DNA於脂雙層上自發展開之研究

Abstract

本研究的目的為開發一拉伸DNA平台，並將其應用於基因圖譜技術。現行的拉伸DNA技術大多利用侷限空間迫使DNA朝無侷限空間展開而達到拉伸效果，其中以奈米通道拉伸法最受大家關注，其優點在於DNA的伸長率佳且DNA的伸長型態為其平衡狀態，利於長時間下的觀察及分析，因此較適合用於基因圖譜的檢測。然而，奈米通道在製作上及操作上皆具有困難度，以整體成本及效益來說，不利於醫療單位的使用。因此我們希望發展一穩定拉伸DNA的平台，且同時具有極佳的伸長效果且低製作成本。 我們沿用侷限空間的概念，將帶負電的DNA吸附於帶正電的脂雙層上，使DNA呈現二維運動行為，並以圖案脂雙層使DNA吸附後於一維方向自發伸展，其中伸展機制是藉由降低系統位能來克服DNA型態變化所產生的熵損失，使DNA伸展型態為系統中最低自由能狀態，即其平衡狀態。然而，實驗上發現氯仿存在於脂雙層內才可觀察到DNA伸展現象，且氯仿含量與DNA伸長率呈正相關，但目前仍無法完全了解其作用。實驗結果顯示lambda DNA的伸長率可達到73%，其相當接近奈米通道拉伸法的極限，本實驗平台之成本遠低奈米通道，較利於基因圖譜檢測應用，有助於未來實際運用於醫療單位。此外，我們也發現不同大小的DNA於相同實驗條件下的伸長率非常接近，此特性能夠將DNA長度轉為基因圖譜檢測特徵之一，使此法應用於基因圖譜檢測能更加準確。

The purpose of this study is to develop a new platform for stretching DNA, and then to apply it to the application of DNA gene mapping. Among the recently developed technologies for DNA gene mapping, using nanochannel to confine and therefore to stretch DNA is the most promising one. DNA confined in nanochannel spontaneously extends to nearly its contour length, and the positions of specific sequences can be readily determined. However, the nanochannel is not only expensive to fabricate but also difficult to operate. Thus, a more efficient, lower-cost, and easy-to-use DNA-stretching platform is much anticipated. Following the concept of confinement, we adsorb DNA on a patterned two-dimensional plane built by cationic lipid bilayers. The pattern consists of periodic shallow trenches, and DNA spontaneously gathers and unravels along the root of the trench wall. The phenomenon is controlled by the competition between the electrostatic interaction and conformational entropy of DNA. However, we also discovered that the existence of chloroform in our system strongly affects the DNA behavior. Although the mechanism is yet to be investigated, the degree of DNA extension was found to increase with chloroform content. For both T4 and Lambda DNA, their extensions were found very close and can be more than 70% of their contour lengths, comparable to the value obtained in nanochannels. Therefore, the platform developed in this study can be considered as a cheaper alternative of nanochannel. The current platform will be tested for gene mapping in the very near future.