利用聚葡萄糖-毛細管電泳分離不同構型dsDNA

Separation of dsDNA with Different Conformations by Dextran-Capillary Electrophoresis

王文政(Weng-Cheng Wang)

指導教授 ： 吳俊弘

淡江大學/理學院/化學學系碩士班/碩士(2005年)

https://doi.org/10.6846/TKU.2005.00793

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。

關鍵字

聚葡萄糖 ； 毛細管電泳 ； DNA分離

並列摘要

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.

並列關鍵字

dextran ； capillary electrophoresis ； DNA separation

參考文獻

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