A simple method for the preparation of the covalently closed circular form of plasmid DNA

We describe a simple, rapid, inexpensive method for isolation of covalently closed circular plasmid DNA. The method involves the electrophoresis of crude DNA preparations in an agarose gel, electrotransfer onto a dialysis membrane and elution of the highly purified circular covalently closed plasmid DNA. Native and recombinant plasmid DNA have been purified by this method and shown to be suitable for restriction enzyme digestion and transformation of bacteria. The yield of this rapid purification procedure makes it a good alternative method to standard centrifugation in cesium chloride ethidium bromide gradients.     

Introduction of new DNA sequences into previously selected regions of a plasmid genome by means of the formation of heteroduplexes

A new method for obtaining the recombinant DNA based on heteroduplex-initiated site-directed insertion of alien nucleotide sequences is proposed. To generate a single-stranded region, plasmid DNA was nicked with restriction endonuclease in the presence of ethidium bromide with subsequent exonuclease III controlled digestion. The inserted DNA sequences flanked by nucleotide sequences complementary to single-stranded region were annealed with plasmid DNA and E. coli cells were transformed by the resulting heteroduplex molecules. The presented data show the possibility to insert as many as 200 nucleotides. The yield of recombinant DNA varied from 16 to 0.7% as the number of nucleotides inserted correspondingly varied from 15 to 200. The site of insertion does not depend crucially on the localization of the restriction site used.     

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.     

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.     

Restriction map of E. coli shuttle plasmid (p# GTE5) with secretive function

The shuttle plasmid (p# GTE5) DNA with secretive function was extracted by the alkali lysozyme method from E. coli RRI strain. Its molecular weight is 4.5 Md and DNA size is 6.9 Kb. Restriction fragments of plasmid was obtained by single and double enzymes complete digestion using five different restriction endonucleases. The restriction map of shuttle plasmid (p# GTE5) was established for the enzymes EcoRI, BglII, pstI, PvuII, and TaqI.     

Rapid isolation of substrate-quality plasmid DNA without CsCl-dye density gradients

The isolation of plasmid DNA produced in transformed bacterial cells is essential for many molecular biology techniques. Two drawbacks to the widely used CsCl-ethidium bromide method of preparation are the need for ultracentrifuge time and the generation of ethidium bromide waste. In this article we describe a method for the quick isolation of plasmid DNA without the use of an ultracentrifuge or ethidium bromide.     

Method for obtaining more-accurate covalently closed circular plasmid-to-chromosome ratios from bacterial lysates by dye-buoyant density centrifugation.

A method that gives high recovery of deoxyribonucleic acid (DNA) from crude bacterial lysates using ethidium bromide-cesium chloride density gradient centrifugation is presented. After Pronase digestion and shearing of the lysate, essentially 100% recovery of chromosomal DNA and a reproducible recovery of covalently closed circular (CCC) plasmid DNA is obtained for a specific plasmid in a given strain. This method should be useful for comparing the CCC plasmid/chromosome ratio of various plasmid-host combinations.     

Microscale method for rapid isolation of covalently closed circular plasmid DNA from group N streptococci

A method for rapid purification of plasmid DNA from lactic streptococci, utilizing microliter quantities of reagents, was developed by combination of a short lysozyme-mutanolysin cell wall digestion with a modification of the Escherichia coli plasmid isolation procedure of McMaster et al. (Anal. Biochem. 109:47-54, 1980). The preparations obtained were highly enriched for covalently closed circular DNA, and the method was applicable to plasmids of at least 40 megadaltons. Centrifugation in CsCl-ethidium bromide density gradients was not required.     

Determination of plasmid copy number by fluorescence densitometry

A simple and reliable method for the determination of plasmid copy numbers by direct fluorescence densitometry of ethidium bromide-stained electrophoretic gels was developed. In developing the method, the following parameters were evaluated and controlled: plasmid DNA trapping in the linear chromosomal DNA, staining-destaining kinetics for ethidium bromide, linearity of the fluorescence response, and the effect of the molecular topology of DNA on ethidium bromide binding to DNA in agarose.     

Microscale method for rapid isolation of covalently closed circular plasmid DNA from group N streptococci.

A method for rapid purification of plasmid DNA from lactic streptococci, utilizing microliter quantities of reagents, was developed by combination of a short lysozyme-mutanolysin cell wall digestion with a modification of the Escherichia coli plasmid isolation procedure of McMaster et al. (Anal. Biochem. 109:47-54, 1980). The preparations obtained were highly enriched for covalently closed circular DNA, and the method was applicable to plasmids of at least 40 megadaltons. Centrifugation in CsCl-ethidium bromide density gradients was not required.     

小鼠pDsRed-Meis1真核表达载体构建及子宫内表达的鉴定

目的构建小鼠Myeloid ectropic viral integration site 1(Meis1)基因和红色荧光蛋白(Red fluorescence protein,RFP)真核表达质粒,为进一步研究Meis1在生殖系统中的功能提供基础。方法设计引物,扩增含Meis1片段的克隆质粒中的meis1基因片段,将其插入真核表达载体pDsRed-N1,重组质粒经酶切鉴定后测序,并转染至小鼠子宫;用Western blot及免疫组织化学方法鉴定外源基因Meis1的表达。结果酶切和测序结果表明,构建的pDsRed-Meis1重组质粒正确;West-ern blot结果显示,外源性Meis1高表达于孕D2的小鼠子宫;转染后冰冻切片结果显示,带红色荧光蛋白的阳性产物表达于孕D4的小鼠子宫内膜及全层;免疫组织化学结果显示,Meis1阳性产物主要表达于孕小鼠子宫上皮、腺体和基质细胞内。结论正确构建了pDsRed-Meis1真核表达质粒,并成功转染及高表达于小鼠子宫,为进一步探讨Meis1在生殖系统中的功能提供了有利工具。     

Rapid construction of sequencing templates by random insertion of antibiotic resistance genes

We describe a novel and handy method for generating a population of templates for sequencing. The method is based on the random insertion of antibiotic resistance gene in plasmid DNA digested by DNase I. The advantages of this approach are the small quantity of DNA necessary for mutagenesis and the complete independence from the restriction map of the plasmid. DNase I digestion provides a random distribution of the insertions, while antibiotic selection provides low background. We also present a convenient PCR-based procedure for the analysis and ordering of obtained insertion mutants.     

An improved strategy for rapid direct sequencing of both strands of long DNA molecules cloned in a plasmid.

A strategy for kilo-base sequencing of a target DNA cloned in plasmid pWR34 is described. A long target DNA is progressively shortened from one end, by digestion with BAL31 nuclease or exonuclease III and nuclease S1, followed by cleaving off the shortened vector DNA. The family of the shortened target DNA molecule is next cloned in between the StuI site on one end, and a cohesive-ended restriction site on the other end, within the polylinker region of pWR34. DNA fragments cloned into this plasmid are sequenced directly by using a synthetic oligonucleotide primer, which binds to one side of the polylinker region using the dideoxynucleotide chain-termination method. The plasmid DNA, easily obtained by adoption of a rapid mini-preparation, is usually pure enough for direct DNA sequencing. Thus, both strands of any DNA several thousand base pairs in length can be completely sequenced (using two different primers) with ease within a short time, without the need for constructing a physical map.     

The use of cetyltrimethylammonium bromide for the rapid isolation from Staphylococcus aureus of relaxable and non-relaxable plasmid DNA suitable for in vitro manipulation

Plasmid DNA was isolated from Staphylococcus aureus by a rapid method that depends on the precipitation of DNA from cleared lysates by cetyltrimethylammonium bromide at low salt concentrations. The method was validated by its ability to provide DNA for restriction analysis of a highly relaxable plasmid species that is not isolated by more traditional techniques. The DNA can be digested with restriction endonucleases and used for transformation without further purification. The method also provides the basis for analysing staphylococcal plasmids that display a high frequency of deletion after transfer. Simple modifications of the technique allow plasmid DNA to be isolated from other bacteria and the rapid purification of DNA samples before in vitro manipulation.     

登革3型病毒prM-E基因重组质粒DNA的构建和真核表达

构建登革 3型病毒 prM E基因的真核表达重组质粒 ,并进行体外表达 ,为登革DNA疫苗的研究奠定基础。用RT -PCR法获得 prM -E基因片段 ,然后将其克隆到真核表达载体中。用电穿孔法将重组质粒DNA转入BHK细胞 ,通过免疫荧光法检测外源基因在真核细胞中的表达。结果 ,通过酶切和序列测定证实了构建的重组质粒DNA含序列正确的 prM- E基因。用免疫荧光法检测到转染了重组质粒DNA的BHK细胞的胞浆中有登革 3型病毒特异蛋白的表达。说明含有登革 3型病毒prM -E基因的真核表达重组质粒可以在BHK细胞中表达 ,该结果为观察该重组质粒的免疫原性奠定了基础。     

Evidence for a chromosomal location of the genes coding for chloramphenicol production in Streptomyces venezuelae.

Of seven chloramphenicol-producing actinomycetes examined, only Streptomyces venezuelae strain 13s contained extrachromosomal DNA detectable by agarose gel electrophoresis and cesium chloride-ethidium bromide density gradient centrifugation. The single 17-megadalton plasmid present in this strain was indistinguishable from plasmid pUC3 previously isolated from mutagenized cultures. Strains selected for their inability to produce chloramphenicol after treatment with acriflavine or ethidium bromide still contained a plasmid that had the same electrophoretic mobility as plasmid pUC3 and yielded similar fragments when digested with restriction endonucleases. By regenerating protoplasts of strain 13s and screening for isolates lacking extrachromosomal DNA, strain PC51-5 was obtained. The absence of plasmid pUC3 sequences in this strain was confirmed by Southern hybridization using 32P-labeled plasmid as a probe. Since the plasmidless strain produced as much chloramphenicol as did the parent strain, pUC3 contains neither structural nor regulatory genes for antibiotic production. Evidence from electrophoretic analysis of BamHI digests of total cellular DNA from wild-type and dye-treated nonproducing progeny indicated that acriflavine caused structural changes in the chromosome.     

A rapid and versatile site-directed method of mutagenesis for double-stranded plasmid DNA

This paper describes a new method for site-directed mutagenesis which allows mutations by deletion, insertion or substitution of large fragments of DNA with more than 50% efficiency and does not require subcloning in a single-stranded (ss) DNA vehicle. The site of mutagenesis is removed from a linearized plasmid DNA by BAL 31 digestion, ss DNA regions are generated by limited exonuclease treatment and the mutated target site is reconstituted by annealing of the plasmid DNA to a 35-70 nucleotide long mutated ss oligodeoxynucleotide containing the desired mutation. The circularized plasmid is finally used to transform directly Escherichia coli competent cells.     

A rapid and simple method for screening large numbers of recombinant DNA clones

A simple and rapid method has been described for the isolation of plasmid, phagemid and phage DNAs. Hundreds of recombinant clones can be screened in one day employing this method. It takes half an hour to prepare plasmid DNA from ten clones, and the DNA prepared from a single colony using this method is of sufficient quality and in sufficient amount to perform at least five restriction digestions. This method eliminates the need for RNase treatment and phenol chloroform extraction if the plasmids are needed only for the restriction digestion. If needed, RNAs can be removed after restriction digestion by adding RNase and incubating for two minutes at room temperature. After RNase treatment and phenol/chloroform extraction, the plasmid DNA serves as a good template for sequencing. The DNA can be stored at -20 degrees C for over eight weeks.     

Stability in Escherichia coli of an antibiotic resistance plasmid from Bacteroides fragilis.

A Bacteroides fragilis strain resistant to penicillin G, tetracycline, and clindamycin was screened for the presence of plasmid deoxyribonucleic acid (DNA). Agarose gel electrophoresis of ethanol-precipitated DNA from cleared lysates of this strain revealed two plasmid DNA bands. The molecular weights of the plasmids were estimated by their relative mobility in agarose gel and compared with standard plasmids with known molecular weights. The molecular weights were 3.40 +/- 0.20 x 10(6) and 1.95 +/- 0.05 x 10(6) for plasmids pBY1 and pBY2, respectively. Plasmid DNA purified by cesium chloride-ethidium bromide gradient centrifugation was used to transform a restriction- and modification-negative strain of Escherichia coli. Penicillin G- and tetracycline-resistant transformants were screened for the presence of plasmid DNA. A plasmid band corresponding to a molecular weight of 1.95 x 10(6) was present in all transformants tested. Curing experiments demonstrated that the plasmid, referred to as pBY22 when present in transformants, was responsible for penicillin G and tetracycline resistance. Plasmid pBY22 was mobilized and transferred to other E. coli strains by plasmid R1drd-19. Stability of pBY22 was examined in different E. coli strains and was shown to be stably maintained in both restriction-negative and restriction-positive strains. Unexpectedly, pBY2 and pBY22 were resistant to digestion by 12 different restriction endonucleases.     

A simple and efficient method for the oligonucleotide-directed mutagenesis using plasmid DNA template and phosphorothioate-modified nucleotide

We have developed a simple and efficient method for oligonucleotide-directed mutagenesis with double-stranded (plasmid) DNA as a template. The template was simply and rapidly prepared by cell lysis and the following DNA denaturation with alkali. The chain elongation was performed with phosphorothioate-modified nucleotide at 37 degrees C. After the selective digestion of original DNA with NciI and exonuclease III, the desired mutated gene was obtained at a high frequency (about 70%).