Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.436127
Title: Precipitation of nucleic acids for selective recovery of plasmid DNA
Author: McHugh, Patrick Michael
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2006
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Abstract:
Nucleic acids are being targeted more commonly as pharmaceutical therapeutic agents, and consequently the ability to purify these biological macromolecules at large scale has taken on increased importance. Although precipitation of nucleic acids plays a key role in existing purification processes, the effects of different process variables are not well understood. In recovery processes, typically supercoiled plasmid DNA is the form of interest, however because other nucleic acid forms (chromosomal DNA, RNA, relaxed plasmid DNA) are very similar to plasmid DNA both structurally and chemically, separation of plasmid DNA from these forms represents a significant challenge. This project studied the precipitation behaviour of different nucleic acids forms in typical clarified lysate streams from E. coli fermentation. Solubilities of individual forms were studied, investigating effects of process variables such as salt type, salt concentration, organic solvents, and polymers. Key findings from single-component studies showed monovalent cations alone to be relatively ineffective agents for precipitation of any of the nucleic acid forms studied. Divalent cations alone were relatively ineffective for precipitation of double- stranded DNA, however they were effective agents for precipitation of RNA and single-stranded DNA, suggesting these salts may be effective for fractional precipitation of these forms. Inclusion of organic solvents or polymers decreased solubilities of all forms in the presence of either monovalent or divalent cations. Single-component findings were extended to investigation of multi-component clarified lysate process streams. To aid in this effort, an analytical anion- exchange HPLC method was developed to quantify different nucleic acid forms from multi-component streams. This analytical method was used to show divalent cations were effective for the fractional precipitation of the majority of single-stranded nucleic acid impurities in clarified lysate, leaving most of the plasmid DNA plus small quantities of chromosomal DNA and RNA impurities in solution. Single-component results were further extended through the development of a novel controlled thermal-denaturation step prior to divalent cation precipitation. This step converted double-stranded DNA forms, with the exception of supercoiled plasmid, to the single-stranded form. Performing thermal denaturation on clarified lysate streams prior to divalent cation precipitation resulted in improved separation of chromosomal and relaxed plasmid DNA forms, leaving highly-purified supercoiled DNA in solution following precipitation. A non-chromatographic purification process for recovery of supercoiled plasmid DNA is proposed based on these results. Findings were further characterised through kinetics of precipitation studies, investigation of precipitation mechanisms, comparison to solubility models, and investigation of plasmid stability.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.436127  DOI: Not available
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