Affinity purification of plasmid DNA directly from crude bacterial cell lysates

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with impractically low DNA yields. We have optimized the procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 microg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required.     

Direct visualization of plasmid DNA in bacterial cells

The direct visualization of plasmid DNA inside Escherichia coli cells is demonstrated using phase-fluorescence microscopy of DAPI (4',6-diamidino-2-phenylindole)-stained bacteria. Small as well as large plasmids could be detected, both in minicells and in cells of larger size. For large plasmids, even single molecules appeared to be within the detection limit. The fluorescence generated from monomers of small plasmids was probably below this limit, and for these plasmids the observed signals may represent aggregates. The distribution of the fluorescence foci might reflect specific plasmid positioning during partition and/or replication.     

Temperature-dependent selective purification of plasmid DNA using magnetic nanoparticles in an RNase-free process

Carboxyl group-functionalized magnetic nanoparticles were used to develop an RNase-free method for plasmid DNA (pDNA) purification directly from RNA-containing crude Escherichia coli lysates. This method takes advantage of differing adsorption behaviors of pDNA and RNA onto magnetic nanoparticle surfaces at different temperatures. Pure pDNA can be isolated between 70 and 80 °C without sacrificing DNA quality and quantity, as evidenced by comparison with that obtained using organic solvents or commercial kits. This RNase-free method is rapid, simple, cost-effective, and environmentally friendly, and it can be easily scaled up for the production of pharmacological-grade pDNA.     

Temperature-dependent selective purification of plasmid DNA using magnetic nanoparticles in an RNase-free process

The efficiency of transformation by electroporation has been known to be compromised by strain dependency. A high efficiency protocol is still lacking for distinct two-hybrid yeast strains of diverse genetic features. Here, we used 0.5 M lithium acetate (LiAc) and 50 mM Tris-HCl with 5 mM EDTA (pH 7.5), i.e., fivefold the standard concentrations, and voltage at 1.0 to develop a protocol which, for the first time, is able to effect an average efficiency of 1.84 × 10⁶ transformants/μg DNA for three commonly used yeast strains committed to two-hybrid screening experiments.     

Large scale purification of plasmid DNA

A simple and rapid procedure for large scale purification of plasmid DNA is described. The procedure consists of two main steps: 1. Alkaline extraction of plasmid DNA (by a slight modification of the method of Birnboim and Doly (1)) and 2. Purification of the crude extract by hydroxyapatite chromatography. The plasmids obtained are biologically active and can be used in gene manipulation experiments.     

Enzymatic purification of plasmid DNA.

A novel method for plasmid DNA purification using enzymes that degrade all major types of contaminating nucleic acids present in crude plasmid DNA mixtures (but not plasmid DNA) has been devised. This method is quick (can be accomplished in two hours), requires no expensive laboratory equipment (an ultracentrifuge is not necessary) and is inexpensive. Plasmid DNA purified by this methodology can be used in a variety of molecular biological techniques including restriction enzyme digestions, subcloning, sequencing, nick translation and end labeling. This plasmid purification technique will be very useful for the molecular biologist performing cloning experiments.     

Application of short monolithic columns for fast purification of plasmid DNA

A simple method for the separation of the four major neutral glycosphingolipids, present in all human tissue, was developed. This gradient normal phase-HPLC method utilises a polyvinyl alcohol bonded stationary phase and an evaporative light-scattering detection (ELSD). Screening pure solvents in a binary gradient elution mode allowed, in a first step, to assess the behaviour of the studied solutes and to select the solvents for further mobile phase optimisation. The proportion of the remaining solvents was defined to reach a maximal resolution. The reduction of the analysis time and the enhancement of the signal were obtained by optimising the gradient slope and the flow-rate. Optimal levels of triethylamine and formic acid (TEA-FA) for the enhancement of the evaporative light scattering detector response were established at 0.1% (v/v). Thus, the optimal conditions for the separation of the four glycosphingolipids was obtained with a gradient elution from a 100% chloroform to a 100% acetone:methanol (90:10 (v/v)) mobile phase at 0.2 ml min-1, using a 10% min-1 gradient slope. Finally, this method was applied to detect the excess of one of the neutral sphingolipids, namely globotriaosylceramide (Gb3) in the urine of patients affected with Fabry disease. A liquid-liquid extraction of the sediments obtained from an aliquot of only ten ml of urine proved sufficient to detect the excess of Gb3 present in both hemizygote and heterozygote patients. In all, the ability of our method to detect abnormal amounts of Gb3 in urinary sediments could allow the diagnosis of weakly symptomatic Fabry patients in large screening programs     

Application of magnetic hydroxyapatite nanoparticles for solid phase extraction of plasmid DNA

We developed a facile method for plasmid DNA (pDNA) extraction from crude Escherichia coli lysate using magnetic hydroxyapatite nanoparticles (MHapNPs) in the presence of polyethylene glycol (PEG)/NaCl. DNA condensation induced by PEG/NaCl is a prerequisite for achieving pronounced DNA recovery. The quality and quantity of MHapNP-purified pDNA under optimal binding buffer conditions (0.5 volume of 20% PEG 8000/2M NaCl) were comparable to those obtained using organic solvents or commercial kits. This MHapNP technique is rapid, simple, cost-effective, and environmentally friendly and has the potential to extract DNA from other cell lysates.     

The use of bacterial minicells to transfer plasmid DNA to eukaryotic cells

The delivery of DNA to mammalian cells is of critical importance to the development of genetic vaccines, gene replacement therapies and gene silencing. For these applications, targeting, effective DNA transfer and vector safety are the major roadblocks in furthering development. In this report, we present a novel DNA delivery vehicle that makes use of protoplasted, achromosomal bacterial minicells. Transfer of plasmid DNA as measured by green fluorescent protein expression was found to occur in as high as 25% of cultured Cos-7 cells when a novel chimeric protein containing the D2-D5 region of invasin was expressed and displayed on the surface of protoplasted minicells. Based on endoplasmic reticulum stress and other responses, protoplasted minicells were non-toxic to recipient eukaryotic cells as a consequence of the transfection process. Taken together, these results suggest that bacterial minicells may represent a novel and promising gene delivery vehicle.     

Affinity purification of plasmid DNA by temperature-triggered precipitation

This protocol presents a new method to purify plasmid DNA using temperature-triggered precipitation. The principle is based on the specific DNA-binding affinity of a bacterial metalloregulatory (MerR) protein to its cognate DNA sequence and the temperature responsiveness of elastin-like protein (ELP). A bifunctional ELP-MerR fusion protein is created to enable the precipitation of plasmid DNA, designed to contain the MerR recognition sequence, by a simple temperature trigger. The protocol covers all stages of the process from the design of ELP-MerR fusion proteins and MerR-binding plasmids, to the isolation of plasmid DNA from Escherichia coli cultures after boiling lysis, the subsequent temperature-triggered precipitation of plasmid DNA-fusion protein complexes and final elution of plasmid DNA by mild heating. This protocol is well suited to laboratory research-scale applications, producing plasmid DNA of better purity and similar yield as one of the most commonly used laboratory methods, standard alkaline lysis (known as the midiprep procedure). The protocol takes approximately 30 min to obtain pure plasmid DNA from cell cultures using the temperature-triggered precipitation method.     

Affinity purification of plasmid DNA by temperature-triggered precipitation

This report describes a new plasmid DNA purification method, which takes advantage of the DNA-binding affinity and specificity of the bacterial metalloregulatory protein MerR, and of the temperature responsiveness of elastin-like proteins (ELPs). Upon increasing the temperature, ELP undergoes a reversible phase transition from water-soluble forms into aggregates, and this property was exploited for the precipitation of plasmid DNA containing the MerR recognition sequence by a simple temperature trigger. In one purification step, plasmid DNA was purified from E. coli cell lysates to a better purity than that prepared by a standard alkaline purification method, with no contaminating chromosomal DNA and cellular proteins. This protein-based approach, in combination with the reversible phase transition feature of ELP, makes the outlined method a promising candidate for large-scale purification of plasmid DNA for sensitive applications such as nonviral gene therapy or DNA vaccines.     

Purifying plasmid DNA from bacterial colonies using the QIAGEN Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.     

Thiophilic interaction chromatography for supercoiled plasmid DNA purification

Supercoiled plasmid DNA was selectively purified from its open circular form by thiophilic interaction chromatography, performed in the presence of high concentrations of water-structuring salts. To identify optimal conditions for purification, various aromatic thioether ligands were coupled to a chromatographic support and screened for their ability to separate plasmid isoforms from each other and from other host cell contaminants, including RNA, genomic DNA, protein, and endotoxins. Selectivity of the chromatographic medium depended on the structure of the ligands, with characteristics of the substituents on the aromatic ring determining the resolution between the different plasmid DNA isoforms. Optimal resolution was obtained with ligands consisting of an thioaromate, substituted with highly electronegative groups. When 2-mercaptopyridine was used as a ligand, the difference in conductivity for eluting open circular and supercoiled plasmid DNA is only 6 mS/cm. However, with 4-nitrothiophol the resolution for plasmid DNA separation on the media increased, resulting in a 20 mS/cm difference. When used in combination with a prior group separation step, these aromatic thioether ligands facilitated the isolation of highly purified supercoiled plasmid DNA, suitable for use in gene therapy and DNA vaccine applications.     

Direct capture of plasmid DNA from non-clarified bacterial lysate using polycation-grafted monoliths

Monolith columns from macroporous polyacrylamide gel were grafted with polycations, poly(N,N-dimethylaminoethyl methacrylate) (polyDMAEMA), (2-(methacryloyloxy)ethyl)-trimethyl ammonium chloride (polyMETA) and partially quaternized polyDMAEMA prepared via treating polyDMAEMA-grafted columns with propylbromide. The polymer grafting degrees varied between 34 and 110%. The polycation-grafted monolithic columns are able to capture plasmid DNA directly from alkaline lysate of Escherichia coli cells. Due to the large pore size in macroporous monoliths the particulate material present in non-clarified feeds did not block the columns. The captured plasmid DNA was eluted with 1M NaCl as particulate-free preparation with significantly reduced content of protein and RNA as compared to the applied lysate.     

Separation of plasmid DNA from protein and bacterial lipopolysaccharides using displacement chromatography

The preparation of plasmid DNA at large scale constitutes a pressing problem in bioseparation. This paper describes a first investigation of displacement chromatography as a means to separate plasmid DNA (4.7 kb) from E. coli lipopolysaccharides and protein (holo transferrin), respectively. Displacement chromatography has advantages in this regard, since the substance mixture is resolved into rectangular zones of the individual components rather than into peaks. Thus a higher total concentration can be maintained in the pooled product fractions. Hydroxyapatite (type I and II) and anion exchange stationary phases were included in the experiments. In addition to a conventional anion exchange column packed with porous particles, the recently introduced continuous bed UNOTM anion exchange column was investigated. No DNA purification was possible with either hydroxyapatite material. Conventional particle based columns in general were not suited to the separation of any two substances varying considerably in molecular mass, e.g. plasmid DNA and standard protein. Presumably, the direct competition for the binding sites, which is essential in displacement chromatography, was restricted by the size dependency of the accessible stationary phase surface area in this case. Better results were obtained with the continuous bed column, in which the adsorptive surface coincides with the walls of the flow through pores. As a result the accessible surface does not vary as much with the size of the interacting molecules as for the conventional stationary phase materials. Sharper transitions were also observed between substance zones recovered from the UNOTM column. The steric mass action model was used to aid method development in case of the anion exchange approach. While further research in obviously necessary, displacement chromatography on continuous bed columns has been shown to be capable of separating plasmid DNA from typical impurities. This revised version was published online in July 2006 with corrections to the Cover Date.     

A procedure for the isolation and purification of plasmid DNA from Rhizobium meliloti

A procedure is described for the isolation and purification of the DNA of plasmids that are indigenous to the agriculturally important nitrogen-fixing bacterium Rhizobium meliloti. The procedure involves the lysis of bacteria with an ionic detergent or a mixture of ionic and nonionic detergents, the extraction of total DNA from precipitated membrane-DNA complexes, the enrichment of supercoiled plasmid DNA by the selective alkaline denaturation of chromosomal DNA, and a further purification of plasmid DNA using cesium chloridepropidium diiodide gradients. This procedure yields pure plasmid DNA in amounts of 30 to 50 μg per liter of a culture of cell density of approximately one A550 unit. The DNA thus obtained has been found to be of sufficient purity to serve as substrate for the most commonly used restriction endonucleases.     

A simple procedure for large-scale purification of plasmid DNA

We report a simple, rapid and reliable procedure for large-scale purification of plasmid DNA from non-amplified bacterial cultures. It is a modification of the boiling method of Holmes and Quigley [Anal. Biochem. 114 (1981) 193-197] and involves gel-filtration chromatography using Sephacryl S-1000 for final purification of plasmid DNA. This method does not require CsCl gradients and the recovered plasmids are free of RNA and chromosomal DNA, are supercoiled, retain their biological activity, and are suitable for restriction analysis.     

Impact of lysine-affinity chromatography on supercoiled plasmid DNA purification

Gene therapy and DNA vaccination cover a variety of applications using viral and non-viral vectors as vehicles of choice for treatment of genetic or acquired diseases. Recently, most therapeutic applications have been performed with non-viral biological agents preparations highly enriched in supercoiled plasmid molecules and it has been concluded that this isoform is more efficient at gene transfection than open circular isoform. This work describes for the first time a new strategy that uses lysine-chromatography to efficiently eliminate Escherichia coli impurities as well as other ineffective plasmid isoforms present in a complex clarified lysate to purify and obtain pharmaceutical-grade supercoiled plasmid DNA. The quality control tests indicated that the levels of impurities in the final plasmid product were below the generally accepted specifications. Furthermore, the delivery of the purified product to eukaryotic cells, the cell uptake and transfection efficiency were also analyzed. The results showed that the transfection efficiency reached with the application of the supercoiled plasmid conformation, purified with lysine-agarose, was higher than the values achieved for other plasmid topologies. Therefore, this study presents a new enabling technology to obtain the completely purified non-viral vector, able to act with good efficiency as gene therapy delivery vehicle in several diseases like cancer.