Nucleic Acids: Innovative Methods for Recovery, Clarification and Purification

Abstract: The importance of nucleic acids in pure form for preparative and analytical perspectives, have increased constantly, demanding the development of new and more efficient methods for their recovery and isolation. This thesis describes a series of different innovative methods for recovery and purification of these biomolecules. In a general overview of a downstream processing, there are several critical steps that may influence the yield and quality of the final product, especially in the lysis, clarification and purification steps. The lysis process is the first critical step, since it can be detrimental to the nucleic acids and it also can release all the content from the cells by its disruption. A new approach was developed in order to minimize this effect and reduce the main host cell contaminants. The electropermeability approach for extraction of small nucleic acids from bacteria avoids complete cell disruption and thereby reduces contaminants for the next stages. The clarification and concentration steps are normally done to remove most of the contaminants from the cell lysis steps. In this thesis an aqueous two-phase system (ATPS) approach based on poly(ethylene glycol)/poly(acrylate)/salt two phase system is described. This method allows the handling of large volumes, which is important in the preparative industrial scale. ATPS resulted in a pure plasmid DNA directly from crude cell lysates. In addition, smaller DNA fragments from Polymerase Chain Reaction (PCR) can be isolated using this method. The regular downstream process is normally finished by chromatographic approaches, which often are the main final purification step. DNA molecules harbour some intrinsic chemical properties that render them suitable for chromatographic separations. These include a negatively charged phosphate backbone as well as a hydrophobic character originating mainly from the major groove of DNA which exposes the base pairs on the surface of the molecule. In addition, single stranded DNA often allows for a free exposure of the hydrophobic aromatic bases. In this thesis, multimodal chromatography (MMC) was evaluated as an alternative tool for complex separations of nucleic acids. MMC embraces more than one kind of interaction between the chromatographic ligand and the target molecules. This resin demonstrated a superiority for DNA isolation, including in the purification of pDNA from crude cell lysate and for DNA fragments from PCR samples.