在液態環境中不同表面固化方式DNA探針之動態行為研究

The Dynamic behavior study of DNA probe in liquid environment by using different immobilization ways

陳學華(Hsueh-Hua Chen)

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(2009年)

https://doi.org/10.6843/NTHU.2009.00355

摘要

固化於基因晶片上的DNA探針之動態行為，是以基因晶片做為研究工具的科學家們所感興趣的問題。常見的固化方式分為兩種：(1)藉由舖在玻片上的poly-L-lysine以正負電荷互相吸引以固化DNA；(2)透過biotin-streptavidin的非共價鍵結使DNA一端固化於玻片上。我們欲鑑別以上兩種固化方式之DNA於溶液中，是否如海草般於溶液中搖擺；或如繩子般平躺於poly-L-lysine表面。這兩種固化方式之DNA探針的動態行為，是影響DNA雜合效率的重要因素。
量子點能夠長時間穩定發光，我們以TIRFM偵測尾端接上量子點的675、1699、2834 bp雙股DNA，其中DNA是以上述兩種不同的固化方式固化於玻片上。透過即時偵測螢光訊號位置與強度隨時間的改變，估計量子點於空間中的位移。為了精確測量量子點位置的改變，本論文建立了單分子追蹤技術。在每秒33張畫面的影像擷取速度下，達到14奈米的系統空間解析度。配合TIRFM產生的漸逝波，其強度具有隨遠離全反射面的方向呈指數衰減的特性，偵測量子點於Z軸的相對位置。
在本實驗結果發現，以poly-L-lysine方式固化於玻片表面，DNA如同繩子般平躺於poly-L-lysine表面，且DNA兩端可以脫離表面的吸引而擺動。以biotin-streptavidin方式鍵結在玻片表面的DNA，則類似海草般，靜止於溶液中，或只來回擺動於特定的二到三個位置。

關鍵字

單分子 ； 全內反射式螢光顯微鏡 ； DNA探針

並列摘要

Abstract
The dynamic behavior of DNA molecules, when immobilized on DNA microarrays, has puzzled the gene chip researchers. In general, there are two types of immobilization methods, i.e. charge-charge interaction by surface coating and immobilize to the glass surface at one end of DNA molecules. In this study, we will attempt to distinguish weather DNA molecules are like seaweed wavering in a solution or like rope sticking to the surface of glass. The two modes have quite different hybridization efficiency.
Qdots can emit the fluorescence continually and stably. The 675 bp, 1699 bp and 2834 bp dsDNA was attached to Qdots in both end. We employ the TIRFM to measure Qdots position and intensity change by time, and estimate the displacement in space. To precise measure the position change of Qdots, we establish the single particle tracking technique, which has the spatial resolution of 18 nm in frame rate 33Hz. To co-operate evanescent wave generated by TIRFM exhibits exponential decay with increasing distance z from the interface of total internal reflection, we detect the relative displacement of Qdots on the Z-axis.
The result shows that DNA do like a rope lie on the poly-L-lysine coating surface, but still can get out of the coating surface then shaking. In contrast, on the one end immobilized of DNA molecules like a seaweed just standstill in the water, or swing in the particular two or three position.

並列關鍵字

single molecule ； TIRF ； DNA probe

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