| 项目编号: | 1505592
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| 项目名称: | UNS: Purification of plasmid DNA using enhanced ultrafiltration systems |
| 作者: | Andrew Zydney
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| 承担单位: | Pennsylvania State Univ University Park
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| 批准年: | 2014
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| 开始日期: | 2015-08-01
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| 结束日期: | 2018-07-31
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| 资助金额: | USD314014
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| 资助来源: | US-NSF
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| 项目类别: | Standard Grant
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| 国家: | US
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| 语种: | 英语
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| 特色学科分类: | Engineering - Chemical, Bioengineering, Environmental, and Transport Systems
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| 英文关键词: | dna
; purification
; supercoiled dna
; membrane system
; membrane
; dna molecule
; individual plasmid isoform
; actual plasmid separation
; different plasmid isoform
; supercoiled plasmid dna
; dna elongation
; dna trapping
; biomolecule purification
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| 英文摘要: | 1505592 (Zydney)
The overall objective of the proposed research is to develop enhanced membrane processes for the purification of supercoiled plasmid DNA, the biologically active form of DNA of interest in therapeutic applications. Experiments will be designed to develop and test novel membranes with unique pore structures specifically designed to elongate the DNA and enhance the overall separation. In addition, small molecules that are known to selectively bind to DNA will be used to fine tune the separation characteristics by altering the elongational flexibility of the DNA molecules. These results will provide fundamental insights and practical guidelines for the development of membrane systems for purification of supercoiled DNA for therapeutic applications.
The proposed research will address both of these issues through: (a) the development and testing of membranes with tapered or gradient pore morphologies that can effectively pre-stretch the DNA and thus minimize DNA trapping at the entrance to the small pores, and (b) the use of selective binding ligands to alter the elongation flexibility and thus enhance the separation between different plasmid isoforms. Membranes with tapered (conical) pores will be produced by differential etching of tracked polycarbonate membranes. Membranes with gradient morphologies will be produced by electrospun nanofibers or by layering membranes with different pore size characteristics into an effective composite membrane. Initial experiments will be performed with solutions of the individual plasmid isoforms, using a series of plasmids with different numbers of base pairs. Experimental results will be analyzed using available theoretical models for polymer elongation, appropriately extended to account for the tapered pore structure. Actual plasmid separations will be performed using linearly-scalable filtration modules operated in a diafiltration mode, with feed and permeate samples analyzed using both agarose gel electrophoresis and affinity chromatography. These studies will provide fundamental insights into the effects of DNA elongation on membrane pore structure and separation as well as practical guidelines for the development of membrane systems for purification of supercoiled DNA for therapeutic applications. In addition, the proposed research program will provide graduate and undergraduate students with hands-on experience dealing with membrane processes and biomolecule purification, skills that are critically needed to support the efforts of the U.S. biotechnology and pharmaceutical industries. |
| 资源类型: | 项目
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| 标识符: | http://119.78.100.158/handle/2HF3EXSE/93913
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| Appears in Collections: | 影响、适应和脆弱性
气候减缓与适应
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| Recommended Citation: | |
Andrew Zydney. UNS: Purification of plasmid DNA using enhanced ultrafiltration systems. 2014-01-01.
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