利用毛細管電泳探討dsDNA之異常電泳遷移行為

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。

關鍵字

毛細管電泳；異常電泳遷移；雙股螺旋DNA

並列摘要

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.

並列關鍵字

capillary electrophoresis ； anomalous electrophoretic migration ； dsDNA

參考文獻

2.van de Ven, T. G. M. Colloid Science, Academic Press, London 1989, Chapter 1.

1.Hunter, R.J. Foundations of Colloid Science, Clarendon Press, Oxford 1989, 1, Chapter6.

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.

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利用毛細管電泳探討dsDNA之異常電泳遷移行為

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