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系統識別號 U0002-3107200516175000
中文論文名稱 利用聚葡萄糖-毛細管電泳分離不同構型dsDNA
英文論文名稱 Separation of dsDNA with Different Conformations by Dextran-Capillary Electrophoresis
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 93
學期 2
出版年 94
研究生中文姓名 王文政
研究生英文姓名 Weng-Cheng Wang
學號 692170078
學位類別 碩士
語文別 中文
口試日期 2005-06-16
論文頁數 129頁
口試委員 指導教授-吳俊弘
委員-鄭建中
委員-陳灝平
中文關鍵字 聚葡萄糖  毛細管電泳  DNA分離 
英文關鍵字 dextran  capillary electrophoresis  DNA separation 
學科別分類 學科別自然科學化學
中文摘要 在本研究中我們利用毛細管電泳技術,結合填充有聚葡萄糖(dextran)的毛細管,可以得到高解析度的雙股DNA (dsDNA)分離效果。聚葡萄糖-毛細管電泳系統 (dextran-CE)可有效分離解析具有相同鹼基對數目,但不同鹼基序列 (sequence)或不同末端構型,如平切端 (blunt end)和黏合端 (sticky end)的dsDNA 片段,並可觀察dsDNA 電泳行為和其隨序列改變的構型之間的關係。
在分離分別經由兩種限制酶(Hae III-平切, Msp I-黏合切)切割的pBR322 DNA 混合樣品的實驗中,我們發現大小相近但末端切口構型不同的DNA,會有不規則電泳析出的現象。另外,我們也分離了同樣都具有平切口的限制酶消化DNA 片段之混合樣品,從所得電泳圖我們發現相同大小DNA 片段可被解析開來,而在某些大小相近的DNA 片段區域,也有不規則電泳析出的現象。我們在平板瓊脂膠(agarose gel)電泳和其他毛細管電泳分離介質(如聚氧乙烯,Poly(ethylene oxide)和聚丙烯醯胺,Polyacrylamide)中並未發現這種現象。在本研究中,我們歸納了DNA 末端切口結構以及特殊DNA 序列,例如A-tracts 和GGGA,以及(G or C)n 序列出現的頻率,如何影響DNA 構型,進而造成上述不規則電泳遷移行為;另外,利用dextran-CE 系統也可以成功地分離具有單點未配對(mismatch)DNA 或凸起(bulge)構型DNA。
英文摘要 In this research capillary electrophoresis (CE) technique combined with dextran-filled capillary was used to achieve high separation resolution for double-strand DNA (dsDNA). Our dextran-CE system could effectively resolve dsDNA fragments with the same size but different sequences, or different end conformations such as blunt ends and sticky ends. The relationship between DNA electrophoretic behaviors and the sequence-altered conformations was also observed.
The respective digests of pBR322 DNA by two restrictions, Msp I and Hae III, would result in DNA fragments with sticky ends and blunt ends. According to the electropherogram of the DNA fragments with different end conformations, DNA fragments with sticky ends usually migrated faster than the ones with similar size and blunt ends. The anomalous DNA elution order was also observed in the experiments for separating DNA fragments generated only by blunt-end cutting enzymes. No anomalous DNA elution order was found in slab agarose gel electrophoresis system or in CE using the other separation mediums such as poly(ethylene oxide) and polyacrylamide. In this research we could conclude that DNA chain end conformations and the existing frequency of some special DNA sequences such as A-tracts, GGGA, and (G or C)n would affect DNA conformation and thus lead to anomalous DNA electrophoretic elution order. Furthermore, we could successfully separate mismatch DNA and bulge DNA by using our dextran-CE system, which has been proved to be an effective method of detecting DNAs with different conformations.
論文目次 目錄
中文摘要..................................................i
英文摘要.................................................ii
目錄....................................................iii
圖表索引.................................................vi
附錄索引..................................................x
第一章、緒論..............................................1
1.1 前言..................................................1
1.2 電泳基本原理..........................................1
1.3 毛細管電泳介紹........................................3
1.3.1 電滲透流原理........................................3
1.3.2 毛細管電泳的分離方法................................5
1.3.3 樣品注入方式........................................7
1.4 分離介質介紹..........................................9
1.5 DNA 片段在膠體中的電泳行為...........................13
1.6 DNA 序列與構型.......................................16
1.7 突變DNA 的偵測.......................................19
1.7.1 基因突變的檢測法...................................21
1.8 本章參考資料.........................................26
第二章、實驗部分.........................................31
2.1 毛細管電泳儀.........................................31
2.2 毛細管部份...........................................31
2.3 DNA 樣品.............................................33
2.3.1 經限制酶切割之雙股DNA 片段樣品.....................33
2.3.2 HPV(human papilloma virus)DNA(24 mer)樣品......37
2.4 緩衝溶液.............................................44
2.5 分離介質.............................................45
2.6 染料分子.............................................46
第三章、結果與討論.......................................47
3.1 利用高解析CE-Dextran 分離dsDNA.......................47
3.1.1 以Dextran 為CE 分離介質............................47
3.1.2 染料分子濃度效應...................................49
3.1.3 電泳溫度效應.......................................50
3.2 dsDNA 不規則電泳析出.................................53
3.2.1 pBR322(Msp I) DNA+ pBR322 (Hae III) DNA............54
3.2.2 φx174 (Hae III) DNA + pBR322 (Hae III) DNA ........56
3.2.3 φx174 (Alu I) DNA + pBR322 (Alu I) DNA.............57
3.2.4 φx174 (Hae III) DNA +φx174 (Alu I) DNA.............59
3.2.5 pBR322 (Hae III) DNA + pBR322 (Alu I) DNA .........60
3.2.6 pBR322 (Hae III) DNA +φx174 (Alu I) DNA............61
3.2.7 φx174 (Hae III) DNA + pBR322 (Alu I) DNA...........62
3.2.8 偵測長度、電場強度、溫度和緩衝溶液組成對於DNA反常析出現象之影響...............................................63
3.2.9 不同分離介質對於DNA 反常析出現象之影響.............65
3.2.10 添加變性試劑對於DNA 反常析出現象之影響............67
3.2.11 質體DNA 經限制酶二次切割實驗......................68
3.3 單點未配對dsDNA 樣品實驗.............................70
3.3.1 染料效應...........................................71
3.3.2 添加離子與分離介質效應.............................72
3.3.3 偵測長度與電場強度效應.............................72
3.3.4 單點未配對dsDNA 之分離.............................74
3.4 總結.................................................74
3.5 本章參考資料.........................................75
圖表索引
圖3-1、dsDNA 電泳圖之Dextran2000k 濃度效應...............77
圖3-2、dsDNA 電泳圖之Dextran 分子量效應..................78
圖3-3、dsDNA 電泳圖之EB 濃度效應.........................79
圖3-4、dsDNA 電泳圖之溫度效應( 20 ℃ ~ 35 ℃)............80
圖3-5、dsDNA 電泳圖之溫度效應( 40 ℃ ~ 55 ℃)............81
圖3-6、Arrhenius plots(1/K vs lnμ) ....................82
圖3-7、Activation Energy(Base pair vs Ea)..............83
圖3-8、混合樣品pBR322(Msp I) DNA + pBR322(Hae III)
DNA 電泳圖...............................................84
圖3-9、 log(1/bp) vs log µ 比較圖(pBR322(Msp I) DNA +
pBR322(Hae III) DNA)....................................85
圖3-10、混合樣品φx174 DNA(Hae III digest) + pBR322(Hae III)
DNA 電泳圖...............................................86
圖3-11、 log(1/bp) vs log µ 比較圖(φx174 DNA(Hae III digest) + pBR322(Hae III))..............................87
圖3-12、混合樣品φx174 DNA(Alu I digest) + pBR322 DNA
(Alu I digest)電泳圖.....................................88
圖3-13、log(1/bp) vs log µ 比較圖(φx174 DNA(Alu I digest) + pBR322 DNA(Alu I digest)) ............................89
圖3-14、混合樣品φx174 DNA(Hae III digest) + φx174 DNA
(Alu I digest)電泳圖.....................................90
圖3-15、log(1/bp) vs log µ 比較圖(φx174 DNA(Hae III digest) + φx174 DNA(Alu I digest))......................91
圖3-16、混合樣品 pBR322 DNA(Hae III digest) + pBR322 DNA
(Alu I digest)電泳圖.....................................92
圖3-17、log(1/bp) vs log µ 比較圖(pBR322 DNA(Hae III digest) + pBR322 DNA(Alu I digest)) ....................93
圖3-18、混合樣品 pBR322 DNA(Hae III digest) + φx174 DNA
(Alu I digest)電泳圖.....................................94
圖3-19、log(1/bp) vs log µ 比較圖( pBR322 DNA(Hae III digest) + φx174 DNA(Alu I digest))......................95
圖3-20、混合樣品φx174 DNA(Hae III digest) + pBR322 DNA
(Alu I digest)電泳圖.....................................96
圖3-21、log(1/bp) vs log µ 比較圖(φx174 DNA(Hae III digest) + pBR322 DNA(Alu I digest)) ....................97
圖3-22、偵測長度效應之電泳比較圖.........................98
圖3-23、分離電場效應之電泳比較圖.........................99
圖3-24、溫度效應之電泳比較圖( I ) ......................100
圖3-25、溫度效應之電泳比較圖( II )......................101
圖3-26、緩衝溶液效應之電泳比較圖........................102
圖3-27、分離介質效應PA(5M-6M)之電泳比較圖...............103
圖3-28、分離介質效應PEO(8M)之電泳比較圖.................104
圖3-29、DNA 瓊脂膠電泳圖................................105
圖3-30、變性試劑效應之電泳比較圖........................106
圖3-31、混合樣品pBR322(Msp I+Alu I) DNA + pBR322(Msp I)
DNA 電泳圖..............................................107
圖3-32、混合DNA 樣品經限制酶二次切割實驗之dsDNA 電泳圖108
圖3-33、不同EB 濃度時(低電場)之電泳圖...................109
圖3-34、不同EB 濃度時(高電場)之電泳圖...................110
圖3-35、添加離子與分離介質效應..........................111
圖3-36、以PA 為分離介質分離單點未配對dsDNA 之電泳圖.....112
圖3-37、偵測長度與電場強度效應之電泳圖..................113
圖3-38、偵測長度及電場強度效應之比較....................114
圖3-39、單點未配對dsDNA 之電泳圖( I )...................115
圖3-40、單點未配對dsDNA 之電泳圖( II ) .................116
附錄索引
附錄3-1、特定序列出現次數比較表(pBR322(Msp I) DNA +
pBR322 (Hae III) DNA)..................................117
附錄3-2、特定序列出現次數比較表(pBR322(Msp I) DNA +
pBR322 (Hae III) DNA....................................118
附錄3-3、特定序列出現次數比較表(φx174(Hae III) DNA +
pBR322 (Hae III) DNA)..................................119
附錄3-4、特定序列出現次數比較表(φx174 (Alu I) DNA +
pBR322 (Alu I) DNA......................................120
附錄3-5、特定序列出現次數比較表(φx174 (Alu I) DNA +
φx174 (Hae III) DNA) ..................................121
附錄3-6、特定序列出現次數比較表(pBR322(Hae III) DNA +
pBR322 (Alu I) DNA......................................122
附錄3-7、特定序列出現次數比較表(pBR322(Hae III) DNA +
pBR322 (Alu I) DNA)....................................123
附錄3-8、特定序列出現次數比較表(pBR322(Hae III) DNA +
φx174 (Alu I) DNA ......................................124
附錄3-9、特定序列出現次數比較表(φx174 (Hae III) DNA +
pBR322 (Alu I) DNA)....................................125
附錄3-10、特定序列出現次數比較表
(pBR322(Msp I) DNA bp 147) ...........................126
附錄3-11、特定序列出現次數比較表
(以GC-rich 觀點討論I).................................127
附錄3-12、特定序列出現次數比較表
(以GC-rich 觀點討論II)................................128
附錄3-13、特定序列出現次數比較表
(以GC-rich 觀點討論III) ..............................129
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