Separation of plasmid DNA topoisomers by multimodal chromatography.

The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases. Here, we describe the separation of supercoiled (sc) and open circular (oc) topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid. The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample.

Quantification of ssDNA Scaffold Production by Ion-Pair Reverse Phase Chromatography.

DNA origami is an emerging technology that can be used as a nanoscale platform in numerous applications ranging from drug delivery systems to biosensors. The DNA nanostructures are assembled from large single-stranded DNA (ssDNA) scaffolds, ranging from hundreds to thousands of nucleotides and from short staple strands. Scaffolds are usually obtained by asymmetric PCR (aPCR) or Escherichia coli infection/transformation with phages or phagemids. Scaffold quantification is typically based on agarose gel electrophoresis densitometry for molecules obtained by aPCR, or by UV absorbance, in the case of scaffolds obtained by infection or transformation. Although these methods are well-established and easy-to-apply, the results obtained are often inaccurate due to the lack of selectivity and sensitivity in the presence of impurities. Herein, we present an HPLC method based on ion-pair reversed-phase (IP-RP) chromatography to quantify DNA scaffolds. Using IP-RP chromatography, ssDNA products (449 and 1000 nt) prepared by aPCR were separated from impurities and from the double stranded (ds) DNA byproduct. Additionally, both ss and dsDNA were quantified with high accuracy. The method was used to guide the optimization of the production of ssDNA by aPCR, which targeted the maximization of the ratio of ssDNA to dsDNA obtained. Moreover, ssDNA produced from phage infection of E. coli cells was also quantified by IP-RP using commercial ssDNA from the M13mp18 phage as a standard.

Purification of Plasmid DNA by Multimodal Chromatography.

Multimodal (MM) chromatography can be described as a chromatographic method that uses more than one mode of interaction between the target molecule and the ligand to achieve a particular separation. Owing to its advantages over traditional chromatography, such as higher selectivity and capacity, its application for the purification of biomolecules with therapeutic interest has been widely studied. The potential of MM chromatography for the purification of plasmid DNA has been demonstrated. In this chapter, a downstream process for the purification of supercoiled plasmid DNA using MM chromatography with two different ligands-Capto™ adhere and PPA HyperCell™-is described. In both the cases, the purification process yields a high purity and highly homogeneous sc plasmid product.

Primary Purification of Plasmid DNA Using Differential Isopropanol Precipitation.

A method for the intermediate recovery of plasmid DNA (pDNA) from alkaline lysates is described that comprises differential isopropanol precipitation steps. In a first low-cut precipitation, a smaller amount of isopropanol (20% v/v) is used so that only high molecular weight RNA precipitates. After solid liquid separation, a high-cut precipitation is performed by bringing isopropanol concentration to 70% v/v to precipitate pDNA. Tests made with lysates show that the differential precipitation increases purity threefold compared to the conventional one-step precipitation at 70% v/v without affecting pDNA recovery (>80%).