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  • 學位論文

於脂雙層上拉伸DNA與其基因圖譜應用之研究

Research of DNA Stretching on Lipid Bilayer and its Application for Rapid Gene Mapping

指導教授 : 謝之真

摘要


近年來許多研究提出利用奈米通道拉伸DNA並希望有助於DNA的分析與醫療應用,然而,奈米通道的製作過程極為困難與昂貴,隨後於操作階段必須將DNA導入奈米通道也是難以控制的過程,整體來說,這些不利因素使奈米通道拉伸法無法普及於醫療服務。 因此本研究主旨是開創一個高效率、低成本、操作簡單的DNA拉伸平台,在不施加外部作用或能量條件下使DNA於脂雙層上進行自發展開並拉伸,同時DNA必須處於平衡狀態,最後將其拉伸結果應用於基因圖譜。實驗上,DNA達平衡狀態時會於側壁夾角根部呈現準一維拉伸狀態,從熱力學觀點來看,這是由於DNA與正電脂質之間的靜電力作用與DNA型態的熵變化所導致的競爭現象,最後系統會處於最低自由能狀態;在此平台中,長度相差3.5倍的λ噬菌體DNA與T4GT7 DNA皆可以達到相對拉伸率0.85,此結果相當於次50nm奈米通道的拉伸率;此外我們成功利用EcoRI、SacI、NheI等限制酵素製作出這兩種不同尺寸DNA的限制圖譜,同時,更進一步運用雙肽核酸成功製作螢光標定λ噬菌體DNA的基因圖譜,其準確率接近200bp。以整體來看,本實驗平台比現行奈米通道拉伸法表現出色且低成本,同時相較於傳統基因圖譜,我們也可以更快速且準確地製作限制圖譜與螢光標定圖譜,所以此平台的應用是非常具有潛力發展為新一代基因圖譜技術。

關鍵字

DNA 奈米通道 脂雙層 基因圖譜 雙肽核酸

並列摘要


Recent studies found that DNA can spontaneously extend when confined in nanochannel with diameters smaller than few tens of nanometers. This phenomenon has been shown to have great potential for DNA analysis. However, fabrication of nanochannels required very complicated and expensive processes. In experiments, it is also very difficult to get DNA into the nanochannels and to perform the following analysis. The goal of this study is to develop a more efficient, low-cost, and user-friendly platform for stretching DNA at equilibrium, and then to apply it to rapid DNA gene mapping. DNA were first adsorbed on cationic lipid bilayers built on a patterned glass surface. The surface pattern consists of periodic shallow trenches, and DNA spontaneously gathers and unravels along the root of the trench walls. The phenomenon is controlled by the subtle competition between the electrostatic interaction of the DNA-lipid complexes and the conformational entropy of DNA. For both T4GT7(166kbp) and lambda DNA(48.5kbp), the relative DNA extension can reach 85% of their contour lengths, comparable to the highest degree of DNA extension obtained in sub-50nm nanochannels. Therefore, our platform can be considered as a much cheaper and easier-to-use alternative of nanochannels. At same time, we have successfully performed restriction mapping of lambda-DNA using the restriction enzymes EcoRI, NheI, and SacI. Furthermore, we also successfully performed gene mapping with bis-PNA tagging and its accuracy could reach 200bp. Since our method is much faster and cheaper than the traditional technologies, it has great potential for rapid gene mapping applications.

並列關鍵字

DNA nanochannel lipid bilayers rapid gene mapping bis-PNA

參考文獻


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