淡江大學覺生紀念圖書館 (TKU Library)
進階搜尋


下載電子全文限經由淡江IP使用) 
系統識別號 U0002-0709201021013700
中文論文名稱 利用毛細管電泳探討dsDNA之異常電泳遷移行為
英文論文名稱 Study of anomalous electrophoretic migration of dsDNA by 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 98
學期 2
出版年 99
研究生中文姓名 許蘭婷
研究生英文姓名 Lan-ting Hsu
學號 696160737
學位類別 碩士
語文別 中文
口試日期 2010-07-19
論文頁數 107頁
口試委員 指導教授-吳俊弘
委員-鄭建中
委員-施增廉
中文關鍵字 毛細管電泳  異常電泳遷移  雙股螺旋DNA 
英文關鍵字 capillary electrophoresis  anomalous electrophoretic migration  dsDNA 
學科別分類 學科別自然科學化學
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.
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 1.5 本章參考資料
1.Hunter, R.J. Foundations of Colloid Science, Clarendon Press, Oxford 1989, 1, Chapter6.
2.van de Ven, T. G. M. Colloid Science, Academic Press, London 1989, Chapter 1.
3.Colyar, C. L.;Chien, N.;Harrison, D. J., "Clinical otential of microchip capillary electrophoresis systems", Electrophoresis 1997, 18, 1733-1741.
4.Cohen, A. S.; Karger, B. L.; Smith, J. A., "High-performance capillary electrophoretic separation of bases, nucleosides, and oligonucleotides: retention manipulation via micellar solution and metal additives", Analytical Chemistry 1987, 59, 1021-1027.
5.Luckey, I. A.; Smith, L. M., "Optimization of electric field strength for DNA sequencing in capillary gel electrophoresis ", Analytical Chemistry 1993, 65, 2841-2850.
6.Slater, G. W.; Mayer, P.; Grossman, P. D., "Diffusion, joule heating, and band broadening in capillary gel electrophoresis of DNA", Electrophoresis 1995, 16, 75-83.
7.Barron, A. E.;Soane, D. S.; Blanch, H. W., "Capillary electrophoresis of DNA in uncross-linked polymer solution", J. Chromatogr. 1993, 652, 3-16.
8.Barron, A. E.;Blanch, H. W.; Soane, D. S., "A transient entanglement coupling mechaniam for DNA separation by capillary electrophoresis in ultradilute polymer solutions", Electrophoresis 1994, 15, 597-615.
9.Heller, C., "Capillary electrophoresis of proteins and nucleic acids in gels and entangled polymer solution", J. Chromatogr. 1995, 698, 19-31.
10.Heller, C., "Finding auniversal low-viscosity polymer for DNA separation, ", Electrophoresis 1998, 19, 1691-1698.
11.Zhang, Jian.; Horvath, Csaba., "Capillary electrophoresis of proteins in dextran-coated columns", electrophoresis 2003, 24, 115-120.
12.Quesada, M. A., "Replaceble polymers in DNA sequencing for capillary eletrophpresis", Current Opinion in Biotechnology 1997, 8, 82-93.
13.Leslie, A. G.; Arnott S.; Chandrasekaran R.; Ratliff RL., "Polymorphism of DNA double helices", J Mol Biol 1980, 143, 49-72.
14.Skeidsvoll J.; Ueland, P. M., "Analysis of double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection using the monomeric dye SYBR Green I", Analytical Biochemistry 1995, 231, 359-365.
15.Atha, D. H., "Characterization of DNA standards by capillary
electrophoresis", Electrophoresis 1998, 19, 1428-1435.
16.Burda, J. V.; Šponer J.; Leszczynski J.; Hobza P., "Interaction of DNA base pairs with various metal cations (Mg2+, Ca2+, Sr2+, Ba2+, Cu+, Ag+, Au+, Zn2+, Cd2+, and Hg2+): nonempirical ab initio calculations on structures, energies, and nonadditivity of the interaction", J. Phys. Chem. B, 1997, 101, 9670–9677.
17.Shui X.; McFail-Isom L.; Hu GG, Williams LD., "The B-DNA dodecamer at high resolution reveals a spine of water on sodium", Biochemistry 1998, 37, 8341-8355.
18.Steven M.; Clark.; Richard A.; Mathies., "Multiplex dsDNA fragment sizing using dimeric intercalation dyes and capillary array electrophoresis: ionic effects on the stability and electrophoretic mobility of DNA-dye complexes", Analytical Chemistry 1997, 69, 355-1363.
19.Olson, W. K.; Zhurkin, V. B., "Twenty years of DNA bending in biological structure and dynamics", Biological structure and dynamics 1996, 2, 341-370.
20.Stellwagen E.; Stellwagen NC., "Determining the electrophoretic mobility and translational diffusion coefficients of DNA molecules in free solution", Electrophoresis, 2002, 23, 2794-2803.
21.Hagerman, P. J., "Sequence dependence of the curvature of DNA: a test of the phasing hypothesis", Biochemistry, 1985, 24, 7033-7037.
22.Goodsell, D. S.; Dickerson, R. E., "Bending and curvature calculations in B-DNA", Nucl. Acids Res., 1994, 22, 5497-5503.
23.Sinden R. R.; Potaman V. N.; Oussatcheva E. A.; Pearson C. E.; Lyubchenko Y. L.; Shlyakhtenko L. S., "Triplet repeat DNA structures and human genetic disease: dynamic mutations from dynamic DNA", J. Biosci., 2002, 27, 53-65.
24.Kiba Y.; Baba Y., " Unusual capillary electrophoretic behavior of triplet repeat DNA", J. Biochem. Biophys., 1999, 41, 143–151.

3.8 章參考資料
1.Gao Y. G.; Sriram M.; Wang A. H., "Crystallographic studies of metal ion-DNA interactions: different binding modes of cobalt(II), copper(II) and barium(II) to N7 of guanines in Z-DNA and a drug-DNA complex", Nucleic Acids Res, 1993, 21, 4093-101.
2.Steven M.; Clark.; Richard A.; Mathies., "Multiplex dsDNA fragment sizing using dimeric intercalation dyes and capillary array electrophoresis: ionic effects on the stability and electrophoretic mobility of DNA-dye complexes", Analytical Chemistry 1997, 69, 355-1363.
3.Wenz H. M., "Capillary electrophoresis as a technique to analyze sequence-induced anomalously migrating DNA fragments", Nucleic Acids Res, 1994, 22, 4002-4008.
4.Berka J.; Pariat Y. F.; Müller O.; Hebenbrock K.; Heiger D.N.; Foret F.; Karger B. L., "Sequence dependent migration behavior of double-stranded DNA in capillary electrophoresis", Electrophoresis, 1995, 3, 377-388.
5.Atha, D. H., "Characterization of DNA standards by capillary
electrophoresis", Electrophoresis 1998, 19, 1428-1435.
6.Paul D. Chastain II.; Evan E. Eichler.; Seongman Kang.; David L. Nelson.; Stephen D. Levene.; Richard R. Sinden., " Anomalous Rapid Electrophoretic Mobility of DNA Containing Triplet Repeats Associated with Human Disease Genest ", Biochemistry, 1995, 34, 16125-16131.
7.Nancy C. Stellwagen1.; Soffia Magnusdottir.; Cecilia Gelfi.;.Pier Giorgio Righetti., " Preferential Counterion Binding to A-Tract DNA Oligomers",J. Mol. Biol, 2001, 305, 1025±1033.
8.Sinden R. R.; Potaman V. N.; Oussatcheva E. A.; Pearson C. E.; Lyubchenko Y. L.; Shlyakhtenko L. S., "Triplet repeat DNA structures and human genetic disease: dynamic mutations from dynamic DNA", J. Biosci., 2002, 27, 53-65.
9.Arakawa, H.; Uetanaka, K.; Tsuji, A., "Enhanced resolution in the capillary electrophoretic separation of double-stranded DNA using dextran sulfate and its application o the analysis og PCR products", Analytical Sciences, 1997, 13, 853-855.
10.Heller, C., "Finding a universal low-viscosity polymer for DNA separation", Electrophoresis, 1998, 19, 1691-1698.
論文使用權限
  • 同意紙本無償授權給館內讀者為學術之目的重製使用,於2013-09-10公開。
  • 同意授權瀏覽/列印電子全文服務,於2013-09-10起公開。


  • 若您有任何疑問,請與我們聯絡!
    圖書館: 請來電 (02)2621-5656 轉 2281 或 來信