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標題: 使用陰離子交換與疏水性薄膜吸附分離質體核酸與核醣核酸
作者: 倪賀生
關鍵字: 質體核酸;核醣核酸;薄膜
出版社: 化學工程學系
Due to the fast progress in biotechnology in recent years, the usage of plasmid DNA becomes broader and broader such as for the purpose of gene therapy. Accordingly, the separation of plasmid DNA from RNA in downstream processing gains more and more attention. In most prevoous studies or some practical processes, chromatographic techniques such as ion exchange, hydrophobic interaction, size exclusion, tripelix affinity, etc. are adopted to purify plasmid DNA. For reducing the mass transfer limitations which are usually encountered in the chromatographic processes, anion exchange and hydrophobic membranes were used to separate plasmid DNA and RNA in this work.
At first, batch and recirculating adsorption operations were conducted. The optimal operating conditions are: adsorption at pH 8 and desorption under 2 M NaCl and pH 12 for strong anion exchange membranes; adsorption at 2 M NaCl and pH 8 and desorption using 10% isopropanol, at pH 8 for C8 hydrophobic membranes. For flow experiment, plasmid DNA and RNA were separated from the mixture of pure plasmid DNA and pure RNA as well as the supernatant from cell broth using the above optimal conditions. For strong anion exchange membranes, a further use of 20% ethanol was employed to elute the plasmid DNA. For C8 hydrophobic membranes, there remained some RNA in the fraction of purified plasmid DNA. Therefore, a further improvement in the elution step is necessary.

本研究藉質體核酸與核醣核酸帶負電性與具疏水性鹼基對之特性,嘗試使用陰離子交換與疏水性薄膜吸附分離質體核酸與核醣核酸,並探討其吸附與脫附效果。本研究主要先分析批式與循環回流操作下的最佳操作條件,所得強陰離子交換薄膜的條件為:吸附於50 mM Tris,pH 8,而脫附於2M NaCl,50 mM Tris,pH 12;C8疏水性薄膜的條件為:吸附於2 M NaCl,10 mM Tris,pH 8,而脫附於10﹪isopropanol,10 mM Tris,pH 8。以循環回流吸附實驗所得之最佳條件進行流動實驗,流動實驗先以混合純質體核酸與純核醣核酸進料,並修正循環回流吸附實驗所得之條件,得到可分離質體核酸與核醣核酸之條件,陰離子交換薄膜部分最後需使用20%乙醇脫附質體核酸,即可分離純化出質體核酸與核醣核酸。疏水性薄膜部分,分離得到之質體核酸內仍含有少量質體核酸,故仍需繼續找出可分離之條件。
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