Large-scale purification of plasmid DNA by fast protein liquid chromatography using a Hi-Load Q Sepharose column.

The large-scale purification of plasmid DNA was achieved using fast protein liquid chromatography on a Hi-Load Q Sepharose column. This method allows for the purification of plasmids starting from crude plasmid DNA, prepared by a simple alkaline lysis procedure, to pure DNA in less than 5 h. In contrast to the previously described plasmid purification methods of CsCl gradient centrifugation or high-pressure liquid chromatography, this method does not require the use of any hazardous or expensive chemicals. More than 100 plasmids varying in size from 3 to 15 kb have been purified using this procedure. A Mono Q Sepharose column was initially used to purify plasmids smaller than 8.0 kb; however, a Hi-Load Q Sepharose column proved more effective with plasmids larger than 8 kb. The loading of plasmids larger than 8 kb on the Mono Q column resulted in a high back pressure and the plasmid DNA could not be eluted from the column. Thus, for routine purification we utilize the Hi-Load Q Sepharose column. Plasmids purified by this method had purity, yield, and transfection efficiency in mammalian cells similar to those of plasmids purified by CsCl density gradient centrifugation.     

An extended boiling method for small-scale preparation of plasmid DNA tailored to long-range automated sequencing

Automated fluorescence sequencing depends on high-quality plasmid DNA, which is conveniently prepared by minipreparation procedures. While those procedures are effective for high-copy number plasmids, purity and yields of low-copy number plasmids are often not sufficient to achieve reasonable sequencing results. Here, we describe a reproducible and cheap procedure for the small-scale preparation of plasmid DNA, which is based on the original Holmes and Quigley protocol, comprising a boiling and two selective precipitation steps. Besides various other modifications, this procedure utilizes polyethylene glycol (PEG) precipitation as a key step to further purify plasmid DNA tailored to automated fluorescence sequencing. Independent of the plasmid size and copy number, the modified procedure yields plasmid DNA, which gives average reading lengths of 800 and more bases with a standard fluorescence cycle sequencing protocol. To demonstrate the efficiency and reproducibility of the method, sequencing data of various human interleukin-6 gene variants cloned in different vectors are presented. This procedure offers an economical alternative to commercial miniprep kits, utilizing silica resins or anion-exchanger matrices and, moreover, is more reliable and consistent with respect to reading lengths and accuracy in automated fluorescence sequencing.     

Electroporation-mediated transfection of mammalian cells with crude plasmid DNA preparations

We designed a simple and reproducible electroporation-mediated transfection procedure with which to screen mammalian expression vector-constructed cDNA libraries. Using a specific chamber composed of five parallel electrodes, the recipient cells can be electroporated separately with 40 plasmid DNA preparations in a single experiment. Over 300 crude plasmids prepared from E. coli (DH-5) carrying a pcD2neo-vector-derived cDNA library were tested. The efficiency of stable transfection by electroporation with crude plasmid DNA preparations was 10-times higher than with the CsCl-purified plasmid DNA. When the crude plasmids were digested with RNase, the efficiency of stable transfection markedly decreased, indicating that the contaminating bacterial RNA in the crude plasmid preparations has a strong carrier effect during the electroporation. Even when salmon sperm DNA or genomic DNA from the recipient cells was used as the carrier of the purified plasmid, the efficiency was not higher than that using the crude preparations. This procedure is useful not only for screening a number of cDNAs but also for routinely introducing biologically active foreign genes into cultured mammalian cells.     

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.     

Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid.

Construction and characterization of a class of multicopy plasmid cloning vehicles containing the replication system of miniplasmid P15A are described. The constructed plasmids have cleavage sites within antibiotic resistance genes for a variety of commonly employed site-specific endonucleases, permitting convenient use of the insertional inactivation procedure for the selection of clones that contain hybrid DNA molecules. Although the constructed plasmids showed DNA sequence homology with the ColE1 plasmid within the replication region, were amplifiable by chloramphenicol or spectinomycin, required DNA polymerase I for replication, and shared other replication properties with ColE1, they were nevertheless compatible with ColE1. P15A-derived plasmids were not self-transmissible and were mobilized poorly by Hfr strains; however, mobilization was complemented by the presence of a ColE1 plasmid within the same cell.     

A rapid microscale technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis

A simple and rapid microscale technique is described for the isolation of plasmid DNA which involves cell lysis with phenol, centrifugation, phenol extraction, ethanol precipitation, and RNase digestion. The plasmid DNA is of suitable purity and quantity for multiple restriction endonuclease digestions and bacterial transformations. This “miniprep” procedure is applicable for a variety of types of plasmids ranging in size from 2900 to 18,400 base pairs (bp) and for a number of Escherichia coli strains. The plasmids are rapidly cleaved by all restriction enzymes (total of 14) tested to date. Recombinant clones have been screened for insertions as small as 10 bp and as large as 5000 bp. The procedure takes ~3 h and has been routinely used to simultaneously analyze 24 candidate clones. This procedure is reliable and useful for rapid screening of recombinant DNA candidates where analysis by restriction endonuclease digestion is necessary.     

Plasmid vector for cloning in Streptococcus pneumoniae and strategies for enrichment for recombinant plasmids

A new plasmid, pLS101, was constructed for use as a vector for cloning in Streptococcus pneumoniae. This plasmid carries two selectable genes, tet and malM, each of which contains two or more restriction sites for cloning. Insertional inactivation of the malM gene allowed direct selection of TcRMal- clones containing recombinant plasmids. Other means of enriching a recipient population for cells containing recombinant plasmids were examined. The effect of removing vector terminal phosphate in attempts to clone heterogeneous DNA fragments, such as those from chromosomal DNA, was to abolish recombinant plasmid establishment altogether, presumably because donor DNA processing during entry into the cell prevented establishment of the hemiligated molecule. However, with homogeneous DNA fragments, such as those from plasmid or viral DNA, vector phosphate removal allowed enrichment for recombinant plasmids. In the cloning of heterogeneous DNA that was homologous to the recipient chromosome (i.e. chromosomal DNA from S. pneumoniae), recovery of recombinant plasmids could be enriched tenfold (relative to the regenerated vector) by the process of chromosomal facilitation of plasmid establishment. This involved an additional passage of the mixed plasmids in which interaction with the chromosome of plasmids containing chromosomal DNA inserts (i.e. recombinant plasmids) increased their frequency of establishment relative to the vector plasmid. An overall strategy for cloning in S. pneumoniae, depending on the nature of the fragment to be cloned, is proposed.     

Concatemer formation of ColE1-type plasmids in mutants of Escherichia coli lacking RNase H activity

rnh mutants harboring pBR322 were found to contain several slowly migrating DNA species when examined by agarose gel electrophoresis. The plasmid DNA from rnh mutants included large molecules, i.e. plasmids two, three or four times the size of a single plasmid unit. That this DNA contained concatemeric plasmid joined in a head-to-tail fashion was determined by digestion with restriction endonucleases that cleaved the monomeric plasmid DNA at a unique site. This treatment resulted in migration of the plasmid DNA at a mobility identical to that of linearized monomeric plasmid by agarose gel electrophoresis. This was confirmed by electron microscopy. Plasmid concatemer formation was detected with several high-copy-number (relaxed type) plasmids but not with low-copy-number (stringent) plasmids. Concatemer formation was dependent on RecA+ and RecF+ functions since several recA and recF mutations abolished concatemer formation. ColE1-type plasmids were previously shown to replicate in rnh mutants in the absence of DNA polymerase I (PolI) activity. This DNA PolI-independent plasmid replication was also examined for its dependence on the recF and recA gene products. rnh- polA(Ts) recF- strains were efficiently transformed with these plasmids at 30 degrees C and 42 degrees C, indicating the presence of DNA PolI-independent replication under recF- conditions. The presence or absence of plasmid replication in rnh- polA- recA(Ts) strains was also examined by measuring the increase in total amounts of plasmid. The results indicated that DNA PolI-independent replication occurred in these triple mutants at 42 degrees C as well as at 30 degrees C. It was concluded that the recombination event giving rise to concatemer formation was not essential for DNA PolI-independent replication in rnh mutants.     

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.     

Simple and rapid method for isolating large plasmid DNA from lactic streptococci.

A procedure for the rapid isolation of plasmid DNA larger than 30 megadaltons from lactic streptococci is described. This protocol can be used on a preparative scale to isolate sufficient quantities of plasmid DNA required for restriction analysis, cloning, or transformation experiments. A scaled-down protocol is very useful for rapidly screening the plasmid content of streptococcal strains. With this methodology, previously undetected large plasmids were observed.     

Transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA

Abstract A method for efficient polyethylene glycol (PEG)-mediated transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA is described. The best conditions found for protoplast regeneration included using 0.45 M sucrose both during the cultivation of the cells and (as an osmotic stabilizer) during their treatment with lysozyme, whereas 0.25 M sodium-succinate was added to the regeneration plates. Under these conditions about 5–10% of input cells regenerated. The highest transformation frequency with plasmid DNA was obtained with a PEG 6000 concentration of 22.5% (w/v). Transforming B. amyloliquefaciens strains with the plasmid pUB110 isolated from B. amyloliquefaciens resulted in 2–4 · 10⁵ transformants/μg DNA, 100–1 000-times as high as with DNA from Bacillus subtilis , suggesting a restriction barrier between the two species. Transformation of B. amyloliquefaciens with plasmids pC194 or pE194 cop -6 gave poor yields and no restriction barrier could be demonstrated for these plasmids. However, by curing pC194 from one of the transformants, a mutant strain compatible to both the plasmids could be isolated, yielding 2–3·10⁴ transformants/μg DNA. Both laboratory and industrial B. amyloliquefaciens strains could be transformed with the procedure.     

Replication, Integration, and Packaging of Plasmid DNA following Cotransfection with Baculovirus Viral DNA

Infection-dependent replication assays have been used to identify numerous putative origins of baculovirus replication. However, plasmid DNA, when cotransfected into insect cells with Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) DNA, replicates independently of any viral sequence in cis (11). Cotransfection of transfer plasmids and baculovirus DNA is a common procedure used in generating recombinant viruses and in measuring the level of gene expression in transient-expression assays. We have examined the fate of a series of vector plasmids in cotransfection experiments. The data reveal that these plasmids replicate following cotransfection and the replication of plasmid DNA is not due to acquisition of viral putative origin sequences. The conformation of plasmid DNA replicating in the cotransfected cells was analyzed and found to exist as high-molecular-weight concatemers. Ten to 25% of the replicated plasmid DNA was integrated into multiple locations on the viral genome and was present in progeny virions following serial passage. Sequence analysis of plasmid-viral DNA junction sites revealed no homologous or conserved sequences in the proximity of the integration sites, suggesting that nonhomologous recombination was involved during the integration process. These data suggest that while a rolling-circle mechanism could be used for baculovirus DNA replication, recombination may also be involved in this process. Plasmid integration may generate large deletions of the viral genome, suggesting that the process of DNA replication in baculovirus may be prone to generation of defective genomes.     

High frequency excision of Ty elements during transformation of yeast.

Yeast (Saccharomyces cerevisiae) transposons (Ty elements) are excised from up to 20% of supercoiled plasmids during transformation of yeast cells. The excision occurs by homologous recombination across the direct terminal repeats (deltas) of the Ty element, leaving behind a single delta in the transforming plasmid. Only the initial transforming plasmid is susceptible to excision, and no high frequency excision is observed in plasmids that have become established in transformed cells or in plasmids that are resident in cells undergoing transformation. High frequency excision from plasmids during yeast transformation is not specific for Ty elements and can be observed with other segments of plasmid DNA bounded by direct repeats. The frequency of Ty excision from supercoiled plasmids is greatly reduced when the host yeast cells contain the rad52 mutation, a defect in double-strand DNA repair. When linear or ligated-linear plasmid DNAs containing a Ty element are used for transformation, few or no excision plasmids are found among the transformant colonies. These results suggest that when a yeast cell is transformed with a supercoiled plasmid, the plasmid DNA is highly susceptible to homologous recombination for a short period of time.     

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.     

大肠杆菌菌蜕装载质粒DNA实验方法的优化

目的:优化大肠杆菌菌蜕装载质粒的效率,并将装载质粒的菌蜕转染抗原提呈细胞,以提高核酸疫苗的递送水平。方法:将质粒pHH43转化大肠杆菌DH5α,制备大肠杆菌菌蜕;优化菌蜕装载质粒时菌蜕、质粒和膜囊的比例,获得更高的装载效率,通过扫描及透射电镜、流式细胞术观察其形态变化及装载效率;将装载质粒的菌蜕与抗原提呈细胞——巨噬细胞RAW264.7和树突状细胞DC2.4共孵育,观察吞噬效果。结果:优化了大肠杆菌菌蜕装载质粒的效率,当菌蜕、质粒、膜囊的比例为7∶10∶4时效率达到最佳,装载DNA效率达98%以上;抗原提呈细胞吞噬装载了质粒的菌蜕,效率达100%。结论:大肠杆菌菌蜕可高效装载核酸疫苗,且高效被抗原提呈细胞捕获,有助于提高核酸疫苗的递送和免疫效果的提高。     

Molecular cloning of complementary DNA: preparation of a plasmid vector with low transformation background

A simple method that allows the rapid preparation of oligo dG-tailed plasmid vectors is presented. The procedure involves purification of the tailed molecules by hybridization to oligo dC-cellulose followed by a stepwise thermal elution. The resulting plasmid is virtually devoid of transformation activity in the absence of oligo dC-tailed DNA fragments. It allows construction of cDNA libraries with as low as 1% of colonies harboring wild-type plasmids.     

Intramolecular recombination during plasmid transformation of Bacillus subtilis competent cells.

We have constructed plasmids carrying direct internal repeats 260-2000 bp long. Monomers of such plasmids transformed Bacillus subtilis competent cells. The efficiency of transformation varied with the square of the length of repeats. The transformed clones harbored either the entire transforming plasmid and the plasmid arising by recombination between the repeats, or only the latter plasmid. Internally-repeated plasmids linearized by in vitro cleavage with restriction endonuclease could transform, yielding clones which exclusively harbored a plasmid resulting from recombination between the repeats. When the transforming plasmid carried repeats which differed slightly, conversion of one repeat into the other could occur. The following model of plasmid transformation accounts for these data: (1) plasmid DNA is cleaved and rendered linear in contact with competent cells; (2) a linear, at least partially double-stranded plasmid molecule is introduced or formed by repair within the cell; (3) a circular viable plasmid is produced by recombination between repeats carried on this molecule; (4) alternatively, a viable plasmid is produced by repairing the cut within one of the repeats by DNA synthesis which uses the other repeat as a template.     

Rapid micropreps and minipreps of Ti plasmids and binary vectors fromAgrobacterium tumefaciens

A simple and versatile procedure has been developed for the isolation of both large helper/Ti plasmids and binary vectors fromAgrobacterium tumefaciens. Using a slightly modified alkaline lysis protocol, intact plasmid can be recovered from cultures grown in standard micro-centrifuge tubes or culture tubes in sufficient yield and purity to allow for restriction analysis on ethidium bromide stained gels of the >200 kb Ti plasmid DNA. Contamination by chromosomal DNA is minimal and there is thus no need for isopycnic gradient purification. This same procedure can be combined with a high temperature treatment (37°C) and antibiotic selection to generate preparations containing binary vector DNA that are virtually free of interfering Ti plasmid DNA. Restriction patterns produced from these binary vector DNA preparations are unambiguous and therefore preliminary screening by Southern hybridization can be eliminated.     

Molecular and genetic studies of an R factor system consisting of independent transfer and drug resistance plasmids

Certain genetic, structural, and biochemical properties of a class 2 R-factor system consisting of the conjugally proficient transfer plasmid I and the naturally occurring non-conjugative tetracycline (Tc) resistance plasmid 219 are reported. I and 219 exist as separate plasmid deoxyribonucleic acid (DNA) species in both Escherichia coli and Salmonella panama, having molecular weights of 42 x 10(6) and 5.8 x 10(6), respectively. The buoyant densities of I and 219 are 1.702 and 1.710 g/cm(3), respectively, in neutral cesium chloride. Although the Tc resistance plasmid is not transmissible in a normal conjugal mating, it is mobilized in a three-component mating by plasmid I and by certain other conjugative plasmids of the fi(+) or fi(-) phenotype. Mobilization does not appear to involve intermolecular recombination between plasmids, and no covalent linkage of resistance markers and fertility functions is observed. Transformation of CaCl(2)-treated E. coli by plasmid DNA is shown to be a useful procedure for studying the biological properties of different plasmid molecular species that have been fractionated in vitro, and for selectively inserting non-self-transmissible plasmids into specific bacterial strains. The effects of tetracycline on the rate of protein synthesis carried out by plasmid 219 were studied by using isolated E. coli minicells into which this plasmid had segregated. Consistent with the results of earlier investigations showing the inducibility of plasmid-mediated Tc resistance in E. coli, the antibiotic was observed to stimulate protein synthesis in minicells carrying the plasmid 219 and totally inhibit (3)H-leucine incorporation by minicells lacking the Tc resistance marker. Five discrete polypeptide species were synthesized by minicells carrying plasmid 219; exposure of minicells or parent bacteria to Tc resulted in specific and reproducible changes in polypeptide synthesis patterns.     

Rescue of a tk-plasmid from transgenic mice reveals its episomal transmission

Summary This communication demonstrates the usefulness of the plamid rescue procedure for recovery of plasmids from transgenic mice. We have microinjected the plasmid pSK1 harbouring the Herpes simplex virus thymidine kinase gene into fertilized mouse oocytes and succeeded in recovering plasmids from newborns by transformation of E. coli either with HindIII cut cellular DNA or with uncut DNA. The majority of the rescued plasmids were indistinguishable from pSK1 by restriction analysis. The rescued plasmids proved to be functionally active in a transient expression assay in mouse Ltk^- cells. The pSK1 DNA sequences were inherited by up to 90% of the second generation progeny mice, which is not in agreement with a Mendelian transmission of heterozygous markers integrated into a single site of the chromosome.These data support the assumption that germ line transmission of non-integrated episomal plasmids can occur.