系統識別號 | U0002-0709201021013700 |
---|---|
DOI | 10.6846/TKU.2010.00233 |
論文名稱(中文) | 利用毛細管電泳探討dsDNA之異常電泳遷移行為 |
論文名稱(英文) | Study of anomalous electrophoretic migration of dsDNA by capillary electrophoresis |
第三語言論文名稱 | |
校院名稱 | 淡江大學 |
系所名稱(中文) | 化學學系碩士班 |
系所名稱(英文) | Department of Chemistry |
外國學位學校名稱 | |
外國學位學院名稱 | |
外國學位研究所名稱 | |
學年度 | 98 |
學期 | 2 |
出版年 | 99 |
研究生(中文) | 許蘭婷 |
研究生(英文) | Lan-ting Hsu |
學號 | 696160737 |
學位類別 | 碩士 |
語言別 | 繁體中文 |
第二語言別 | |
口試日期 | 2010-07-19 |
論文頁數 | 107頁 |
口試委員 |
指導教授
-
吳俊弘(cwu@mail.tku.edu.tw)
委員 - 鄭建中(cccheng@mail.ncyu.edu.tw) 委員 - 施增廉(tlshih@mail.tku.edu.tw) |
關鍵字(中) |
毛細管電泳 異常電泳遷移 雙股螺旋DNA |
關鍵字(英) |
capillary electrophoresis anomalous electrophoretic migration dsDNA |
第三語言關鍵字 | |
學科別分類 | |
中文摘要 |
在本論文中我們開發了聚葡萄醣-毛細管電泳分析方法對dsDNA進行高解析分離,如此,我們可以偵測到DNA之間的微小電泳行為差異,進而探討dsDNA序列對其構型所造成的影響。 根據文獻,DNA序列中含有A-track會使其構型產生彎曲,而導致DNA異常偏慢電泳遷移行為;而具有三重覆序列"CNG"的DNA因其構型較柔軟,會導致DNA異常偏快電泳遷移行為。我們計畫藉由此聚葡萄醣-毛細管電泳分析技術尋找其它可能導致DNA異常偏快電泳遷移的序列。 我們以兩種限制酶(Hae III以及Alu I)分別對三種質體DNA(φx174、pBR322、pACPm)做切割(digest),所得六組DNA片段樣品兩兩混合成十三種組合樣品,再進行電泳分離實驗。將電泳結果以log(μ) (μ為DNA電泳遷移率)對log(1/bp) (bp為DNA鹼基對數目)作圖,依DNA大小分段進行線性廻歸而得到相關係數大於0.99以上的線性關係,電泳遷移行為與線性方程式吻合的DNA定義為正常電泳遷移DNA,電泳遷移率比其線性方程式預測值大者,定義為異常偏快電泳遷移DNA,若電泳遷移率比其線性方程式預測值較小者,則定義為異常偏慢電泳遷移DNA。我們在電泳緩衝溶液中添加不同濃度的溴化乙錠染料(EB),不同種類以及濃度的多價陽離子,或不同種類以及濃度的醣類,分析其對DNA異常遷移百分率的影響程度,探討DNA在不同環境下的構型改變,以及發現可能導致DNA異常電泳遷移行為的序列。根據本論文的實驗結果,我們歸納了幾種可能導致DNA異常偏快電泳行為的序列。 |
英文摘要 |
In this thesis we have developed a Dextran-Capillary electrophoresis (Dextran-CE) based analytical method for the high resolution separation of dsDNA. In this way we are able to detect the minute difference in DNA electrophoretic migration behavior and furthermore to investigate the effect of DNA sequence on the DNA conformation. According to literatures, DNA sequence carrying A-tracks would result in bending conformation and cause anomalously slow electrophoretic migration behavior. On the other hand, DNA containing triplet repeat sequence “CNG” would have flexible conformation and thus shows anomalously rapid electrophoretic migration behavior. We plan to discover the other sequences which could lead to DNA anomalously rapid electrophoretic migration. Two restriction enzymes, Hae III and Alu I, were used to digest three plasmid DNAs, φx174, pBR322, and pACPm respectively, and every two of the subsequent six sets of restriction fragment samples were mixed to give 13 mixtures, which were then subjected to Dextran-CE separation experiments. The DNA base pair number (bp) and its corresponding measured electrophoretic mobility (μ) were plotted in the log(1/bp) versus log(μ) figure. Linear equations with correlation coefficients larger than 0.99 were obtained when we applied linear regression fittings to the data in different DNA size ranges. The DNA with electrophoretic mobility consisting with, larger than, or smaller than the linear prediction value was defined as normally, rapidly or slowly electrophoretic migrating DNA respectively. With the addition of different concentrations of ethidium bromide, various cations, or sacchrides into the CE run buffer and separation medium we were able to analyze their influences on the degree of DNA anomalous electrophoretic migration, to investigate the conformational change of DNA under different environments, and to discover the sequences which could induce the anomalous electrophoretic migration of DNA. According to the experimental results of the this thesis, we have inferred some DNA sequences which could possibly cause anomalous electrophoretic migration of DNA. |
第三語言摘要 | |
論文目次 |
目錄 中文摘要 I 英文摘要 II 目錄 IV 圖片索引 VII 表格索引 X 第一章 緒論 1 1.1 前言 1 1.2 毛細管電泳分離原理 2 1.2.1 毛細管電泳介紹 3 1.2.2 毛細管電泳偵測方法 10 1.2.3 分離介質介紹 12 1.3 DNA片段在膠體中的電泳行為 15 1.4 DNA序列與構型 18 1.4.1 雙螺旋構型 18 1.4.2 染料對dsDNA構型的影響 20 1.4.3 陽離子對dsDNA構型的影響 21 1.4.4 特定序列之偏慢因子對dsDNA構型的影響 22 1.4.5 特定序列之偏快因子對dsDNA構型的影響 23 1.5 本章參考資料 24 第二章 實驗部分 27 2.1 毛細管電泳實驗 27 2.2 實驗條件 27 2.3 電泳緩衝溶液及分離介質之製備 28 2.3.1 TB 緩衝溶液(TB Buffer) 28 2.3.2 添加陽離子之TB buffer 29 2.3.3 添加EB之TB陽離子buffer 29 2.3.4 添加醣類之TB (2000μM Mg2++3μg/mL)buffer 29 2.3.5 分離介質 30 2.4 藥品 30 2.5 毛細管PA(polyacrylamide)塗覆 34 2.6 DNA樣品 36 2.6.1 經限制酶切割之雙股DNA片段樣品 36 2.6.2 DNA經Gel/PCR DNA Fragment Extration Kit純化步驟 38 2.7 歸納導致DNA電泳析岀偏快的序列 40 2.8 將ssDNA anneal成dsDNA 48 2.9 磷酸化步驟 51 2.10 去磷酸化步驟(CIP) 51 第三章 結果與討論 52 3.1 定義dsDNA之異常及正常電泳遷移 52 3.2 DNA異常電泳遷移百分率測量之標準偏差 53 3.3 異常析出行為之dsDNA 55 3.4 利用CE-Dextran分離dsDNA 57 3.4.1 電泳溫度對異常析出dsDNA之影響 57 3.5 異常析出之dsDNA在各條件下的效應 59 3.5.1 離子濃度效應 59 3.5.2 離子種類效應 59 3.5.3 染料(EB)濃度效應 61 3.5.4 離子與染料競爭效應 62 3.5.5 醣類效應 62 3.6 異常析出之序列樣品實驗 63 3.7 總結 67 3.8 章參考資料 68 圖片索引 圖3-1、染料濃度變化之電泳圖 70 圖3-2-1、利用染料濃度變化定義DNA電泳遷移行為 71 圖3-2-2、利用Sr2+濃度變化定義DNA電泳遷移行為 72 圖3-3-1、溫度變化20~30℃,(pBR322(Alu I )DNA+ pBR322 (Hae III )DNA)樣品組合之電泳圖 73 圖3-3-2、溫度變化35~50℃,(pBR322(Alu I )DNA+ pBR322 (Hae III )DNA)樣品組合之電泳圖 74 圖3-4、20~50℃之log µ對log(1/bp)圖 75 圖3-5、電泳遷移率(log μ)對鹼基對(log1/bp)做圖(pBR322(Alu I )DNA+φX174(Alu I )DNA) 76 圖3-6、電泳遷移率(log μ)對鹼基對(log1/bp)做圖(pBR322(Hae III)DNA)+φX174(Hae III)DNA) 77 圖3-7、電泳遷移率(log μ)對鹼基對(log1/bp)做圖(pBR322(Alu I)DNA+φX174(Hae III)DNA) 78 圖3-8、電泳遷移率(log μ)對鹼基對(log1/bp)做圖(pBR322(Hae III)DNA+φX174(Alu I)DNA) 79 圖3-9、電泳遷移率(log μ)對鹼基對(log1/bp)做圖(φX174(Hae III)DNA+φX174(Alu I)DNA) 80 圖3-10、電泳遷移率(log μ)對鹼基對(log1/bp)做圖(pBR322(Hae III)DNA+pBR322(Alu I)DNA) 81 圖3-11、電泳遷移率(log μ)對鹼基對(log1/bp)做圖(pACPm(Alu I)DNA+pBR322(Alu I)DNA) 82 圖3-12、電泳遷移率(log μ)對鹼基對(log1/bp)做圖(pACPm(Alu I)DNA+pBR322(Hae III)DNA) 83 圖3-13、電泳遷移率(log μ)對鹼基對(log1/bp)做圖(pACPm(Hae III)DNA+pBR322(Hae III)DNA) 84 圖3-14、電泳遷移率(log μ)對鹼基對(log1/bp)做圖(pBR322(Alu I)DNA+pACPm(Hae III)DNA) 85 圖3-15、電泳遷移率(log μ)對鹼基對(log1/bp)做圖(pACPm(Hae III)DNA+φX174(Alu I)DNA) 86 圖3-16、電泳遷移率(log μ)對鹼基對(log1/bp)做圖(pACPm(Hae III)DNA+φX174(Hae III)DNA) 87 圖3-17、電泳遷移率(log μ)對鹼基對(log1/bp)做圖(pACPm(Hae III)DNA+ pACPm(Alu I)DNA) 88 圖3-18 Mg2+濃度變化之電泳圖 89 圖3-19、Mg2+濃度變化對dsDNA之電泳遷移率 90 圖3-20、二價鹼土族離子對dsDNA之異常遷移百分率關係圖 91 圖3-21、二價過渡金屬離子對dsDNA之異常遷移百分率關係圖 92 圖3-22、三價離子對dsDNA之異常遷移百分率關係圖 93 圖3-23、染料分子濃度對dsDNA之異常遷移百分率關係圖 94 圖3-24、染料分子與Sr2+離子濃度之競爭效應對dsDNA之異常遷移 百分率關係圖 95 圖3-25、染料分子與Mg2+離子濃度之競爭效應對dsDNA之異常遷移百分率關係圖 96 圖3-26、各種醣類對dsDNA之異常遷移百分率關係圖 97 圖3-27、醣類濃度對dsDNA之異常遷移百分率關係圖 98 圖3-28、pACPm(Hae III Digest)之偏快bp43 DNA相關樣品之電泳圖 99 圖3-29、pACPm(Hae III Digest)之偏快bp57 DNA相關樣品之電泳圖 100 圖3-30、偏快以及正常去磷酸根bp57 DNA之相關電泳圖 101 圖3-31、CNG去磷酸化樣品與Digest樣品之電泳圖( I ) 102 圖3-32、CNG及去磷酸化樣品與Digest樣品之電泳圖( II ) 103 圖3-33、CNG及pBR322(Alu I)bp57 DNA磷酸化樣品(即CNG_ p和 N57_ p)5之相關電泳圖 104 圖3-34、pACpm(Hae III)bp43 DNA磷酸化樣品(即43_ p)之相關電泳圖 105 圖3-35、pACpm(Hae III)bp57 DNA磷酸化樣品(即57_ p)之相關電泳圖 106 圖3-36、pBR322 DNA(Alu I Digest)去磷酸化樣品之相關電泳圖 107 表格索引 表1-1 (CNG)10和標準DNA(20~160bp)之電泳遷移時間 23 表2-1 藥品總覽 31 表2-2 三種不同DNA質體以兩種不同酵素切割所得片段大小 36 表2-3 在8種正常或偏快的小DNA(a)中統計不同四鹼基序列出現次數 41 表2-4 以不含偏快序列DNA(a)篩選之可能偏快序列 44 表2-5-1 八個可能偏快四鹼基序列(a)在200bp以下的偏快DNA(b)序列中出現之統計 45 表2-5-2 八個可能偏快四鹼基序列(a)在200bp以下的偏快DNA(b)序列中出現之統計 46 表2-6 八個可能偏快四鹼基序列(a)在200~500bp的偏快DNA(b)序列中出現之統計 47 表2-7 八個可能偏快四鹼基序列在57個偏快(a)DNA中曾出現之比例 48 表3-1-1 正常電泳遷移DNA片段之μra(5次測量結果) 54 表3-1-2 異常電泳遷移DNA片段之μra(5次測量結果) 55 表3-2-1 DNA片段之電泳遷移行為歸納( I ) 56 表3-2-2 DNA片段之電泳遷移行為歸納( II ) 57 表3-3 溫度變化(20℃~50℃)時dsDNA之異常遷移百分率 58 |
參考文獻 |
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