Generation of chromosomal DNA during alkaline lysis and removal by reverse micellar extraction

The separation of structurally related impurities from pharmaceutical plasmid DNA by highly scalable purification techniques is a challenge for biochemical engineering. Next to RNA, proteins, and lipopolysaccharides, the chromosomal DNA of the plasmid replicating host has to be removed. Here, we describe the application of reverse micellar extraction for the separation of chromosomal from plasmid DNA. By applying different procedures for alkaline lysis, bacterial lysates with different amounts of chromosomal DNA were generated. A reverse micellar extraction step enabled us to deplete the concentration of this impurity below the required level of 50 mg g⁻¹ of plasmid DNA with almost complete plasmid recovery.     

Concentrated,digestible DNA after hydroxylapatite chromatography with cetylpyridinium bromide precipitation

A method for the direct extraction of the DNA from the unfavorable phosphate eluant of hydroxylapatite chromatography is described. The DNA—reversibly precipitated with the cationic detergent cetylpyridinium bromide—can be subjected to further enzymatic manipulations within minutes. This method is applied to the rapid separation of pBR322 plasmid from the chromosomal DNA.     

Purification and separation of various plasmid forms by exclusion chromatography

A chromatographic method for the rapid isolation of preparative amounts of plasmid DNA without the use of cesium chloride centrifugation is described. The protocol uses the alkaline extraction procedure and an exclusion column of Fractogel TSK 75S. From a clear lysate it is possible to obtain plasmid DNA completely free of proteins, RNA, and chromosomal DNA. From partially purified plasmid the procedure allows the separation of the different forms. This technique was successfully applied to different plasmids ranging in size from 2.9 to 17.5 MDa. It is a preparative method yielding easily 500 micrograms of pBR322 from 1 liter of amplified culture. The plasmid is suitable for topoisomerase I, topoisomerase II, and EcoRI assays.     

One-step separation of IgG subclasses of DNA hydrolyzing autoantibodies: a study on sera of patients with systemic lupus erythematosus and primary antiphospholipid syndrome by histidine ligand affinity chromatography

A method based on histidine ligand affinity chromatography has been utilized for the separation of DNA hydrolyzing autoantibodies from sera of patients suffering from systemic lupus erythematosus and primary antiphospholipid syndrome using the gel, histidyl-aminohexyl-sepharose. The separation of autoantibodies was carried out under mild chromatographic conditions. Human IgG subclass distribution in the different fractions separated on the column was studied by enzyme-linked immunosorbent assay. The purified DNA hydrolyzing autoantibodies were shown to hydrolyze plasmid DNA.     

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.     

A rapid electrophoretic method for the measurement of plasmid copy number

A rapid and inexpensive method for the measurement of copy number of small plasmids, ranging from 8.7 to 13 kb, in under 1 ml of liquid culture is described. The method involves whole cell lysis, electrophoretic separation of plasmid and chromosomal DNA followed by relative densitometric measurement of each, to give an estimation of the plasmid copies per chromosome. Results can be obtained in under 8 h and the method proved to be reproducible, fast and ideal for processing large numbers of samples from batch or continuous culture.     

Natural Transformation of Acinetobacter calcoaceticus by Plasmid DNA Adsorbed on Sand and Groundwater Aquifer Material

It is known that plasmid DNA and linear duplex DNA molecules adsorb to chemically purified mineral grains of sand and to particles of several clay fractions. It seemed desirable to examine whether plasmid DNA would also adsorb to nonpurified mineral materials taken from the environment and, particularly, whether adsorbed plasmid DNA would be available for natural transformation of bacteria. Therefore, microcosms consisting of chemically pure sea sand plus buffered CaCl₂ solution were compared with microcosms consisting of material sampled directly from a groundwater aquifer (GWA) plus groundwater (GW) with respect to the natural transformation of Acinetobacter calcoaceticus by mineral-associated DNA. The GWA minerals were mostly sand with inorganic precipitates and organic material plus minor quantities of silt and clay (illite and kaolinite). The amount of plasmid DNA which adsorbed to GWA (in GW) was about 80% of the amount which adsorbed to purified sand (in buffered CaCl₂ solution). Plasmid DNA adsorbed on sand transformed A. calcoaceticus significantly less efficiently than did plasmid DNA in solution. In contrast, the transformation by sand-adsorbed chromosomal DNA was as high as that by DNA in solution. In GWA/GW microcosms, the efficiency of transformation by chromosomal DNA was similar to that in sand microcosms, whereas plasmid transformation was not detectable. However, plasmid transformants were found at a low frequency when GWA was loaded with both chromosomal and plasmid DNA. Reasons for the low transformation efficiency of plasmid DNA adsorbed to mineral surfaces are discussed. Control experiments showed that the amounts of plasmid and chromosomal DNA desorbing from sand during incubation with a cell-free filtrate of a competent cell suspension did not greatly contribute to transformation in sand microcosms, suggesting that transformation occurred by direct uptake of DNA from the mineral surfaces. Taken together, the observations suggest that plasmid DNA and chromosomal DNA fragments which are adsorbed on mineral surfaces in a sedimentary or soil habitat may be available (although with different efficiencies for the two DNA species) for transformation of a naturally competent gram-negative soil bacterium.     

A rapid alkaline extraction procedure for screening recombinant plasmid DNA.

A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.     

Polyethyleneglycol-induced transformation of Bacillus subtilis protoplasts by bacterial chromosomal DNA

Summary Bacillus subtilis protoplasts, which in the presence of polyethyleneglycol (PEG) are transformed by plasmid DNA (Chang and Cohen 1979) can also be transformed under these conditions by chromosomal DNA. Transformation in this case occurs at a much lower frequency, not fully accounted for by the heterogeneity of this DNA. Another unexpected feature of the transformation studied, which may explain why it previously went unnoticed, is that DNA concentrations higher than 1–2 g/ml decrease the yield of transformants, without showing signs of general toxicity.PEG-induced protoplasts (PIP) transformation for chromosomal markers operates normally with protoplasts prepared from a non-transformable bacterial mutant. The evidence indicates that both native linear and plasmid DNAs must somehow be forced into the cells as a result of PEG action. Denatured chromosomal DNA however is almost inactive in PIP transformation. No competition between chromosomal and plasmid DNAs could be detected, when the DNA tested as inhibitor was in tenfold excess.     

Fate of cloned bacteriophage T4 DNA after phage T4 infection of clone-bearing cells

Plasmid pBR322 replication is inhibited after bacteriophage T4 infection. If no T4 DNA had been cloned into this plasmid vector, the kinetics of inhibition are similar to those observed for the inhibition of Escherichia coli chromosomal DNA. However, if T4 DNA has been cloned into pBR322, plasmid DNA synthesis is initially inhibited but then resumes approximately at the time that phage DNA replication begins. The T4 insert-dependent synthesis of pBR322 DNA is not observed if the infecting phage are deleted for the T4 DNA cloned in the plasmid. Thus, this T4 homology-dependent synthesis of plasmid DNA probably reflects recombination between plasmids and infecting phage genomes. However, this recombination-dependent synthesis of pBR322 DNA does not require the T4 gene 46 product, which is essential for T4 generalized recombination. The effect of T4 infection on the degradation of plasmid DNA is also examined. Plasmid DNA degradation, like E. coli chromosomal DNA degradation, occurs in wild-type and denB mutant infections. However, neither plasmid or chromosomal degradation can be detected in denA mutant infections by the method of DNA--DNA hybridization on nitrocellulose filters.     

Cloning, sequencing and expression of the xylanase gene from a Bacillus subtilis strain B10 in Escherichia coli

Bacillus subtilis strain B10 was isolated for degumming of ramie blast fibers, and a fragment of 642-bp was amplified from chromosomal DNA by using primers directed against the sequence of Bacillus subtilis xylanase gene given in GenBank. The positive clones were screened on the selected LB agar plates supplemented with xylan by Congo-red staining method. The recombinant plasmid from one positive clone was used for further analysis and DNA sequencing. The gene sequence is different from the reported xylanase gene sequence in sites of two base pairs. The recombinant plasmid was expressed in Escherichia coli, and xylanase activity was measured. The xylanase distribution in extracellular, intracellular and periplasmic fractions were about 22.4%, 28.0% and 49.6%, respectively. The xylanase had optimal activity at pH 6.0 and 50 degrees C.     

Rapid preparation of total nucleic acids from E. coli for multi-purpose applications

Separate protocols are commonly used to prepare plasmid DNA, chromosomal DNA, or total RNA from E. coli cells. Various methods for the rapid preparation of plasmid DNA have been developed previously, but the preparation of the chromosomal DNA and total RNA are usually laborious. We report here a simple, fast, reliable, and cost-effective method to extract total nucleic acids from E. coli by direct lysis of the cells with phenol. Five distinct and sharp bands, which correspond to chromosomal DNA, plasmid DNA, 23S rRNA, 16S rRNA, and a mixture of small RNA, were observed when analyzing the prepared total nucleic acids on a regular 1-2% agarose gel. The simple and high-quality preparation of the total nucleic acids in a single tube allowed us to rapidly screen the recombinant plasmid, as well as to simultaneously monitor the change of the plasmid copy number and rRNA levels during the growth of E. coli in the liquid medium.     

Preparation of plasmid DNA by sequential enzymatic digestion.

A new method for the preparation of plasmid DNA from Escherichia coli, sequential enzymatic digestion, is described. The method is based on sequential and selective enzymatic digestion of all components of E. coli except for the supercoiled plasmid DNA. The key enzymes are exonuclease I and exonuclease III that specifically hydrolyze linear chromosomal DNA and are unable to attack supercoiled plasmid DNA under controlled conditions. Isolated plasmid DNA can be sequenced and digested with restriction enzymes.     

Comparison of different methods of introducing a label into the DNA of microorganisms

Different methods of isotope introduction into plasmid and chromosomal DNA have been compared. The efficiency of label introduction into DNA was estimated by the results of DNA--DNA hybridisation and by the thermostability of hybrid duplexes. Using the enzymatic methods of label introduction levels of DNA specific radioactivity and label binding in homologous and heterologous reactions were close. DNA labeled by the method of enzymatic methylation forms more thermostable hybrid duplexes than "nick-translation" DNA. The advantages of enzymatic methods of label introduction for creating a microorganism reference DNA bank are discussed.     

Study of the influence of plasmids on the arbitrary primer polymerase chain reaction fingerprint of Escherichia coli strains

Abstract To study the effect of plasmids on the arbitrary primer-polymerase chain reaction fingerprint of bacterial strains, the Escherichia coli strains DH5, Top10, and W3110 were transformed with plasmids of different sizes: respectively, pUC19, pCEP and two clinically important plasmids carrying resistance to several antibiotics. Total DNA, i.e. both chromosomal and plasmid DNA, was prepared from transformed cells by boiling the cell suspensions and by phenol-chloroform extraction; chromosomal DNA was prepared by the same methods from the non-transformed, plasmid-free strains; plasmid DNA of pUC19 was purchased; plasmid DNA of pCEP was purified from the transformed strains by caesium chloride density gradient centrifugation. Arbitrarily primed polymerase chain reaction was carried out for all of these preparations. Amplification carried out independently with three different primers resulted in similar patterns for the chromosomal preparations whether or not plasmid was present. Amplification of plasmid DNA gave different patterns, characterized by fragments larger than those obtained when total or chromosomal DNA were used as the target. These data illustrate that the plasmids studied here do not influence the chromosomal arbitrarily primed PCR fingerprint, although plasmids alone are amplified in the absence of chromosomal DNA. Experiments comparing different relative concentrations of plasmid and chromosomal DNA indicate that under natural conditions the amount of chromosomal DNA per cell is sufficient to inhibit observable amplification of the plasmid(s) present.     

Detection of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> by novel chromosomal polymerase chain reaction primers

A new sensitive and specific method for the detection of Erwinia amylovora was developed. The method is based on the detection of a chromosomal DNA sequence specific for this bacterial species and enables detection of E. amylovora pathogenic strains, including recent isolates that lack plasmid pEA29 and thus cannot be detected by the previously popular PCR methods based on the detection of this plasmid. A species-specific random amplified polymorphic DNA (RAPD) marker was identified, cloned, and sequenced, and sequence characterized amplified region (SCAR) primers for specific PCR were developed. The E. amylovora specific sequence, 1269 bp long, was amplified in polymerase chain reaction and detected with electrophoresis in agarose gel stained with ethidium bromide. Amplification with other bacterial species did not produce any PCR product detectable by electrophoresis. Matching of the E. amylovora specific sequence to chromosomal DNA was confirmed by computer analysis of the E. amylovora genome. A consistent sensitivity limit of the method was 3 CFU/reaction, and in some cases it was possible to detect 0.6 CFU/reaction. Due to its high sensitivity and specificity, our method of E. amylovora detection is currently the most reliable, taking into account that the reliability of PCR methods based on plasmid pEA29 has been compromised by the isolation of pathogenic E. amylovora strains that lack this plasmid.     

A rapid screening procedure for staphylococcal plasmids

A method has been developed for rapidly screening representatives of all currently recognized species of the genus Staphylococcus for the presence of plasmid DNA. The isolated plasmid DNA is substantially free from contaminating chromosomal and relaxed plasmid DNA. The method will detect plasmids in strains grown on various types of solid or liquid culture media and is convenient enough for routine epidemiological studies.     

The replication origin of <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis>

The putative replication origin of Azotobacter vinelandii was cloned as an autonomously replicating fragment after ligation to an antibiotic resistance cartridge. The resulting plasmids could be isolated and labelled by Southern hybridisation with the antibiotic resistance cartridge as probe and also visualised by electron microscopy. These plasmids integrated into the chromosome after a few generations, even in the recA mutant of A. vinelandii. The integrated copy of the plasmid was re-isolated from the chromosome and the DNA and its subfragments were cloned in the plasmid vector pBR322. A 200-bp DNA fragment was sufficient to allow the replication of pBR322 in an Escherichia coli polA strain. Electron microscopic analysis of this plasmid showed that replication initiated mostly within the A. vinelandii DNA fragment. The nucleotide sequence of the putative replication origin and its flanking regions was determined. In the sequence of the 200-bp fragment many of the distinctive features found in other replication origins are lacking. A greater variation from the consensus DnaA binding sequence was observed in A. vinelandii. Direct sequencing of the relevant genomic fragment was also carried after amplifying it from A. vinelandii chromosomal DNA by PCR. This confirmed that no rearrangements had taken place while the cloned fragment was resident in E. coli. It was shown by hybridisation that the 200-bp chromosomal origin fragment of A. vinelandii was present in three other field strains of Azotobacter spp.     

Simple method for extracting plasmid DNA from lactic acid bacteria

Rapid screening and large-scale plasmid DNA isolation procedures are described for lactic acid bacteria, using glass beads to break cells. The rapid screening procedure allows one to obtain plasmid DNA pellets in less than 1 h. This method has been successfully tested on various bacteria from the genera Lactococcus, Leuconostoc, Lactobacillus, Pediococcus, Streptococcus, Enterococcus and Propionibacterium. This procedure yields plasmid DNA with minor chromosomal and plasmid DNA-degraded form contaminations.