Large-scale isolation of plasmid DNA using cetyltrimethylammonium bromide

A rapid procedure for the large-scale isolation of plasmid DNA is described. The method utilizes cetyltrimethylammonium bromide to precipitate the plasmid following extraction of DNA by lysozyme digestion and boiling. The plasmid is then purified by passing through the spin column pZ523. The purity and yield of the plasmid obtained with this method is similar to that isolated by cesium chloride-ethidium bromide gradient centrifugation. The method does not involve any phenol-chloroform extractions and takes five to six hours for completion after growth of the bacterial cells. The plasmid obtained is amenable to digestion with various restriction endonucleases, can be used for cloning with high efficiency and is also suitable as template for dideoxy sequencing.     

Enzymatic purification of plasmid DNA.

A novel method for plasmid DNA purification using enzymes that degrade all major types of contaminating nucleic acids present in crude plasmid DNA mixtures (but not plasmid DNA) has been devised. This method is quick (can be accomplished in two hours), requires no expensive laboratory equipment (an ultracentrifuge is not necessary) and is inexpensive. Plasmid DNA purified by this methodology can be used in a variety of molecular biological techniques including restriction enzyme digestions, subcloning, sequencing, nick translation and end labeling. This plasmid purification technique will be very useful for the molecular biologist performing cloning experiments.     

The involvement of RNA in the initiation of DNA synthesis in mammalian cells. Artifacts arising during caesium-salt density-gradient centrifugation which simulate a covalent attachment of RNA to newly synthesized DNA.

Artifacts were encountered during caesium salt density gradient centrifugation which simulated results expected if newly synthesized DNA is covalently attached to RNA. Newly synthesized DNA (in baby hamster kidney cells, BHK-21/C13), pulse-labelled with [3H]thymidine for 10 min at temperatures below 25 degrees C, banded at a greater buoyant density than mature [14C]DNA (heat-denatured nucleic acids) when centrifuged to equilibrium in caesium chloride gradients. Some of the newly synthesized RNA, labelled with [3H]uridine, banded at a buoyant density slightly greater than DNA in caesium sulphate gradients. These results were not obtained when nucleic acids were pulse-labelled at 37 degrees C, nor when samples were heat-denatured in the presence of formaldehyde. This suggests that nucleic acids can aggregate during centrifugation; this is discussed in relation to the molecular size of the DNA.     

Characterization of DNA degradation using direct current conductivity and dynamic dielectric relaxation techniques

The purpose of this study was to evaluate DNA degradation upon thermal heating using dielectric relaxation and direct current (DC) conductivity methods. Herring sperm DNA, human growth hormone (HgH) plasmid DNA, and secreted alkaline phosphatase (SEAP) plasmid DNA were used as the examples. DNA was heated at 80°C for 1 hour. The dielectric relaxation spectra as a function of the applied field frequency were measured for HgH DNA at 0.5 hours and at 1 hour. The frequency range covered was from 10 kHz to 100 kHz. The DC conductivity measurements were made for all 3 kinds of DNA at 4 time points: 0 hours, 0.5 hours, 0.75 hours, and 1 hour. At each time point the DC conductivity was measured for each sample as a function of concentration via water dilution. The results show that the dielectric relaxation method is less sensitive in characterizing heat-driven DNA degradation. Conversely, DC conductivity is very sensitive. The semiquantitative dependence of the conductivity upon heating suggests that DNA degradation involves more than plasmid DNA nicking. Double strand and single strand breaks may also occur. In addition, herring sperm DNA, HgH DNA, and SEAP DNA, though similar in their DC conductivity functional forms upon dilution, exhibit significant differences in their responses to sustained heating.     

Rapid purification of plasmid DNA by a single centrifugation in a two-step cesium chloride-ethidium bromide gradient

A procedure for rapid, preparative purification of plasmid DNA is described and compared with a conventional equilibrium centrifugation method. A discontinuous, two-step CsCl-ethidium bromide gradient is used, with the starting position of the plasmid-containing extract being at the bottom of the tube. During centrifugation in a fixed angle rotor, covalently closed circular plasmid DNA is separated from contaminating protein, RNA, and chromosomal DNA in 5 hr. Plasmids purified by this method are considerably less contaminated with RNA than when purified by a 48-hr equilibrium run in a homogeneous gradient, as determined by agarose gel electrophoresis and 5'-end-labeling studies. Plasmid DNA purified in two-step gradients can be used directly for restriction endonuclease analysis and DNA sequencing.     

Fate of plasmid DNA in transformation of Bacillus subtilis protoplasts

Summary Polyethylene glycol-treated protoplasts of B. subtilis can be transformed by plasmid DNA at very high frequencies (Chang and Cohen 1979). From analysis of plasmid mediated transformation of transformation-deficient mutants it appeared that mutants, reduced in the transformation by plasmid DNA in the competent state, were plasmid transformation-proficient when transformed as protoplasts. By means of CsCl-gradient centrifugation of re-extracted plasmid DNA it could be demonstrated that plasmid DNA enters the protoplasts in the double-stranded form. In addition, sucrose gradient centrifugation of the re-extracted plasmid DNA showed that the entered DNA is predominantly present as covalently closed circular DNA. The efficiency of plasmid transformation in protoplasts was found to be close to one (each plasmid molecule having entered into the protoplasts gives rise to a transformed cell). This is in good agreement with the observation that little, if any, damage is done to this DNA during or after entry into protoplasts.     

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.     

Rapid preparation of covalently closed circular DNA by acridine yellow affinity chromatography

Covalently closed circular DNA can be isolated rapidly from cell lysates in a two-step process. Hydroxylapatite chromatography to prepurify the plasmid DNA from contaminating protein and RNA is followed by a step gradient elution of covalently closed circular (CCC) plasmid DNA from an acridine yellow affinity column. This procedure results in CCC DNA of a purity comparable to that obtained from ethidium bromide-CsCl gradients without lengthy centrifugation and free of contaimination by intercalating dye. Up to 250 μg of CCC pBR 322 can be isolated from 500 ml of bacterial culture in 4–6 h.     

Rapid methods for the study of both stable and unstable plasmids in Pseudomonas

Rapid methods for the analysis of degradative plasmids in Pseudomonas are described. Bacterial lysates prepared with alkaline sodium dodecyl sulphate were vigorously stirred in the presence of antifoam agent. Samples were subjected to agarose gel electrophoresis to detect plasmid DNA. After a short period of centrifugation on a sucrose gradient, the lysates yielded plasmid DNA of sufficient purity for restriction endonuclease digestion without further treatment. The methods have proved most useful in the extraction of previously undetected plasmid DNA, some of which appears to be unstable.     

Purification of supercoiled DNA of plasmid col E1 by RPC-5 chromatography

Col E1 DNA can be purified to a high degree by RPC-5 chromatography of a partially purified cell lysate with a very shallow linear NaCl gradient at pH 7.8. Electron micrographs demonstrated that the purest fractions were composed of 93% supercoiled (form I) DNA and 7% open circular (form II) DNA. The actual chromatography can be accomplished in 13–14 h and is designed for the production of several milligrams of plasmid DNA.     

Characterization of the plasmid DNAs of Rhodopseudomonas capsulata

Presence of extrachromosomal DNa in Rhodopseudomonas capsulata strain BH9 was shown by the appearance of a satellite band in a dye-buoyant density gradient. Radioactively labelled DNA was prepared from this satellite band and examined on a 5–20% sucrose gradient. Three radioactive peaks with sedimentation coefficients of 100 S, 94 S, and 58–64 S, respectively, were consistently observed. Analysis of these sedimentation coefficients suggested that there are two species of plasmid DNA with molecular sizes of 94×10⁶ daltons (named pBH91) and 74×10⁶ daltons (named pBH92). The 58–64 S peak is attributed to open circular molecules. DNAs from each peak of the sucrose gradient were examined by electronmicroscopy, and the results agree closely with those of the sucrose gradient analysis. Reassociation kinetics of the plasmid DNA was also followed. Addition of total DNA of strain BH9 increased the renaturation rate of the plasmid DNA. It was calculated from the magnitude of the increase that approximately 10% of the BH9 total DNA may hybridize with the plasmid sequences. DNA prepared from the gene transfer agent (GTA) produced by R. capsulata increases the renaturation rate of the plasmid to the same extent as total DNA isolated from the GTA producing strain, Y262.     

Detection of nonintegrated plasmid deoxyribonucleic acid in the folded chromosome of Escherichia coli: physiochemical approach to studying the unit of segregation.

Physiocochemical evidence presented indicates plasmid deoxyribonucleic acid (DNA) can associate with host chromosome without linear insertion of the former into the latter. This conclusion is based on the observation that covalently closed circular (CCC) plasmid DNA can cosediment with undegraded host chromosome in a neutral sucrose gradient. When F plus bacteria are lysed under conditions that preserve chromosome, approximately 90% of CCC F sex factor plasmid (about 1% of the total DNA) is found in folded chromosomes sedimenting at rates between 1,500 and 4,000s. The remaining 10% of the CCC F DNA sediments at the rate (80S) indicative of the free CCC plasmid form. Reconstruction experiments in which 80S, CCC F DNA is added to F plus or F minus bacteria before cell lysis show that exogenous F DNA does not associate with folded chromosomes. In F plus bacteria, F plasmid is harbored at a level of one or two copies per chromosomal equivalent. In bacteria producing colicin E1, the genetic determinant of this colicin, the Col E1 plasmid, is harbored at levels of 10 to 13 copies per chromosomal equivalent; yet, greater than 90% of these plasmids do not cosediment with the 1,800S species of folded chromosome. However, preliminary evidence suggests one or two Col E1 plasmids may associate with the 1,800S folded chromosome. Based on evidence presented in this and other papers, we postulate F plasmid can link to folded chromosome because the physicochemical structure of the plasmid resembles a supercoiled region of the chromosome and, therefore, is able to interact with the ribonucleic acid that stabilizes the folded chromosome structure. Implications of this model for F plasmid replication and segregation are discussed.     

Plasmid chromosome isolation: An improved batch procedure for large plasmids

A one-step, batch fractionation procedure for the isolation of Escherichia coli plasmids with molecular weights up to at least 1.3 × 10⁸ has been devised. A 15-ml lysate of 5 × 10¹⁰ bacteria is gently prepared and underlain with 15 ml of CsCl-ethidium bromide solution in a 30-ml centrifuge bottle. During an 18- to 24-h 100,000g centrifugation in an anglehead rotor, an aggregate of dense cellular debris including almost all of the host DNA pellets. Plasmid chromosomes are banded in the supernatant isopycnic gradient and are detected by fluorescence of their intercalated ethidium bromide. Plasmid recoveries of 40–70% are achieved. The preparations are pure enough to be used for plasmid circumference measurements by electron microscopy.     

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.     

Normalization of soil DNA extraction for accurate quantification of target genes by real-time PCR and DGGE

The analysis of microbial communities in environmental samples requires accurate and reproducible methods for extraction of DNA from sample matrices that have different physical and chemical characteristics. Even with the same sample type, variations in laboratory methods can result in different DNA yields. To circumvent this problem, we have developed an easy and inexpensive way to normalize the quantities of DNA that involves the addition of an internal standard prepared from plasmid DNA. The method was evaluated by comparing DNA yields using different DNA extraction procedures, after which the DNA was used for microbial community analysis by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S ribosomal RNA (rRNA) and for quantification of 16S rRNA gene copy numbers in environmental samples by real-time PCR. Our results show that use of the internal standard allows normalization of the resulting data and more accurate quantification of gene copy numbers in soil samples. These methods should also have broad application for various other types of environmental samples.     

Large scale sequencing projects using rapidly prepared double-stranded plasmid DNA.

We have developed a simple rapid plasmid DNA mini-preparation method which yields DNA of sufficient quality to be used in large scale sequencing projects. The method, which is a modification of the alkaline method of Birnboim and Doly (1979), requires less than two hours. We have eliminated the use of organic extractions, RNase digestion and alkaline denaturation of the DNA for annealing of the primer. The proportion of supercoiled plasmid DNA obtained is close to 100%. Greater than 80% of the clones yield at least 500 bp of sequence information per primer. The sequencing reactions from these double-stranded templates can be done on both strands using the universal and reverse sequence primers with the usual two reactions per primer, one to read close to the primer and one to read far from it. Thus, each clone yields at least 1 kb of sequence information. The preparation of the templates and the sequencing reactions can be done in less than three hours so that the sequencing gel can be run the same day.     

用ribozyme抑制增殖细胞核抗原的表达对HaLa细胞增殖的影响

增殖细胞核抗原(PCNA)是DNA聚合酶δ的辅助蛋白,它是细胞染色体DNA复制所必需的。人工设计的ribozyme具有可特异地切割PCNA mRNA的性质,将此ribozyme的自修剪体内表达质粒导入HeLa细胞,从细胞总RNA中分离相应部分能在体外切割靶RNA片段,证明此表达质粒在细胞内能表达出有活性的ribozyme分子。与对照相比,导入ribo-zyme表达质粒的HeLa细胞进入S期的时间从12 h推迟到20 h,而突变ribozyme的对照表明反义抑制对细胞进入S期的影响较小(推迟到15 h)。证明该ribozyme能有效抑制He-La细胞DNA复制,同时亦证明PCNA对于细胞DNA复制及细胞周期进程的重要性。     

Enrichment and visualization of small replication units from cultured mammalian cells

DNA from cultured Chinese hamster cells has been fractionated to yield a population of DNA enriched for replicating molecules. Molecules containing replication structures were analyzed by electron microscopy, and replicon size was estimated. The enrichment procedure takes advantage of single-stranded regions characteristic of replicating molecules, and the greater affinity of mercuric ion for single-stranded rather than native DNA. After interaction with low concentrations of HgCl2, DNA with bound mercury is separated from the bulk of the DNA by virtue of its increased buoyant density in an isopycnic Cs2SO4 gradient. When DNA from cells labeled with [3H]thymidine for 45 s is interacted with HgCl2 and banded in Cs2SO4, the DNA with the highest specific activity is found in a dense region of the gradient. The high specific activity DNA behaves kinetically like nascent DNA since the radioactivity can be chased into main band if the cells are incubated for a further 2 h in excess unlabeled thymidine. Electron microscope analysis of the DNA in the enriched fraction confirmed that it contains a substantial fraction of molecules with replication structures. The level of enrichment is about 25-fold compared to unfractionated DNA or DNA taken from the main band of the Hg++/Cs2SO4 gradient. Of the replicating molecules visualized, 85% possessed a single replication structure. All molecules with multiple replication forms contained replicon sizes less than 5 micron, ranging from 0.2 to 4.5 micron. Replicon size was determined by measuring the distance from the center of one replication structure to the center of the adjacent replication structure on the same molecule. The replicons observed in this study are far smaller than can be detected by DNA fiber autoradiography and are in the same size range as the very small replication units reported in embryonic systems.