Research of DNA Stretching Mechanism on Lipid Bilayer and Its Behavior Under Confinement

• Main Authors: Hou-Jun Guo, 郭厚均
• Other Authors: Chih-Chen Hsieh
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/40007021649627071092

碩士 === 國立臺灣大學 === 化學工程學研究所 === 104 === We have recently developed a DNA optical gene mapping platform. The working principle of the platform relies on the phenomenon that DNA can be adsorbed and spontaneously extended along a groove covered with cationic lipid bilayers. The physical gene map can then be readily obtained using nick-translating method. This new platform has great potential because of its low cost and easy operation; however, the reason why DNA can spontaneously extend along the grooves is not clear and it has to be investigated in order to optimize the platform. Since DNA extends only along the grooves, it suggests there exists an electrostatic energy well for DNA. Two possible sources of this well are postulated: (1) the steric effect drives cationic lipids to aggregate on curved surface, (2) the geometry effect allows DNA to interact with more cationic lipids on the curved surface. In order to examine our postulations, we observed DNA behavior on three sets of lipid bilayers (A) DOPC/DOTAP, (B) DOPC/EPC and (C) DOPE/EPC, in which the ratio of the area of the headgroup to that of the tail varies gradually from larger than one to smaller than one. The experimental results show both the streic and the geometry effect exist. However, the geometry effect is always in favor of DNA extension while the steric effect can either enhance or undermine the phenomenon. We have also investigated the effect of the concentration of the positively charged lipids and found higher concentration always help DNA extend. In order to quantify the geometry effect, we have derived the electrostatic potential of DNA on curved surface and found the theoretical prediction is in quantitative agreement with the experimental results. We have also calculated lipid distribution due to steric effect and found that a modest variation of lipid concentration in conjunction with the geometry effect can lead to drastic change on DNA extension. Last but not least, we want to measure the width of the DNA confinement using atomic force microscopy and compare our results with the prediction between DNA extension and the confinement width proposed by de Gennes and Odijk. However, since the lateral diffusion of the lipid bilayer is too fast, we cannot find DNA on lipid bilayer using normal AFM probes. We plan to solve this problem in the future by employing lipids with transition temperatures higher than the room temperature so that lipid bilayer can remain in gel phase during experiments.