A Rapid and Economic In-House DNA Purification Method Using Glass Syringe Filters

Background

Purity, yield, speed and cost are important considerations in plasmid purification, but it is difficult to achieve all of these at the same time. Currently, there are many protocols and kits for DNA purification, however none maximize all four considerations.

Methodology/Principal Findings

We now describe a fast, efficient and economic in-house protocol for plasmid preparation using glass syringe filters. Plasmid yield and quality as determined by enzyme digestion and transfection efficiency were equivalent to the expensive commercial kits. Importantly, the time required for purification was much less than that required using a commercial kit.

Conclusions/Significance

This method provides DNA yield and quality similar to that obtained with commercial kits, but is more rapid and less costly.     

Purification of plasmids by triplex affinity interaction.

Production of pharmaceutical grade plasmid DNA is an important issue in gene therapy. We developed a method for affinity purification of plasmids by triple helix interaction. This method is based on sequence-specific binding of an oligonucleotide immobilized on a large pore chromatography support to a target sequence on the plasmid. Using design criteria derived from thermodynamic data, we produced a 15mer target sequence which binds strongly to the affinity support under mildly acidic conditions. Plasmid DNA was purified from clarified Escherichia coli lysate by incubation with the affinity beads at pH 5.0 and high NaCl concentration. After extensive washing of the beads, purified plasmid DNA was eluted with alkaline buffer. The purified plasmid showed no RNA or cell DNA contamination in HPLC analysis and total protein concentration was reduced considerably. Due to its mechanical stability and porosity this support can be used in a continuous affinity purification process, which has a high potential for scale up.     

Characterization of an oligonucleotide-binding fluorescent ligand for application in affinity purification of dsDNA

The fluorescent probe PO-PRO-3 was investigated as a potential ligand for the affinity immobilization and purification of genomic or plasmid DNA fragments. Affinities and mechanisms for PO-PRO-3 binding to superhelical and linearized pUC 18 plasmid DNA were examined through measurement of binding isotherms, continuous-variation analysis, and DNA titrations. In addition, the effects of DNA conformation, protein and RNA contaminants, solvent polarity, and ionic strength are examined with the aim of optimizing binding and elution conditions and of assisgning limits to the range of applicability of the affinity purification. (c) 1995 John Wiley & Sons, Inc.     

Characterization of the diatomite binding domain in the ribosomal protein L2 from E. coli and functions as an affinity tag

The ribosomal protein L2, a constituent protein of the 50S large ribosomal subunit, can be used as Si-tag using silica particles for the immobilization and purification of recombinant proteins (Ikeda et al. (Protein Expr Purif 71:91–95, 2010); Taniguchi et al. (Biotechnol Bioeng 96:1023–1029, 2007)). We applied a diatomite powder, a sedimentary rock mainly composed with diatoms silica, as an affinity solid phase and small ubiquitin-like modifier (SUMO) technology to release a target protein from the solid phase. The L2 (203–273) was the sufficient region for the adsorption of ribosomal protein L2 on diatomite. We comparatively analyzed the different adsorption properties of the two deleted proteins of L2 (L2 (1–60, 203–273) and L2 (203–273)) on diatomite. The time required to reach adsorption equilibrium of L2 (203–273) fusion protein on diatomite was shorter than that of L2 (1–60, 203–273) fusion protein. The maximum adsorption capacity of L2 (203–273) fusion protein was larger than that of L2 (1–60, 203–273) fusion protein. In order to study whether the L2 (203–273) can function as an affinity purification tag, SUMO was introduced as one specific protease cleavage site between the target protein and the purification tags. The L2 (203–273) and SUMO fusion protein purification method was tested using enhanced green fluorescent protein as a model protein; the result shows that the purification performance of this affinity purification method was good. The strong adsorption characteristic of L2 (203–273) on diatomite also provides a potential protein fusion tag for the immobilization of enzyme.     

Rapid purification of Ti plasmids from Agrobacterium by ethidium bromide treatment and phenol extraction

An efficient method is described for the purification of Ti plasmid DNA from Agro-bacterium. The procedure is based on the relative binding capacity of ethidium bromide to supercoiled plasmid DNA and linear DNA and on the high solubility of ethidium bromide in phenol. Following treatment with ethidium bromide, more than 87% of linear chromosomal DNA and most of the RNA was present in the phenol phase, while 91% of Ti plasmid DNA was recovered from the aqueous phase. The Ti plasmid DNA was sufficiently pure for restriction endonuclease analysis and cloning. The procedure is simple, fast and provides eight times higher yield than the standard isopycnic ultracentrifugation method.     

Interaction of Human Replication Protein A with DNA Duplexes Containing Gaps of Varying Size

Replication protein A (RPA) is a heterotrimeric protein that has high affinity for single-stranded (ss) DNA and is involved in DNA replication, repair, and recombination in eukaryotic cells. Photoaffinity modification was employed in studying the interaction of human RPA with DNA duplexes containing various gaps, which are similar to structures arising during DNA replication and repair. A photoreactive dUMP derivative was added to the 3" end of a gap-flanking oligonucleotide with DNA polymerase , and an oligonucleotide containing a 5"-photoreactive group was chemically synthesized. The 5" end predominantly interacted with the large RPA subunit (p70) regardless of the gap size, whereas interactions of the 3" end with the RPA subunits depended both on the gap size and on the RPA concentration. Subunit p32 was mostly labeled in the case of a larger gap and a lower RPA concentration. The results confirmed the model of polar RPA–DNA interaction, which has been advanced earlier.     

In situ DNA-RNA hybridization using in vivo bromodeoxyuridine-labeled DNA probe

Summary An in vivo 5-bromodeoxyuridine (BrdUrd) labeled DNA probe was used for in situ DNA-RNA hybridization. BrdUrd was incorporated into plasmid DNA by inoculating E. coli with Luria-Bertani (LB) culture medium containing 500 mg/L of BrdUrd. After purification of the plasmid DNA, specific probes of the defined DNA fragments, which contained the cloned insert and short stretches of the vector DNA, were generated by restriction endonuclease. The enzymatic digestion pattern of the BrdUrd-labeled plasmid DNA was the same as that of the non-labeled one. BrdUrd was incorporated in 15%–20% of the total DNA, that is, about 80% of the thymidine was replaced by BrdUrd. Picogram amounts of the BrdUrd-labeled DNA probe itself and the target DNA were detectable on nitrocellulose filters in dot-blot spot and hybridization experiments using a peroxidase/diaminobenzidine combination. The BrdUrd-labeled DNA probe was efficiently hybridized with both single stranded DNA on nitrocellulose filters and cellular mRNA in in situ hybridization experiments. Through the reaction with BrdUrd in single stranded tails, hybridized probes were clearly detectable with fluorescent microscopy using a FITC-conjugated monoclonal anti-BrdUrd antibody. The in vivo labeling method did not require nick translation steps or in vitro DNA polymerase reactions. Sensitive, stable and efficient DNA probes were easily obtainable with this method.     

Isolation and separation of α-amylase inhibitors I-1 and I-2 from seeds of ragi (Indian finger millet, Eleusine coracana) by metal chelate affinity precipitation

The concept of immobilized metal affinity chromatography (IMAC) was integrated with affinity precipitation for the single step isolation of -amylase inhibitors I-1 and 1-2 from the seeds of ragi (Indian finger millet, Eleusine coracana). -Amylase inhibitor I-1 was purified 13-fold with a yield of 84%, using Cu(II) loaded thermosensitive metal chelate copolymer of N-isopropylacrylamide (NIPAM) and 1-vinyl imidazole (VI). The protein also showed trypsin inhibitory activity. The binding of the protein to the copolymer was strongly pH dependent. -Amylase inhibitor I-2 was recovered in the supernatant as unprecipitated protein with significant purification and constituted 27% of the total inhibitor power. The yield with respect to inhibitor I-2 was around 85%. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed significant purification of inhibitor I-1 and indicated evident separation of the two proteins on metal chelate affinity precipitation.     

DNA purification by triple-helix affinity precipitation

Recent advances in DNA-based medicine (gene therapy, genetic vaccination) have intensified the necessity for pharmaceutical-grade plasmid DNA purification at comparatively large scales. In this contribution triple-helix affinity precipitation is introduced for this purpose. A short, single-stranded oligonucleotide sequence (namely (CTT)(7)), which is capable of recognizing a complementary sequence in the double-stranded target (plasmid) DNA, is linked to a thermoresponsive N-isopropylacrylamide oligomer to form a so-called affinity macroligand (AML). At 4 degrees C, i.e., below its critical solution temperature, the AML binds specifically to the target molecule in solution; by raising the temperature to 40 degrees C, i.e., beyond the critical solution temperature of the AML, the complex can be precipitated quantitatively. After redissolution of the complex at lower temperature, the target DNA can be released by a pH shift to slightly alkaline conditions (pH 9.0). Yields of highly pure (plasmid) DNA were routinely between 70% and 90%. Non-specific co- precipitation of either the target molecule by the non-activated AML precursor or of contaminants by the AML were below 7% and presumably due to physical entrapment of these molecules in the wet precipitate. Ligand efficiencies were at least 1 order of magnitude higher than in triple-helix affinity chromatography.     

Purification of DNA from agarose gels

Summary We describe a rapid and easily reproducible modification of the freeze-squeeze method of separating DNA from agarose gels. Our method involves slicing out the agarose gel portion which contains the DNA of interest, freezing this gel slice at –20°C, then centrifuging the frozen slice in a filtration unit which contains a cellulose acetate filter. The agarose is retained on the filter and the filtrate contains the DNA. DNA purified in this manner could be completely digested with restriction endonucleases and completely ligated with DNA ligase, without further purification. The percentages of recovery for various sizes of linear and plasmid double-stranded DNA ranged from 57 to 69%. The procedure takes less than 30 minutes to perform.     

Optimization of fermentation conditions for an <Emphasis Type="Italic">Escherichia coli</Emphasis> strain engineered using the response surface method to produce a novel therapeutic DNA vaccine for rheumatoid arthritis

Background

Fermentation condition optimization and nutrients screening are of equal importance for efficient production of plasmid DNA vaccines. This directly affects the downstream purification and final quality and yield of plasmid DNA vaccines. The present study aimed to optimize the fermentation conditions for high-throughput production of therapeutic DNA vaccine pcDNA-CCOL2A1 by engineered Escherichia coli DH5α, using the response surface method (RSM).

Results

We hypothesized that optimized fermentation conditions significantly increase the yield of pcDNA-CCOL2A1 therapeutic DNA vaccine, a novel DNA vaccine for treating rheumatoid arthritis (RA). Single-factor analysis was performed to evaluate the optimal basal culture medium from LB, 2?×?YT, TB, M9 (Glycerol) and M9 (Glucose), respectively. Thereafter, the Plackett-Burman design (PBD) was used to ascertain the three most significant factors affecting the vaccine yields, followed by the paths of steepest ascent to move to the nearest region of maximum response. Initial screening through the PBD revealed that the most key factors were peptone, mannitol, and inoculum concentration. Subsequent use of RSM was further optimized for the production of therapeutic DNA vaccine pcDNA-CCOL2A1 through Box-Behnken design (BBD). The final optimized fermentation conditions were as follows: peptone, 25.86 g/L; mannitol, 8.08 g/L; inoculum concentration, OD?=?0.36. Using this statistical experimental design, the yield of therapeutic DNA vaccine pcDNA-CCOL2A1 markedly increased from 223.37 mg/L to339.32 mg/L under optimal conditions, and a 51.9% increase was observed compared with the original medium.

Conclusions

The present results provide a basis for further production of high-quality and high-yield therapeutic DNA vaccine pcDNA-CCOL2A1 in pilot-scale and even industrial-scale.

     

Polyclonal Antibodies in Microplates to Predict the Maximum Adsorption Activities of Enzyme/Mutants from Cell Lysates

With microplate-immobilized polyclonal antibodies against a starting enzyme or its active mutant bearing consistent accessible epitopes, the maximum activity of an adsorbed enzyme/mutant (Vs) was predicted for comparison to recognize weakly-positive mutants. Rabbit antisera against Escherichia coli alkaline phosphatase (ECAP) were fractionated with 33% ammonium sulfate to yield crude polyclonal antibodies for conventional immobilization in 96-well microplates. The response curve of the activities of ECAP/mutant adsorbed by the immobilized polyclonal antibodies to protein quantities from a cell lysate was fit to an approximation model to predict Vs. With 0.4 μg crude polyclonal antibody for immobilization, Vs was consistent for ECAP in cell lysates bearing fourfold differences in its apparent specific activities when its abundance was greater than 0.9%. The ratio of Vs of the mutant R168K to that of ECAP was 1.5?±?0.1 (n?=?2), consistent with that of their specific activities after affinity purification. Unfortunately, the prediction of Vs with polyclonal antibodies that saturated microplate wells was ineffective to Pseudomonas aeruginosa arylsulfatase bearing less than 2% specific activity of ECAP. Therefore, with microplate-immobilized polyclonal antibodies to adsorb enzyme/mutants from cell lysates, high-throughput prediction of Vs was practical to recognize weakly-positive mutants of starting enzymes bearing fairly-high activities.     

DNA Microarray for Rapid Detection of Mitochondrial DNA Haplotypes of Chum Salmon

For use in genetic stock identification, we developed an oligonucleotide (DNA) microarray hybridization method for rapid and accurate detection of nucleotide sequence variations in 20 previously identified variable nucleotide sites in about 500 bp within the 5 half of the control region of mitochondrial DNA of chum salmon (Oncorhynchus keta). The method includes immobilization of synthesized oligonucleotides containing respective polymorphic sites on a glass slide precoated with polycarbodiimide resin, a 2-hour hybridization with DNA microarray of biotinylated polymerase chain reaction fragments spanning the 5 variable portion followed by short washing, and visualization of hybridization signals by conventional ABC method and scanner-assisted computation of signal intensity on a computer. The entire process of hybridization and detection was completed within 4 hours. The resulting DNA microarray could detect all of the single nucleotide mutations and therefore could be used to identity the sequence variations defining 30 mtDNA haplotypes of chum salmon as revealed previously by nucleotide sequence analysis.     

Oligonucleotide directed mutagenesis: Selection of mutants by hemimethylation of GATC-sequences

Summary We have developed a selection procedure for mutants obtained by oligonucleotide directed mutagenesis based on asymmetrical A-methylation of GATC-sequences in the duplex DNA. The method involves the construction of gapped duplexes of circular single-stranded phage DNA. An oligonucleotide, complementary to part of the gap except for a single mismatch, is hybridized to the gapped duplex DNA and the remaining single stranded regions are filled-in enzymatically. When the template is undermethylated, the yield of mutants is almost, solely dependent on the priming efficiency of the oligonucleotide. The approach was used to introduce an ATCG transversion in the nut L region of phage . Under optimal conditions, about 50–60% of the transformants were of the mutant genotype. Although situated adjacent to a known nut L mutation, the present mutation was phenotypically silent. The possibility of screening for mutants by means of a coupled, easily detectable marker was also investigated.Abbreviations bp base pairs - RF replicative form - ssDNA single stranded DNA - Ap gene carbenicillin resistance gene - EtBr ethidium bromide - O.D. optical density - Kb kilobases - P_L major leftward promoter of phage      

The harnessing of peptide–monolith constructs for single step plasmid DNA purification

The availability of synthetic peptides has paved the way for their use in tailor-made interactions with biomolecules. In this study, a 16mer LacI-based peptide was used as an affinity ligand to examine the scale up feasibility for plasmid DNA purification. First, the peptide was designed and characterized for the affinity purification of lacO containing plasmid DNA, to be employed as a high affinity ligand for the potential capturing of plasmid DNA in a single unit operation. It was found there were no discernible interactions with a control plasmid that did not encode the lacO nucleotide sequence. The dissociation equilibrium constant of the binding between the 16mer peptide and target pUC19 was 5.0 ± 0.5 × 10⁻⁸ M as assessed by surface plasmon resonance. This selectivity and moderated affinity indicate that the 16mer is suitable for the adsorption and chromatographic purification of plasmid DNA. The suitability of this peptide was then evaluated using a chromatography system with the 16mer peptide immobilized to a customized monolith to purify plasmid DNA, obtaining preferential purification of supercoiled pUC19. The results demonstrate the applicability of peptide–monolith supports to scale up the purification process for plasmid DNA using designed ligands via a biomimetic approach.     

Incision of trivalent chromium [Cr(III)]-induced DNA damage by Bacillus caldotenax UvrABC endonuclease

Some hexavalent chromium [Cr(VI)]-containing compounds are lung carcinogens. Once within cells, Cr(VI) is reduced to trivalent chromium [Cr(III)] which displays an affinity for both DNA bases and the phosphate backbone. A diverse array of genetic lesions is produced by Cr including Cr–DNA monoadducts, DNA interstrand crosslinks (ICLs), DNA–Cr–protein crosslinks (DPCs), abasic sites, DNA strand breaks and oxidized bases. Despite the large amount of information available on the genotoxicity of Cr, little is known regarding the molecular mechanisms involved in the removal of these lesions from damaged DNA. Recent work indicates that nucleotide excision repair (NER) is involved in the processing of Cr–DNA adducts in human and rodent cells. In order to better understand this process at the molecular level and begin to identify the Cr–DNA adducts processed by NER, the incision of CrCl₃ [Cr(III)]-damaged plasmid DNA was studied using a thermal-resistant UvrABC NER endonuclease from Bacillus caldotenax (Bca). Treatment of plasmid DNA with Cr(III) (as CrCl₃) increased DNA binding as a function of dose. For example, at a Cr(III) concentration of 1 μM we observed 2 Cr(III)–DNA adducts per plasmid. At this same concentration of Cr(III) we found that 17% of the plasmid DNA contained ICLs (0.2 ICLs/plasmid). When plasmid DNA treated with Cr(III) (1 μM) was incubated with Bca UvrABC we observed 0.8 incisions/plasmid. The formation of endonuclease IV-sensitive abasic lesions or Fpg-sensitive oxidized DNA bases was not detected suggesting that the incision of Cr(III)-damaged plasmid DNA by UvrABC was not related to the generation of oxidized DNA damage. Taken together, our data suggest that a sub-fraction of Cr(III)–DNA adducts is recognized and processed by the prokaryotic NER machinery and that ICLs are not necessarily the sole lesions generated by Cr(III) that are substrates for NER.     

Oligonucleotide based magnetic bead capture of Onchocerca volvulus DNA for PCR pool screening of vector black flies

Background

Entomological surveys of Simulium vectors are an important component in the criteria used to determine if Onchocerca volvulus transmission has been interrupted and if focal elimination of the parasite has been achieved. However, because infection in the vector population is quite rare in areas where control has succeeded, large numbers of flies need to be examined to certify transmission interruption. Currently, this is accomplished through PCR pool screening of large numbers of flies. The efficiency of this process is limited by the size of the pools that may be screened, which is in turn determined by the constraints imposed by the biochemistry of the assay. The current method of DNA purification from pools of vector black flies relies upon silica adsorption. This method can be applied to screen pools containing a maximum of 50 individuals (from the Latin American vectors) or 100 individuals (from the African vectors).

Methodology/Principal Findings

We have evaluated an alternative method of DNA purification for pool screening of black flies which relies upon oligonucleotide capture of Onchocerca volvulus genomic DNA from homogenates prepared from pools of Latin American and African vectors. The oligonucleotide capture assay was shown to reliably detect one O. volvulus infective larva in pools containing 200 African or Latin American flies, representing a two-four fold improvement over the conventional assay. The capture assay requires an equivalent amount of technical time to conduct as the conventional assay, resulting in a two-four fold reduction in labor costs per insect assayed and reduces reagent costs to $3.81 per pool of 200 flies, or less than $0.02 per insect assayed.

Conclusions/Significance

The oligonucleotide capture assay represents a substantial improvement in the procedure used to detect parasite prevalence in the vector population, a major metric employed in the process of certifying the elimination of onchocerciasis.     

Monitoring the (photo)genotoxicity of photosensitizer drugs: direct quantitation of single-strand breaks in deoxyribonucleic acid using an oligonucleotide chip

Oligonucleotide chip-based assays can be a sample-thrifty, time-saving, routine tool for evaluation of chemical-induced DNA strand breaks. This article describes a novel approach using an oligonucleotide chip to determine photosensitizer-induced DNA single-strand breaks. Surface coverage of fluorophore-labeled oligonucleotides on silicon dioxide chip surfaces was determined on alkaline phosphatase digestion. Fluorescence maxima (at 520 nm) of the solutions were converted to molar concentrations of the fluorescein-modified oligonucleotide by interpolation from a predetermined standard linear calibration curve. The photosensitizing activity of chlorpromazine and triflupromazine toward DNA single-strand breaks was then studied at different drug doses and also as a function of photoirradiation time. Photoinduced single-strand breaks calculated using the method described here agreed with values predicted by theoretical extrapolation of the single-strand breaks obtained for plasmid DNAs from agarose gel electrophoresis, and thereby indirectly validated the chip-based assays. Under UV irradiation (93.6 kJ/m²) chlorpromazine (0.08 mM) was found to have significant photogenotoxicity. However, triflupromazine did not exhibit any (photo)genotoxicity over the concentration range studied (0.04–0.20 mM). The method developed will be useful for quantitative screening of drug genotoxicity in terms of induction of breaks in DNA.