以此推測DNA在玻片表面之形態。另外我們也以同樣的方式觀察以poly-L-lysine固化之DNA，並以統計的方法比較兩種固化方式的差異。

關鍵字

單分子 ； 全內反射螢光顯微鏡 ； DNA ； 布朗運動

並列摘要

Abstract

The dynamical behavior of DNA, which was immobilized on DNA microarray, have puzzled the gene chip researchers. In general, there are two types of immobilization methods, i.e. charge-charge interaction by poly-L-lysine coating and covalent binding to the glass surface at the one end of DNA. In this study, we aim to distinguish that either DNA is like a seaweed wavering in the solution (wavering mode) or like a rope sticking on the surface (sticking mode) of glass. The two modes have quite different hybridization efficiency.
Because evanescent wave generated by TIRFM exhibits exponential decay with increasing distance z from the interface, the signal intensity excited by evanescent wave is thus sensitive to the change of the location of the signal emmitor along z direction. Therefore, the TIRFM is adequate to gauge the vertical location of the fluorescence dye. We measure the TIRF signal of Cy3 labeled 20-bp DNA molecules which was immobilized on the coverslip via a biotin-streptavidin. To determine which mode that the behavior DNA are belong to,,we record the time trace of the single Cy3 fluorescence intensity to see if there is a changes in intensity corresponding to the vertical displacement. We also performed the same measurement for the other type of immobilization methods, i.e. charge-charge interaction by poly-L-lysine coating.
In this study, we will also report the theoriotical estimated size of fluorescence signal produced by the wavering of dye in the exponentially decayed evanescent excitation field. The amount and the time characteristic of the wavering is determined by Brownian motion. This wavering signal should be larger enough to stand out from the noise of a steaty single dye.

並列關鍵字

single molecule ； TIRFM

參考文獻

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