於漸縮微流道以電場拉伸吸附於脂雙層上DNA之研究

Abstract

本研究探討如何利用非均勻的電場將吸附於脂質雙層膜上之DNA拉伸。近年來，開發DNA快速基因定位技術一直是重要的研究課題。在快速基因定位技術所使用的直接線性分析法中，穩定並有效地拉伸DNA是提升其準確度的關鍵。為了達到這個目的我們提出先將帶負電的DNA吸附在帶正電的脂質雙層膜上，再利用非均勻的電場以表面電泳的方式將DNA拉伸。這個方法不同一般利用非均勻的流場或電場將DNA拉伸之處，在於DNA被限制於二維平面而不是三維空間。侷限於二維平面下的DNA具有較大的平衡型態。在拉伸的過程中，二維型態的DNA也比三維型態下的DNA損失較少亂度。基於這些因素，我們預期拉伸二維型態下的DNA要比拉伸三維型態下的DNA容易很多。本實驗在漸縮微流道中進行，非均勻的電場是利用改變微流道的寬度來產生。 在實驗的觀察中，我們發現DNA雖然能夠被拉伸至其輪廓長度的70~75%，但並不是因為非均勻電場的效應。原本預期DNA會因電場梯度而產生拉伸的現象並沒有出現。我們觀察到當DNA在脂雙層上移動時，會出現拉伸到一定程度後快速鬆弛的現象，造成DNA在漸縮處無法有效地受到電場梯度的作用。我們在有漸縮和非漸縮的微流道部位中，比較不同脂質雙層膜之帶電程度、電場強度和電場梯度下的DNA拉伸型態，並對不同拉伸型態所造成的伸長量和出現機率來討論DNA在脂雙層上的動態行為。此外，在分析DNA拉伸時，我們觀察到過去文獻中沒有提到過的拉伸型態-Tethered型，Tethered型出現時為DNA的末端會吸附於脂雙層上。我們檢驗DNA末端為黏接端或是鈍端對脂雙層的作用，發現原本具有兩黏接端的DNA會較容易產生Tethered型拉伸。

We perform DNA stretching on supported lipid bilayer (SLB) set on the surface of glass by electric field and electric field gradient. This study is inspired by the recent development of the technology for direct DNA gene mapping. It has received much attention because it enables efficient determination of useful genomic information from DNA. However, the ability to stably and efficiently stretch DNA is the key to the success of this technology. The proposed method is different from typical DNA stretching techniques because in our case DNA is adsorbed on a lipid bilayer, and therefore DNA stretching happens on 2-dimensional (2D) plane, not in 3-dimensional (3D) space. DNA confined in 2D has a larger equilibrium size and also relaxes slower. In addition, a DNA molecule loses less entropy if it is stretched from 2D configuration relative to from 3D configuration. Therefore, it is expected that stretching DNA on 2D plane will be easier than stretching DNA in 3D space. Our experiments were performed in a microfluidic channel, and the field gradient was generated by a microcontraction. We find that DNA can be stretched to 70~75% of its contour length. Surprisingly, however, DNA stretching is not caused by the electric field gradient but by other mechanisms. We have observed three different types of DNA stretching. DNA can be hooked by a fixed post, hooked by mobile imprints or tethered to stretch. The formal two have been reported in literatures, while the later is first found in this study. The presence of tethered DNA has been found to be caused by the overhangs of our DNA. We have also studied how the average DNA extension and the probability distribution vary with the electric field strength for different types of DNA stretching. The influence of the charge density of lipid bilayer to DNA stretching has also been investigated. The cause of the failure to stretch DNA by electric field gradient is still unclear. However, DNA has often been observed to rapidly retract once it is stretched over a certain degree. We believe this is strongly related to the breakdown of our expectation, and more investigation is required to resolve this mystery.