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.     

重组质粒pUDK-HGF 的中试纯化工艺

pUDK-HGF是携带人肝细胞生长因子的裸质粒,目前已进入I期临床试验,因此需要大量符合药学规格的质粒DNA。文中建立了pUDK-HGF中试规模纯化制备的新工艺。流程包括：发酵、离心收获菌体、碱裂解、超滤浓缩碱裂解液、Sephacryl S-1000层析除去RNA并更换缓冲液、plasmidselect捕获超螺旋质粒DNA、琼脂糖凝胶6BFF除盐。新工艺可获得浓度为2.0 mg/mL、纯度在1.70以上的裸质粒原液,符合相关质量标准,并避免使用动物源性的酶及有毒试剂。     

Purification of supercoiled plasmid DNA using chromatographic processes

The interest in purifying injectable-grade plasmid DNA has increased with the development of gene therapy and DNA vaccination technologies. In this paper we develop a method for purifying a 4.8 kb plasmid based on chromatographic processes. An NaCl gradient was optimized on a Q Sepharose column and plasmid was eluted at 800-820 mM NaCl in a broad peak. Supercoiled plasmid was isolated after a final Sepharcryl S1000 SF gel filtration step. Final plasmid preparation was depleted of proteins and RNA, as revealed by the BCA assay and 1% agarose gel electrophoresis.     

Hot spot for Tn1000 insertions in cloned repressor gene of the L phage

Insertions of Tn1000 into a cloned fragment of the L-phage genome comprising the repressor gene were prepared. Repressor activities produced by plasmids with insertions were assayed in vivo. As a result, the repressor gene was localized within 0.5 kb near one end of the cloned fragment. Transposon insertions were nonrandomly clustered within the repressor gene and in its close vicinity. An analysis of supercoiled plasmid DNA with the nuclease S1 revealed no distortion of the secondary structure of DNA in this region.     

Production of lentiviral vectors in suspension cells using low proportion of supercoiled circular plasmid DNA

The supercoiled circular (SC) topology form of plasmid DNA has been regarded to be advantageous over open circular or linearized analogue in transfection and expression efficiency, and therefore are largely demanded in the biopharmaceutical manufacturing. However, production of high-purity SC plasmid DNA would result in high manufacturing cost. The effect of SC proportion in plasmid DNA on the quality of packaged lentiviral vectors has never been reported. In this study, we established an efficient system for production of high-titer lentiviral vectors using suspension HEK293SF cells in serum-free media, and the lentiviral titer was not associated with the proportion of SC plasmid DNA. Plasmids DNA with different proportion of SC, open-circular, and linearized forms were prepared using the thermal denaturation method, and were transfected to adherent HEK293T or suspension HEK293SF cells for packaging of lentiviral vectors. The titer of lentiviral vectors from HEK293T cells, but not from HEK293SF cells, was significantly impaired when the proportion of SC plasmid DNA decreased from 60–80% to 30–40%. Further decrease of SC plasmid proportion to 3% led to a dramatic reduction of lentiviral titer no matter the packaging cell line was. However, lentiviral vectors from HEK293SF cells still showed a high titer even when the proportion of SC plasmid DNA was 3%. This study demonstrated that extremely high proportion of SC plasmid DNA was not required for packaging of high-titer lentiviral vector in HEK293SF cells, at least under our manufacturing process.     

Hot spot for Tn1000 insertions in cloned reprossor gene of the L phage

Insertions of Tn1000 into a cloned fragment of the L-phage genome comprising the repressor gene were prepared. Repressor activities produced by plasmids with insertions were assayed in vivo. As a result, the repressor gene was localized within 0.5 kb near one end of the cloned fragment. Transposon insertions were nonrandomly clustered within the repressor gene and in its close vicinity. An analysis of supercoiled plasmid DNA with the nuclease S1 revealed no distortion of the secondary structure of DNA in this region.     

Solid-phase assembly of DNA duplexes from synthetic oligonucleotides

A new method of rapid and efficient assembly of extended DNA duplexes in solid phase was developed. Subassemblies of separately annealed oligonucleotides were stepwise hybridized to each other on a solid support. Two types of supports with anchor oligonucleotide were tested: Fractosil-1000 with oligo-dT sequence and Sephacryl S-500 with an oligonucleotide bound via CNBr-activation procedure. Sephacryl S-500 turned out to be the support of choice since all enzymatic reactions of the assembly procedure (phosphorylation, ligation, restriction enzyme digestion) could be efficiently performed with DNA immobilized on Sephacryl S-500 particles.     

Purification and characterization of UV endonucleases I and II from murine plasmacytoma cells

Three endonucleases from murine plasmacytoma cells that specifically nick DNA which was heavily irradiated with ultraviolet (UV) light were resolved by Sephacryl S-200 column chromatography. Two of these, UV endonucleases I and II, were purified extensively. UV endonuclease I appears to be a monomeric protein with a molecular mass of 43 kDa; UV endonuclease II has an S value of 2.9 S, with a corresponding molecular mass estimated at 28 kDa. Both enzymes act as a class I AP endonuclease, cleaving phosphodiester bonds via a beta-elimination mechanism, so as to form an unsaturated deoxyribose at the 3' terminus. Both have thymine glycol DNA glycosylase activity and their substrate specificities generally appear to be overlapping but not identical. UV endonuclease I acts on both supercoiled and relaxed DNAs, whereas II acts only on supercoiled DNA. Both enzymes are active in EDTA, but have different optima for salt, pH, and Triton X-100. Each enzyme is also present in cultured diploid human fibroblasts.     

Dynamics of cruciform extrusion in supercoiled DNA: use of a synthetic inverted repeat to study conformational populations.

An inverted repeat has been created in a plasmid by ligation of two 13 nucleotide synthetic oligonucleotides into the cloning vector pAT153. The resulting recombinant plasmid, pIRbke8, is hypersensitive to cleavage by the single-strand-specific nuclease S1, and to modification by the single-strand-selective reagent bromoacetaldehyde, when the plasmid is negatively supercoiled. The new inverted repeat is a stronger S1 site than those derived from pBR322, but, in contrast to the ColE1 and phi X174 RF inverted repeats, these repeats share a similar temperature dependence. The kinetics of EcoRI cleavage at the centre of the synthetic inverted repeat have been studied in supercoiled and linear molecules. It is found that in the supercoiled molecule this target is not refractory to EcoRI cleavage to an extent which is greater than the resolution of the experiment. We conclude that in this molecule the cruciform is in a dynamic equilibrium with the regular duplex, in which the cruciform constitutes a relatively small subpopulation of conformational species.     

A simple procedure to obtain plasmids suitable for transfecting mammalian cell lines

A simple procedure to obtain plasmid preparations, suitable for transfecting mammalian cell lines using a calcium phosphate co-precipitation technique, is described. The protocol is based on the purification of plasmid DNA by double gel-filtration chromatography on Sephacryl S-1000 and additional slight modifications to the original transfection procedure. The purity of plasmid preparation was verified by analytical methods. The resulting preparation efficiently transfected NIH-3T3 cells.The authors are with the National Center for Scientific Research, Molecular Biology Department, Biotechnology Branch, POB 6880, Havana, Cuba.     

Binding and purification of plasmid DNA using multi-layered carbon nanotubes

We propose a new method for the separation of nucleic acids using multi-layered carbon nanotubes (CNTs) as an adsorbent. According to agarose gel electrophoresis, oxidized water-stable CNTs adsorb certain forms of nucleic acids, such as high molecular weight RNA, chromosomal DNA, linear and denatured forms of plasmid DNA. However, CNTs do not adsorb supercoiled form of plasmid DNA. Nucleic acids bound to CNTs can be readily removed by centrifugation whereas supercoiled plasmid DNA remains in solution. Upon the addition of divalent metal ions supercoiled plasmid DNA forms relatively stable complexes with CNTs due to chelation. Thus, new details about association of nucleic acids with CNTs were revealed and stoichiometry of the complexes was estimated. Our results can be used for fine purification of supercoiled plasmid DNA for gene therapy applications as well as manipulation of nucleic acids for biosensor design.     

An altered DNA conformation detected by S1 nuclease occurs at specific regions in active chick globin chromatin

The single-stranded activity of S1-nuclease cleaves globin chromatin in red cell nuclei in specific regions. The cleavages are observed only in tissues in which the globin genes are active, and they "switch" to reflect the switching pattern of globin-gene expression in embryonic and adult red cells. The positions of the S1 cleavages in the beta- and alpha-globin chromatin correspond to the general region of known DNAase I-hypersensitive sites, but can be distinguished in detail. When DNA segments containing these regions are subcloned into pBR322 and the supercoiled molecules are treated with S1, similar sites are cleaved in the purified supercoiled (but not linear) recombinant plasmid DNA. However, the dominant S1 cutting sites are shifted in the plasmid vis-a-vis the chromatin. We believe that some aspect of DNA sequence is translated into an altered DNA structure in chromatin and that it is this altered structure that is recognized by s1 nuclease and possibly by certain chromosomal proteins. Several physical properties reflected in the S1 digestion of supercoiled plasmids suggest a mechanism for generating differences in daughter cells during development.     

Transformation ofStreptomyces avermitilis by plasmid DNA

Summary Polyethylene glycol (PEG) efficiently mediated the transformation ofStreptomyces avermitilis protoplasts by plasmid DNA to yield 10⁷ transformants per g of plasmid DNA. Under conditios in which the maximum transformation frequency was observed, the cotransformation frequency exceeded 10%. The number of transformants increased linearly with the amount of DNA and number ofS. avermitilis protoplasts. Relaxed and supercoiled, but not linear DNA transformed protoplasts efficiently. Dimethyl sulfoxide (DMSO)-mediated transformation of protoplasts was 1000-fold less efficient. PEG and, less efficiently, DMSO also mediated the transformation of whole cells ofS. avermitilis by DNA.     

Screening of l‐histidine‐based ligands to modify monolithic supports and selectively purify the supercoiled plasmid DNA isoform

The growing demand of pharmaceutical‐grade plasmid DNA (pDNA) suitable for biotherapeutic applications fostered the development of new purification strategies. The surface plasmon resonance technique was employed for a fast binding screening of l ‐histidine and its derivatives, 1‐benzyl‐l ‐histidine and 1‐methyl‐l ‐histidine, as potential ligands for the biorecognition of three plasmids with different sizes (6.05, 8.70, and 14 kbp). The binding analysis was performed with different isoforms of each plasmid (supercoiled, open circular, and linear) separately. The results revealed that the overall affinity of plasmids to l ‐histidine and its derivatives was high (K_D > 10⁻⁸ M), and the highest affinity was found for human papillomavirus 16 E6/E7 (K_D = 1.1 × 10⁻¹⁰ M and K_D = 3.34 × 10⁻¹⁰ M for open circular and linear plasmid isoforms, respectively). l ‐Histidine and 1‐benzyl‐l ‐histidine were immobilized on monolithic matrices. Chromatographic studies of l ‐histidine and 1‐benzyl‐l ‐histidine monoliths were also performed with the aforementioned samples. In general, the supercoiled isoform had strong interactions with both supports. The separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration in the eluent, in both supports, but a lower salt concentration was required in the 1‐benzyl‐l ‐histidine monolith because of stronger interactions promoted with pDNA. The efficiency of plasmid isoforms separation remained unchanged with flow rate variations. The binding capacity for pDNA achieved with the l ‐histidine monolith was 29‐fold higher than that obtained with conventional l ‐histidine agarose. Overall, the combination of either l ‐histidine or its derivatives with monolithic supports can be a promising strategy to purify the supercoiled isoform from different plasmids with suitable purity degree for pharmaceutical applications. Copyright © 2015 John Wiley & Sons, Ltd.     

Fractionation of DNA with Sephacryl S-1000

In this study the application of gel filtration for purification of heterogeneous DNA is described. The fractionation of partial restriction enzyme digests of bacterial chromosomal DNA on a Sephacryl S-1000 -column is easy and rapid. Simultaneously intact chromosomal DNA and low molecular weight substances are eliminated in the run. The method is also applicable to the purification of plasmid DNA, as has been previously reported (3). Thus we are able to get pure DNA with yields over 80%.     

Plasmid purification by phenol extraction from guanidinium thiocyanate solution: development of an automated protocol.

We have developed a novel plasmid isolation procedure and have adapted it for use on an automated nucleic acid extraction instrument. The protocol is based on the finding that phenol extraction of a 1 M guanidinium thiocyanate solution at pH 4.5 efficiently removes genomic DNA from the aqueous phase, while supercoiled plasmid DNA is retained in the aqueous phase. S1 nuclease digestion of the removed genomic DNA shows that it has been denatured, which presumably confers solubility in the organic phase. The complete automated protocol for plasmid isolation involves pretreatment of bacterial cells successively with lysozyme, RNase A, and proteinase K. Following these digestions, the solution is extracted twice with a phenol/chloroform/water mixture and once with chloroform. Purified plasmid is then collected by isopropanol precipitation. The purified plasmid is essentially free of genomic DNA, RNA, and protein and is a suitable substrate for DNA sequencing and other applications requiring highly pure supercoiled plasmid.     

A long polypyrimidine/polypurine tract induces an altered DNA conformation on the 3'' coding region of the adjacent myosin heavy chain gene.

A long (147 base pairs), natural A.T rich polypyrimidine/polypurine tract has been found 55 base pairs downstream of a chicken embryonic myosin heavy chain (MHC) gene. Analysis at the nucleotide level of nicks induced by S1 and Neurospora crassa nucleases indicate that this long interrupted polypyrimidine/polypurine tract exists in an alternate DNA structure in vitro at pH 4.5 and pH 7.5 in both supercoiled and linear plasmid DNA. The polypyrimidine/polypurine tract induces this alternate structure upon at least 200 base pairs of its 5' flanking DNA, and thus extends into the 3' coding and non-coding regions of the neighboring MHC gene. The different nicking patterns induced by the nucleases S1 and N. crassa on each strand of this alternate structure suggests that the polypyrimidine/polypurine tract may form heteronomous DNA. When this long polypyrimidine/polypurine tract is present in a supercoiled plasmid at low pH, a new and as yet undefined S1 hypersensitive DNA alteration was detected near the center of this tract.     

Interarm interaction of DNA cruciform forming at a short inverted repeat sequence

A novel interarm interaction of DNA cruciform forming at inverted repeat sequence was characterized using an S1 nuclease digestion, permanganate oxidation, and microscopic imaging. An inverted repeat consisting of 17 bp complementary sequences was isolated from the bluegill sunfish Lepomis macrochirus (Perciformes) and subcloned into the pUC19 plasmid, after which the supercoiled recombinant plasmid was subjected to enzymatic and chemical modification. In high salt conditions (200 mM NaCl, or 100-200 mM KCl), S1 nuclease cut supercoiled DNA at the center of palindromic symmetry, suggesting the formation of DNA cruciform. On the other hand, S1 nuclease in the presence of 150 mM NaCl or less cleaved mainly the 3'-half of the repeat, thereby forming an unusual structure in which the 3'-half of the inverted repeat, but not the 5'-half, was retained as an unpaired strand. Permanganate oxidation profiles also supported the presence of single-stranded part in the 3'-half of the inverted repeat in addition to the center of the symmetry. Both electron microscopy and atomic force microscopy have detected a thick protrusion on the supercoiled DNA harboring the inverted repeat. We hypothesize that the cruciform hairpins at conditions favoring triplex formation adopt a parallel side-by-side orientation of the arms allowing the interaction between them supposedly stabilized by hydrogen bonding of base triads.     

A novel chromatographic procedure for purification of bacterial plasmids

A new, rapid procedure for purifying bacterial plasmids with high recovery is described. The sequence of operations consists essentially of treatment with alkali, ribonuclease, and proteinase K, followed by chisam extraction and gel filtration on Sephacryl S-1000, and finally a precipitation step using isopropanol at room temperature. The method gives rather good yields of plasmid DNA of high purity, and lends itself to scaling up.     

Partial characterization of nucleoids and nucleoid-plasmid complexes from Salmonella typhimurium.

Nucleoids from Salmonella typhimurium strain LT2 consist of supercoiled deoxyribonucleic acid structures that are ribonuclease labile sedimenting at 1,700S. More than 90% of the covalently closed circular deoxyribonucleic acid of a cryptic plasmid harbored by this strain cosediments with the host's 1,700S nucleoids.