亞精胺影響下DNA構形與DNA碎片分佈之研究

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姓名

賴仕修(Shih-Hsiu Lai) 查詢紙本館藏

畢業系所

光電科學與工程學系

論文名稱

亞精胺影響下DNA構形與DNA碎片分佈之研究
(Effects of Spermidine on DNA Conformation and the Distributions of DNA Fragmentation)

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摘要(中)

本論文研究在凝聚物-亞精胺影響下去氧核醣核酸(DNA)之構形變化。由電泳實驗可知，依據不同的亞精胺濃度，DNA 可分為非凝聚態、凝聚態以及複現態。為了比較此三種狀態下DNA 構形之異同，我們使用原子力顯微鏡(AFM)直接觀測DNA 分子。在非凝聚態下，DNA 呈現花狀結構；而複現態下，DNA 呈現樹枝狀結構。
此外，我們藉由DNA 碎片大小之分佈來預測其原始之結構。DNA 斷碎實驗是由DNA 分子與螢光染劑(YOYO-1)接合及亞精胺之影響下，光解作用使之斷碎，再由電泳分析其碎片大小分佈。經由相互比較有或沒有亞精胺影響之碎片分佈，我們發現a) 在亞精胺的影響下，加速DNA 的斷碎過程，b) 在初期的斷碎過程中，有亞精胺影響之斷碎實驗擁有較長DNA 分子的存在，c) 在複現態下，DNA 碎片分佈形式類似於一Lognormal 分佈。由以上的發現，推測高階結構之DNA 分子影響了其斷碎的過程。

摘要(英)

The conformation of DNA in the presence of condensing agent, spermidine is studied. The non-condensed, condensed, and reentrant states of DNA in different concentrations of spermidine are determined by the gel electrophoresis. The goal of this study is to compare the conformations of DNA in these three states. The atomic force microscope (AFM) is used to probe the DNA directly. The flower-shaped and the branch-like structures of DNA are observed in the non-condensed and the reentrant state, respectively.
We also estimate the structure of DNA from the distributions of the DNA fragments.The light induced fragmentation of DNA in the presence of fluorescent dye, YOYO-1 and spermidine is analyzed by the gel electrophoresis. By the comparison of the distributions in
the presence and absence of spermidine, it is found that a) spermidine enhances the fragmentation process, and b) in the presence of spermidine, the existence of the longer DNA fragments is observed during the early fragmentation process, and c) the form of the distribution of DNA fragments in the reentrant state is similar to the Lognormal distribution. These findings suggest that the higher order structures of DNA molecules are involved in the fragmentation process.

關鍵字(中)

★ 複現
★ DNA 構形
★ 亞精胺
★ 原子力顯微鏡
★ 斷碎

關鍵字(英)

★ AFM
★ reentrant
★ DNA conformation
★ spermidine
★ fragmentation

論文目次

Abstract………………………………………………………………………………………ii
Contents………………………………………………………………………………………iv
List of figures………………………………………………………………………………vii
List of tables…………………………………………………………………………………x
1 Chapter 1 Introduction…………………………………………………………………1
1.1 Conformation of DNA and Its Biological Meaning……………………………………1
1.1.1 Structure of DNA………………………………………………………………3
1.1.2 Introduction to Spermidine………………………………………………………5
1.1.3 Condensation and Reentrant State of DNA ……………………………………7
1.2 Fragmentation Experiments……………………………………………………………8
1.2.1 Fragmentation of DNA by Irradiation…………………………………………9
1.2.2 Fragmentation of DNA Induced by Light………………………………………10
1.2.3 The Form of the Distributions…………………………………………………10
1.3 Overview of This Thesis……………………………………………………………12
2 Chapter2 Materials and Methods……………………………………………………14
2.1 Overview……………………………………………………………………………14
2.2 Introduction to YOYO-1……………………………………………………………14
2.3 DNA Sample Preparation……………………………………………………………16
2.3.1 Samples for Illumination Experiments…………………………………………16
2.3.2 Samples Observed under the AFM……………………………………………17
2.4 Experimental Setups…………………………………………………………………19
2.5 Observation Methods and Procedures………………………………………………21
2.5.1 Gel Electrophoresis……………………………………………………………21
2.5.1.1 Introduction to Gel Electrophoresis……………………………………21
2.5.1.2 Procedure of Gel Electrophoresis……………………………………22
2.5.2 AFM…………………………………………………………………………24
2.5.2.1 Introduction to the AFM……………………………………………24
2.5.2.2 Operation Procedure of the AFM………………………………………28
2.6 Simulation Method……………………………………………………………………29
2.7 Summary and Discussions……………………………………………………………31
3 Chapter 3 Results and Discussions…………………………………………………32
3.1 Overview……………………………………………………………………………32
3.2 Condensation and Reentrant State of DNA in Gel Electrophoresis…………………32
3.3 The Conformation of DNA Observed under the AFM………………………………35
3.3.1 The Conformation of DNA in the Presence of Spermidine……………………35
3.3.2 The Conformation of DNA-YOYO Complex in the Presence of
Spermidine……………………………………………………………………44
3.3.3 Summary for the Conformation of the DNA and DNA-YOYO Complex in the
Presence of Spermidine………………………………………………………48
3.4 Illumination Experiments Results……………………………………………………49
3.4.1 The Effect of YOYO in the Illumination Experiments…………………………49
3.4.2 The Distributions of Fragmentation of DNA…………………………………50
3.4.3 Total Intensity Problem in the Illumination Experiments………………………52
3.4.4 Discussion of the Fragmentation Distributions…………………………………55
3.4.5 DNA Fragments Observed under the AFM……………………………………61
4 Chapter 4 Conclusions………………………………………………………………64
5 Reference………………………………………………………………………………66
6 Appendix ………………………………………………………………………………70
A. Sample Preparation for Illumination Experiments……………………………………70
B. Sample Preparation Observed under the AFM………………………………………71
C. Gel Preparation ………………………………………………………………………73
D. Gel Electrophoresis……………………………………………………………………74
E. Programs………………………………………………………………………………75

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指導教授

陳志強、楊宗勳
(Chi-Keung Chan、Tsung-Hsun Yang)

審核日期

2007-7-24

推文