538 results on '"DNA, Single-Stranded isolation & purification"'
Search Results
202. Application of denaturant gradient gel electrophoresis for the analysis of the porcine gastrointestinal microbiota.
- Author
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Simpson JM, McCracken VJ, White BA, Gaskins HR, and Mackie RI
- Subjects
- Aging, Animals, DNA, Bacterial genetics, DNA, Ribosomal genetics, DNA, Single-Stranded isolation & purification, Diet, Ecosystem, Feces microbiology, Genetic Variation, Polymerase Chain Reaction methods, RNA, Ribosomal, 16S genetics, Swine growth & development, Bacteria genetics, Bacteria isolation & purification, Digestive System microbiology, Electrophoresis methods, Swine microbiology
- Abstract
The porcine gastrointestinal tract (GIT) microbiota has been studied to increase production efficiency, improve product quality, and help attempt to reduce disease. During the developmental period from birth through weaning, the intestinal microbiota undergoes a rapid ecological succession. There is interest in developing a monitoring technique that allows for analysis of bacterial population levels and shifts within the pig intestine. The objective of this study was to determine if denaturant gradient gel electrophoresis (DGGE) could be effectively applied to measure changes in bacterial populations of the pig GIT, as influenced by age, diet or compartment. Bacterial genetic diversity was determined using DGGE analysis of the V3 region of 16S rDNA PCR products (approximately 200 bp) obtained from primers specific for the domain Bacteria. Protocol development included optimization of: DNA extraction procedures, PCR amplification, removal of PCR artifacts, and optimization of gel preparation and image capture. DGGE analysis revealed diverse bacterial populations between pigs of different ages and among individual gut compartments. Comparison of fecal DNA from different aged pigs revealed several unique PCR product bands indicating the presence of unique bacterial populations. Comparison of different gut compartments demonstrated that bacterial populations were most similar (C, value > 50%) within a single compartment and between adjacent ones. Thus, DGGE can be used to examine bacterial diversity and population shifts in the pig GIT.
- Published
- 1999
- Full Text
- View/download PDF
203. Separation of double- and single-stranded DNA restriction fragments: capillary electrophoresis with polymer solutions under alkaline conditions.
- Author
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Liu Y and Kuhr WG
- Subjects
- DNA chemistry, DNA, Single-Stranded chemistry, Hydrogen-Ion Concentration, Osmolar Concentration, Polymers chemistry, Reproducibility of Results, Restriction Mapping, Solutions, DNA isolation & purification, DNA, Single-Stranded isolation & purification, Electrophoresis, Capillary methods
- Abstract
Capillary electrophoresis in buffers containing hydroxyethyl cellulose (HEC) was used to separate double- and single-stranded DNA restriction fragments under neutral and alkaline conditions in epoxy-coated capillaries. It was found that better resolution was achieved using highly entangled HEC solutions for a narrow range of DNA fragment sizes, while lower resolution was obtained over a wide separation range using diluted HEC solutions. Optimal resolution of these DNA fragments was obtained using buffers containing 0.5% HEC at pH 11 with plate numbers exceeding 3 x 10(6) plates/m. It was also found that the diffusion coefficients and electrophoretic mobilities of DNA fragments decreased with increasing pH. This may indicate a more extended DNA conformation and, therefore, enhancement of transient entanglement coupling between DNA and HEC polymers under alkaline condition. At pH 12, ss-DNA were well separated in entangled HEC solutions; however, the resolution of ss-DNA was significantly decreased in diluted polymer solution.
- Published
- 1999
- Full Text
- View/download PDF
204. Osmotically driven radial diffusion of single-stranded DNA fragments on an agarose bed as a convenient measure of DNA strand scission.
- Author
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Sestili P and Cantoni O
- Subjects
- Animals, Cell Nucleus chemistry, Cell Nucleus ultrastructure, DNA, Single-Stranded drug effects, DNA, Single-Stranded isolation & purification, Diffusion, Electrophoresis methods, Humans, Hydrogen-Ion Concentration, Indicators and Reagents, Mammals, Nucleic Acid Denaturation, Osmolar Concentration, Sepharose, U937 Cells, tert-Butylhydroperoxide pharmacology, DNA Damage, DNA, Single-Stranded chemistry
- Abstract
The present study describes the development and characterization of a novel technique, the alkaline-halo assay, for the assessment of DNA single strand breakage in mammalian cells. This technique allows the measurement of DNA lesions at the single cell level and presents the additional advantages of being rapid, sensitive, virtually costless and environmentally friendly, because it does not require the use of isotopes. The alkaline halo assay involves a series of sequential steps in which the cells are first treated, then embedded in melted agarose and spread onto microscope slides that are incubated for 2 min at ice-bath temperature to allow complete geling. The slides are then incubated for 20 min in a high salt alkaline lysis solution, for an additional 15 min in a hypotonic alkaline solution and, finally, for 10 min in ethidium bromide. Under these conditions, single-stranded DNA fragments spread radially from the nuclear cage and generate a fluorescent image that resembles a halo concentric to the nucleus remnants. The area of the halos increased at increasing levels of DNA fragmentation and this process was associated with a progressive reduction of areas of the nuclear remnants. These events were conveniently monitored with a fluorescence microscope and quantified by image processing analysis. The sensitivity of the alkaline-halo assay, which is based on the osmotically driven radial diffusion of single-stranded DNA fragments through agarose pores, is remarkably similar to that of the widely used alkaline elution and comet assays.
- Published
- 1999
- Full Text
- View/download PDF
205. Sequence analysis of plasmid pKJ50 from Bifidobacterium longum.
- Author
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Park MS, Shin DW, Lee KH, and Ji GE
- Subjects
- Amino Acid Sequence, Base Sequence, Cloning, Molecular, DNA Replication, DNA, Single-Stranded isolation & purification, DNA-Binding Proteins genetics, Genes, Bacterial, Genetic Vectors, Molecular Sequence Data, Open Reading Frames, Protein Structure, Secondary, Proteins genetics, RNA, Bacterial genetics, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Trans-Activators genetics, Bacterial Proteins, Bifidobacterium genetics, DNA Helicases, Plasmids genetics
- Abstract
The complete nucleotide sequence of a plasmid, pKJ50, isolated from an intestinal bacterium, Bifidobacterium longum KJ, has been determined. The plasmid was analysed and found to be 4960 bp in size with a G+C content of 61.7 mol%. Computer analysis of sequence data revealed three major ORFs encoding putative proteins of 31.5 (ORFI), 24.5 (ORFII) and 38.6 kDa (ORFIII). ORFI encodes a protein with a pI of 10.18 and shows relatively high amino acid sequence similarity (more than 60%) with several plasmid replication proteins from Gram-positive and -negative bacteria. Southern blot analysis showed that pKJ50 accumulates an ssDNA intermediate, suggesting that it replicates by a rolling-circle mechanism. Upstream of ORFI, three sets of repeated sequences resembling iteron structures of related plasmids were identified. ORFIII encodes a protein with a pI of 10.97. It also shows a high level of amino acid sequence similarity with some plasmid mobilization proteins. Upstream of ORFIII, a 12 bp stretch resembles an oriT DNA sequence with inverted repeats identical to those found in conjugative plasmids. Hydropathy plot analysis of ORFII, encoding an acidic protein (pI = 4.95), suggests it is a transmembrane protein. Several interesting palindromic sequences, repeat sequences and hairpin-loop structures around ORFI, which might confer regulatory effects on the replication of the plasmid, were also noted. Reverse transcriptase PCR (RT-PCR) and in vitro translation confirmed the expression of ORFI and ORFII. RT-PCR produced amplified DNA fragments of the expected sizes, corresponding to ORFI and ORFII. However, no RT-PCR product corresponding to ORFIII was obtained. In vitro translation showed protein bands of the expected sizes, corresponding to each ORF. A shuttle vector capable of transforming Bifidobacterium animalis MB209 was constructed by cloning pKJ50 and a chloramphenicol resistance gene into pBR322.
- Published
- 1999
- Full Text
- View/download PDF
206. Effect of p53 tumor suppressor on nucleotide excision repair in human colon carcinoma cells treated with 4-nitroquinoline 1-oxide.
- Author
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Seo YR, Lee SH, Han SS, and Ryu JC
- Subjects
- 4-Nitroquinoline-1-oxide, Carcinogens, Carcinoma metabolism, Colonic Neoplasms metabolism, Comet Assay, DNA, Single-Stranded isolation & purification, Dose-Response Relationship, Drug, Enzyme-Linked Immunosorbent Assay, Fluorescein-5-isothiocyanate, Humans, Tumor Cells, Cultured, Tumor Suppressor Protein p53 isolation & purification, Tumor Suppressor Protein p53 pharmacology, Carcinoma drug therapy, Colonic Neoplasms drug therapy, DNA Damage drug effects, DNA Repair drug effects, DNA, Single-Stranded drug effects, Tumor Suppressor Protein p53 therapeutic use
- Abstract
In probing the mechanism of nucleotide excision repair (NER) in response to 4-nitroquinoline 1-oxide (4NQO)-induced DNA damage, the effect of p53 tumor suppressor was investigated. The effect of p53 protein on the repair of damaged DNA was examined by comet assay. Expression of p53 and p21(Waf1/Cip1) proteins was measured by the Enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry, respectively. Compared to RKO cells having the wild-type p53 gene, increased cytotoxicity by 4NQO was observed in RKOmp53 cells with a mutation in p53 protein. DNA single strand breaks (SSB), indicative of the DNA repair, were considerably increased in 4NQO-treated RKO cells. Also, the expression of p53 and p21 proteins was significantly increased in 4NQO-treated RKO cells. In RKOmp53 cells, no effect of 4NQO on p21 expression was observed. Our findings suggest that 4NQO-induced NER is p53-dependent and involves up-regulation of its downstream regulator, p21(Waf1/Cip1) proteins.
- Published
- 1999
207. PCR-based assays for strand-specific measurement of DNA damage and repair. I. Strand-specific quantitative PCR.
- Author
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Grimaldi KA, Bingham JP, and Hartley JA
- Subjects
- Animals, Base Sequence, Biotinylation, Cell Adhesion, Cells, Cultured, Cisplatin toxicity, DNA drug effects, DNA isolation & purification, DNA, Single-Stranded genetics, DNA, Single-Stranded isolation & purification, Electrophoresis, Agar Gel methods, Humans, Indicators and Reagents, Introns, Molecular Sequence Data, Mutagenesis, Mutagens toxicity, DNA genetics, DNA Damage, DNA Repair, Genes, ras, Polymerase Chain Reaction methods
- Published
- 1999
- Full Text
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208. In vitro base excision repair assay using mammalian cell extracts.
- Author
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Frosina G, Cappelli E, Fortini P, and Dogliotti E
- Subjects
- Animals, Base Sequence, CHO Cells, Cell Culture Techniques methods, Cricetinae, DNA, Circular chemistry, DNA, Circular isolation & purification, DNA, Circular metabolism, DNA, Single-Stranded chemistry, DNA, Single-Stranded isolation & purification, DNA, Single-Stranded metabolism, Escherichia coli enzymology, Genetic Techniques, Mammals, N-Glycosyl Hydrolases, Oligodeoxyribonucleotides chemistry, Plasmids metabolism, Restriction Mapping methods, Uracil-DNA Glycosidase, DNA Damage, DNA Glycosylases, DNA Repair, Plasmids chemistry, Tissue Extracts metabolism
- Published
- 1999
- Full Text
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209. Assessment of hepatitis C virus sequence complexity by electrophoretic mobilities of both single-and double-stranded DNAs.
- Author
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Wang YM, Ray SC, Laeyendecker O, Ticehurst JR, and Thomas DL
- Subjects
- Cloning, Molecular, DNA, Single-Stranded chemistry, DNA, Single-Stranded isolation & purification, DNA, Viral chemistry, DNA, Viral isolation & purification, Entropy, Hepatitis C blood, Hepatitis C virology, Humans, Molecular Sequence Data, Polymerase Chain Reaction methods, Polymorphism, Single-Stranded Conformational, Prospective Studies, Reverse Transcriptase Polymerase Chain Reaction methods, Sensitivity and Specificity, DNA, Single-Stranded genetics, DNA, Viral genetics, Genetic Variation, Hepacivirus genetics
- Abstract
To assess genetic variation in hepatitis C virus (HCV) sequences accurately, we optimized a method for identifying distinct viral clones without determining the nucleotide sequence of each clone. Twelve serum samples were obtained from seven individuals soon after they acquired HCV during a prospective study, and a 452-bp fragment from the E2 region was amplified by reverse transcriptase PCR and cloned. Thirty-three cloned cDNAs representing each specimen were assessed by a method that combined heteroduplex analysis (HDA) and a single-stranded conformational polymorphism (SSCP) method to determine the number of clonotypes (electrophoretically indistinguishable cloned cDNAs) as a measure of genetic complexity (this combined method is referred to herein as the HDA+SSCP method). We calculated Shannon entropy, incorporating the number and distribution of clonotypes into a single quantifier of complexity. These measures were evaluated for their correlation with nucleotide sequence diversity. Blinded analysis revealed that the sensitivity (ability to detect variants) and specificity (avoidance of false detection) of the HDA+SSCP method were very high. The genetic distance (mean +/- standard deviation) between indistinguishable cloned cDNAs (intraclonotype diversity) was 0.6% +/- 0.9%, and 98.7% of cDNAs differed by <2%, while the mean distance between cloned cDNAs with different patterns was 4.0% +/- 3.2%. The sensitivity of the HDA+SSCP method compared favorably with either HDA or the SSCP method alone, which resulted in intraclonotype diversities of 1.6% +/- 1.8% and 3.5% +/- 3.4%, respectively. The number of clonotypes correlated strongly with genetic diversity (R2, 0.93), but this correlation fell off sharply when fewer clones were assessed. This HDA+SSCP method accurately reflected nucleotide sequence diversity among a large number of viral cDNA clones, which should enhance analyses to determine the effects of viral diversity on HCV-associated disease. If sequence diversity becomes recognized as an important parameter for staging or monitoring of HCV infection, this method should be practical enough for use in laboratories that perform nucleic acid testing.
- Published
- 1998
- Full Text
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210. Migration of single-stranded DNA in polyacrylamide gels during electrophoresis.
- Author
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Pluen A, Tinland B, Sturm J, and Weill G
- Subjects
- Diffusion, Electric Conductivity, Solutions, Acrylic Resins, DNA, Single-Stranded isolation & purification, Electrophoresis, Polyacrylamide Gel methods
- Abstract
By using fluorescence recovery after photobleaching (FRAP) and electric birefringence, the migration of single-stranded DNA in polyacrylamide gels and orientation as a response to an electric pulse were investigated. Electrophoretic mobility is in good agreement with the model of biased reptation including fluctuations. The determination of the electrophoretic mobility in solution, mu0, allows an estimation of the gel pore diameter seen by the molecule. As previously observed for double-stranded DNA, the electric birefringence results from two processes: the alignment of the molecule along the electric field and the elongation of the primitive path in the gel, for long single-stranded DNA (>2000 bases). The combination of results obtained with the two techniques allows us to propose experimental conditions to improve the separation of single-stranded DNA with pulsed field techniques.
- Published
- 1998
- Full Text
- View/download PDF
211. The differentiation of Phytophthora species that are pathogenic on potatoes by an asymmetric PCR combined with single-strand conformation polymorphism analysis.
- Author
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Scott DL, Clark CW, Fyffe AE, Walker MD, and Deah KL
- Subjects
- Base Sequence, DNA Primers genetics, DNA, Fungal genetics, DNA, Fungal isolation & purification, DNA, Single-Stranded genetics, DNA, Single-Stranded isolation & purification, Molecular Sequence Data, Phytophthora classification, Plant Diseases microbiology, Solanum tuberosum microbiology, Species Specificity, Virulence genetics, Phytophthora genetics, Phytophthora pathogenicity, Polymerase Chain Reaction methods, Polymorphism, Single-Stranded Conformational
- Abstract
The goal of this study was to develop a polymerase chain reaction (PCR) capable of differentiating Phytophthora species that are pathogenic on potatoes using a single primer pair. To achieve this objective, primers were derived from conserved regions flanking variable sequences in the internal transcribed spacer 1 (ITS1) of Phytophthora species. One primer pair produced a 140 bp product from P. infestans, P. erythroseptica and P. nicotianae. The PCR products were purified and used in an asymmetric PCR (A-PCR) protocol to generate single-strand DNA (ssDNA). The ssDNA of the Phytophthora potato pathogens reproducibly migrated in non-denaturing polyacrylamide gels in a species-specific manner.
- Published
- 1998
- Full Text
- View/download PDF
212. Formation of plasmid DNA strand breaks induced by low-energy ion beam: indication of nuclear stopping effects.
- Author
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Chen Y, Jiang B, Chen Y, Ding X, Liu X, Chen C, Guo X, and Yin G
- Subjects
- DNA, Bacterial isolation & purification, DNA, Bacterial metabolism, DNA, Single-Stranded isolation & purification, DNA, Single-Stranded metabolism, DNA, Single-Stranded radiation effects, Elementary Particle Interactions, Escherichia coli, Linear Energy Transfer, Models, Theoretical, Monte Carlo Method, Nitrogen adverse effects, Plasmids metabolism, DNA Damage radiation effects, DNA, Bacterial radiation effects, Ions, Plasmids radiation effects
- Abstract
Plasmid pGEM 3zf(+) was irradiated by nitrogen ion beam with energies between 20 and 100 keV and the fluence kept as 1x10(12)ions/cm2. The irradiated plasmid was assayed by neutral electrophoresis and quantified by densitometry. The yields of DNA with single-strand and double-strand breaks first increased then decreased with increasing ion energy. There was a maximal yield value in the range of 20-100 keV. The relationship between DNA double-strand breaks (DSB) cross-section and linear energy transfer (LET) also showed a peak-shaped distribution. To understand the physical process during DNA strand breaks, a Monte Carlo calculation code known as TRIM (Transport of Ions in Matter) was used to simulate energy losses due to nuclear stopping and to electronic stopping. It can be assumed that nuclear stopping plays a more important role in DNA strand breaks than electronic stopping in this energy range. The physical mechanisms of DNA strand breaks induced by a low-energy ion beam are also discussed.
- Published
- 1998
- Full Text
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213. Use of the restriction enzyme AvaI and exo- Bst polymerase in strand displacement amplification.
- Author
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Milla MA, Spears PA, Pearson RE, and Walker GT
- Subjects
- DNA Primers, DNA, Bacterial isolation & purification, DNA, Single-Stranded isolation & purification, Nucleic Acid Denaturation, DNA Polymerase I genetics, Deoxyribonucleases, Type II Site-Specific genetics, Exodeoxyribonucleases genetics, Geobacillus stearothermophilus enzymology, Geobacillus stearothermophilus genetics
- Published
- 1998
- Full Text
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214. Single-stranded DNA production from phagemids containing GC-rich DNA fragments.
- Author
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Kuczek K, Kotowska M, Wiernik D, and Mordarski M
- Subjects
- DNA, Fungal genetics, DNA, Recombinant genetics, DNA, Viral genetics, Oligodeoxyribonucleotides chemistry, Sequence Analysis, DNA, Streptomyces genetics, Bacteriophage M13 genetics, DNA, Single-Stranded isolation & purification, Plasmids genetics
- Published
- 1998
- Full Text
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215. Analysis of two multiplexed short tandem repeat systems using capillary electrophoresis with multiwavelength fluorescence detection.
- Author
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Isenberg AR, Allen RO, Keys KM, Smerick JB, Budowle B, and McCord BR
- Subjects
- DNA, Single-Stranded chemistry, DNA, Single-Stranded isolation & purification, Humans, Nucleic Acid Denaturation, Spectrometry, Fluorescence, Electrophoresis, Capillary methods, Repetitive Sequences, Nucleic Acid
- Abstract
A series of experiments was performed to analyze the utility of capillary electrophoresis (CE) with multiwavelength detection capabilities for multiplex typing of short tandem repeat loci. Characteristics of the sieving polymer, hydroxyethylcellulose, which affect resolution of single strand (ss) DNA fragments were examined. Additionally, the effects of denaturant in the polymer system, separation voltage, and analysis temperature were studied to ascertain their effects on DNA separations and capillary lifetime. The use of elevated run temperature (60 degrees C) was found to improve sizing precision, to increase the lifetime of capillaries (100 runs or more per capillary), and to provide runtimes of under 20 min. Finally, 100 individual human DNA samples were typed successfully using CE. The average resolution obtained was 1.4 bases for a 200 base fragment with a standard deviation of sizing of 0.2 bases, allowing all alleles examined to be distinguished clearly.
- Published
- 1998
- Full Text
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216. S1 mapping using single-stranded DNA probes.
- Author
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Viville S and Mantovani R
- Subjects
- DNA, Single-Stranded genetics, DNA, Single-Stranded isolation & purification, Gene Expression Regulation, Nucleic Acid Hybridization, RNA, Messenger genetics, RNA, Messenger isolation & purification, DNA Probes genetics, DNA Probes isolation & purification, Molecular Probe Techniques, Restriction Mapping methods, Single-Strand Specific DNA and RNA Endonucleases
- Published
- 1998
- Full Text
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217. Mechanical separation of the complementary strands of DNA.
- Author
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Essevaz-Roulet B, Bockelmann U, and Heslot F
- Subjects
- Bacteriophage lambda, Base Composition, Biophysical Phenomena, Microscopy, Atomic Force, Nucleic Acid Denaturation, Optics and Photonics, Rotation, Thermodynamics, Biophysics, DNA, Single-Stranded isolation & purification, DNA, Viral, Micromanipulation methods
- Abstract
We describe the mechanical separation of the two complementary strands of a single molecule of bacteriophage lambda DNA. The 3' and 5' extremities on one end of the molecule are pulled progressively apart, and this leads to the opening of the double helix. The typical forces along the opening are in the range of 10-15 pN. The separation force signal is shown to be related to the local GC vs. AT content along the molecule. Variations of this content on a typical scale of 100-500 bases are presently detected.
- Published
- 1997
- Full Text
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218. Improved single-strand DNA sizing accuracy in capillary electrophoresis.
- Author
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Rosenblum BB, Oaks F, Menchen S, and Johnson B
- Subjects
- Algorithms, Electrophoresis, Capillary statistics & numerical data, Evaluation Studies as Topic, Molecular Weight, Nucleic Acid Denaturation, Pyrrolidinones, Temperature, Urea, DNA, Single-Stranded chemistry, DNA, Single-Stranded isolation & purification, Electrophoresis, Capillary methods
- Abstract
Interpolation algorithms can be developed to size unknown single-stranded (ss) DNA fragments based on their electrophoretic mobilities, when they are compared with the mobilities of standard fragments of known sizes; however, sequence-specific anomalous electrophoretic migration can affect the accuracy and precision of the called sizes of the fragments. We used the anomalous migration of ssDNA fragments to optimize denaturation conditions for capillary electrophoresis. The capillary electrophoretic system uses a refillable polymer that both coats the capillary wall to suppress electro-osmotic flow and acts as the sieving matrix. The addition of 8 M urea to the polymer solution, as in slab gel electrophoresis, is insufficient to fully denature some anomalously migrating ssDNA fragments in this capillary electrophoresis system. The sizing accuracy of these fragments is significantly improved by the addition of 2-pyrrolidinone, or increased capillary temperature (60 degrees C). the effect of these two denaturing strategies is additive, and the best accuracy and precision in sizing results are obtained with a combination of chemical and thermal denaturation.
- Published
- 1997
- Full Text
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219. Isolation and characterization of replication protein A (RP-A) from tobacco cells.
- Author
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Garcia-Maya MM and Buck KW
- Subjects
- Cells, Cultured, DNA Replication, DNA, Single-Stranded isolation & purification, DNA, Single-Stranded metabolism, DNA-Binding Proteins pharmacology, DNA-Directed DNA Polymerase drug effects, DNA-Directed DNA Polymerase metabolism, Replication Protein A, DNA Helicases isolation & purification, DNA Helicases metabolism, DNA-Binding Proteins isolation & purification, DNA-Binding Proteins metabolism, Plants, Toxic, Nicotiana chemistry
- Abstract
Replication protein A (RP-A) was isolated from tobacco suspension cells and purified to near homogeneity by a procedure involving isolation of protoplasts, preparation of nuclei, nuclear lysis, binding to a column of single-stranded (ss) DNA cellulose and elution at different salt concentrations. The purified protein contained three subunits with molecular masses of 70, 34 and 14 kDa, and was free from nuclease activity. Tobacco RP-A had a high affinity for ssDNA. Binding competition experiments indicated only a weak affinity for double-stranded DNA and no detectable affinity for ssRNA. Photochemical cross-linking experiments indicated that the 70 kDa subunit has the ssDNA-binding activity. Tobacco RP-A was able to stimulate the activity of a tobacco alpha-like DNA polymerase about 4-fold. This is the first isolation of RP-A from a plant and the procedure may be generally applicable to other plant species.
- Published
- 1997
- Full Text
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220. Fractionation, phosphorylation and ligation on oligonucleotide microchips to enhance sequencing by hybridization.
- Author
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Dubiley S, Kirillov E, Lysov Y, and Mirzabekov A
- Subjects
- Base Composition, Base Sequence, DNA chemistry, DNA Primers, DNA, Single-Stranded isolation & purification, Electrophoresis, Polyacrylamide Gel instrumentation, Electrophoresis, Polyacrylamide Gel methods, Globins genetics, Humans, Molecular Sequence Data, Phosphorylation, Polymerase Chain Reaction, DNA, Single-Stranded chemical synthesis, Nucleic Acid Hybridization methods, Oligodeoxyribonucleotides chemical synthesis, Oligodeoxyribonucleotides isolation & purification
- Abstract
Oligonucleotide microchips are manufactured by immobilizing presynthesized oligonucleotides within 0.1 x 0.1 x 0.02 mm or 1 x 1 x 0.02 mm polyacrylamide gel pads arranged on the surface of a microscope slide. The gel pads are separated from each other by hydrophobic glass spacers and serve as a kind of 'microtest tube' of 200 pl or 20 nl volume, respectively. Fractionation of single-stranded DNAs is carried out by their hybridization with chip pads containing immobilized 10mers. DNA extracted separately from each pad is transferred onto a sequencing chip and analyzed thereon. The chip, containing a set of 10mers, was enzymatically phosphorylated, then hybridized with DNA and ligated in a site-directed manner with a contiguously stacked 5mer. Several cycles of successive hybridization-ligation of the chip-bound 10mers with different contiguously stacked 5mers and hybridized with DNA were carried out to sequence DNA containing tetranucleotide repeats. Combined use of these techniques show significant promise for sequence comparison of homologous regions in different genomes and for sequence analysis of comparatively long DNA fragments or DNA containing internal repeats.
- Published
- 1997
- Full Text
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221. Efficient preparation of single-stranded DNA for in vitro selection.
- Author
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Kujau MJ and Wölfl S
- Subjects
- DNA, Single-Stranded chemistry, Polymerase Chain Reaction, DNA, Single-Stranded isolation & purification
- Abstract
In vitro selection of aptamers requires the reliable enzymatic preparation of large amounts of (+) single-stranded DNA molecules. This can be achieved by selective enzymatic digest of 5'-phosphorylated (-) strands from PCR products, a method already widely used in sequencing of PCR products. Here we present an adaptation of this method to prepare large pools of single-stranded DNA molecules for in vitro selection.
- Published
- 1997
- Full Text
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222. Deoxyribonuclease treatment improves the homogeneity of single-stranded DNA preparations.
- Author
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Aliev TK, Panina AA, Korobko VG, and Varfolomeyev SD
- Subjects
- Bacteriophages genetics, Electrophoresis, Agar Gel, Escherichia coli enzymology, Escherichia coli genetics, Nucleic Acid Denaturation, Plasmids, Transformation, Genetic, DNA, Single-Stranded isolation & purification, DNA, Viral isolation & purification, Deoxyribonucleases chemistry
- Abstract
The isolation of single-stranded (ss) phagemid DNA using standard protocols often results in impure preparations, which contain undesirable quantities of chromosomal and/or double-stranded (ds) phagemid DNA. Here we report a simple and efficient method for elimination of virtually all dsDNA by incubation of phagemid viral particles with deoxyribonuclease I. In addition to analyzing the ratio of linear-to-circular topological forms of ssDNA after deoxyribonuclease I treatment, we verified that no decrease in transformation efficiency occurred and demonstrated that ssDNA molecules covered by capsid proteins remained intact following such treatment.
- Published
- 1997
- Full Text
- View/download PDF
223. A single vector cloning, mutagenesis and expression system.
- Author
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Towler EM, Stebbins JW, and Debouck C
- Subjects
- Amino Acid Sequence, Bacteriophage M13 genetics, Bacteriophage lambda genetics, Base Sequence, DNA, Single-Stranded biosynthesis, DNA, Single-Stranded isolation & purification, Gene Expression, Molecular Sequence Data, Replication Origin genetics, Cloning, Molecular methods, Escherichia coli genetics, Genetic Vectors genetics, Mutagenesis, Site-Directed
- Abstract
We describe a multipurpose Escherichia coli vector, pOTSf1blue, that can be utilized for high efficiency subcloning, epitope-tagged protein overexpression, authentic protein overexpression and efficient mutagenesis.
- Published
- 1996
- Full Text
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224. Directional random oligonucleotide primed (DROP) global amplification of cDNA: its application to subtractive cDNA cloning.
- Author
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Hampson IN, Hampson L, and Dexter TM
- Subjects
- Blotting, Northern, Cell Line, DNA, Complementary isolation & purification, DNA, Single-Stranded isolation & purification, DNA-Directed RNA Polymerases genetics, Promoter Regions, Genetic, Viral Proteins, Cloning, Molecular, DNA Primers, DNA, Complementary genetics, Polymerase Chain Reaction methods
- Abstract
We describe a method of global PCR amplification of cDNA such that the strand sense is maintained. The products of this process are random primed fragments ranging in size from 100 to 500 bp which facilitates uniform PCR amplification of total cDNA. Directional incorporation of a T7 RNA polymerase initiator/promoter sequence allows efficient synthesis of total sense RNA from this material and the use of a biotinylated primer permits the separation of single-stranded cDNA. Isolation of these products from different cell types provides a renewable source of target single-stranded cDNA and driver RNA from limited cell numbers and we demonstrate their use for subtractive hybridisation cloning of differentially expressed cDNAs.
- Published
- 1996
- Full Text
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225. Fork-like DNA templates support bypass replication of lesions that block DNA synthesis on single-stranded templates.
- Author
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Hoffmann JS, Pillaire MJ, Lesca C, Burnouf D, Fuchs RP, Defais M, and Villani G
- Subjects
- 2-Acetylaminofluorene, Animals, Antineoplastic Agents, Base Sequence, CHO Cells, Carcinogens, Cisplatin, Cricetinae, DNA, Single-Stranded isolation & purification, Molecular Sequence Data, Templates, Genetic, DNA Adducts, DNA Damage, DNA Replication, DNA, Single-Stranded chemistry, DNA, Single-Stranded metabolism, Nucleic Acid Conformation
- Abstract
DNA replication is an asymmetric process involving concurrent DNA synthesis on leading and lagging strands. Leading strand synthesis proceeds concomitantly with fork opening, whereas synthesis of the lagging strand essentially takes place on a single-stranded template. The effect of this duality on DNA damage processing by the cellular replication machinery was tested using eukaryotic cell extracts and model DNA substrates containing site-specific DNA adducts formed by the anticancer drug cisplatin or by the carcinogen N-2-acetylaminofluorene. Bypass of both lesions was observed only with fork-like substrates, whereas complete inhibition of DNA synthesis occurred on damaged single-stranded DNA substrates. These results suggest a role for additional accessory factors that permit DNA polymerases to bypass lesions when present in fork-like DNA.
- Published
- 1996
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226. Integration of the DNA of a novel filamentous bacteriophage VSK from Vibrio cholerae 0139 into the host chromosomal DNA.
- Author
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Kar S, Ghosh RK, Ghosh AN, and Ghosh A
- Subjects
- Bacteriophages isolation & purification, Bacteriophages ultrastructure, Blotting, Southern, Chromosomes genetics, DNA, Bacterial analysis, DNA, Single-Stranded analysis, DNA, Single-Stranded isolation & purification, DNA, Viral analysis, Genome, Microscopy, Electron, Restriction Mapping, Vibrio cholerae genetics, Bacteriophages genetics, DNA Replication, Vibrio cholerae virology, Virus Integration, Virus Replication
- Abstract
An unusual filamentous bacteriophage, VSK, containing single-stranded, circular DNA as its genome was isolated from Vibrio cholerae 0139 strains P07 and B04. Unlike other single-stranded DNA phages, VSK can integrate its genome into the chromosome of the host and enter into a lysogenic state. The double-stranded replicative form (RF) of the single-stranded phage DNA was isolated. A restriction map of the VSK RF DNA was constructed using HaeII, AvaII, ClaI and XbaI. By Southern blot analysis of the chromosomal DNA of the lysogen using labeled phage DNA as probe, the attachment site (attP) on the viral genome was also identified.
- Published
- 1996
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227. Nuclear antigens, as DNA, of DSB389 MAb and some other anti-desmin monoclonal antibodies.
- Author
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Kamei H
- Subjects
- Adenocarcinoma immunology, Adenocarcinoma pathology, Antibody Specificity, Antigens, Neoplasm immunology, Antigens, Nuclear, Cetrimonium, Cetrimonium Compounds pharmacology, DNA isolation & purification, DNA, Neoplasm immunology, DNA, Single-Stranded isolation & purification, Detergents pharmacology, Enzyme-Linked Immunosorbent Assay, Fluorescent Antibody Technique, Indirect, HeLa Cells drug effects, HeLa Cells immunology, Humans, Immunoblotting, Intermediate Filaments immunology, Intermediate Filaments ultrastructure, Nuclear Proteins isolation & purification, Octoxynol pharmacology, Tumor Cells, Cultured drug effects, Tumor Cells, Cultured immunology, Antibodies, Monoclonal immunology, DNA immunology, DNA, Single-Stranded immunology, Desmin immunology, Nuclear Matrix immunology, Nuclear Proteins immunology
- Abstract
The anti-desmin monoclonal antibodies (MAbs) DSB389, AC54, and DSB860 recognize intermediate filaments (IFs) and nuclear antigens that appear granular, locate around chromosomes, and are insoluble following 0.5% Triton X-100 and cetyltrimethylammonium bromide (CTAB) extraction. Nuclear antigens of the MAbs were searched for in an IFs-deficient clone of SW13 cells. Reactive materials specific to DSB389, AC54, and DSB860 MAbs were trapped at the top of the gel of SDS-agarose-PAGE. The reactivity of the materials disappeared after treatment with DNase I. The reactivity, or trapping of this material at the top of the gel, required previous heat treatment of the sample before application to the gel. The MAbs recognized both single-stranded and double-stranded DNA in ELISA. These results indicate that at least the main nuclear antigens of DSB389, DSB860 and AC54 MAbs are DNA.
- Published
- 1996
- Full Text
- View/download PDF
228. An improved method of alkaline sucrose density gradient sedimentation to detect less than one lesion per 1 Mb DNA.
- Author
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Yamada K, Kameyama Y, and Inoue S
- Subjects
- Alkalies, DNA drug effects, DNA radiation effects, Mutagens pharmacology, Nucleic Acid Denaturation, Sucrose, Sulfuric Acid Esters pharmacology, Ultraviolet Rays adverse effects, X-Rays adverse effects, Centrifugation, Density Gradient methods, DNA Damage, DNA, Single-Stranded isolation & purification
- Abstract
We improved alkaline sucrose density gradient sedimentation to detect very long single-strand DNA at the megabase level (from less than 1 to about 4 Mb). Hitherto, these have not sedimented correctly due to some artifacts. One artifact was aggregation of sticky DNA and proteins formed in the gradient. Then, in some gradients, biphasic distribution was observed, the major peak of which was reasonable as a result of random scission by X-rays, but the minor, fast-sedimenting population was another artifact resulting from incomplete denaturation of the DNA. We mainly reduced the centrifugal force and used a solution for cell lysis with a high concentration of salt. By means of this procedure, DNA single-strand breaks induced by relatively low doses of X-rays and subsequent repair processes can be measured in human fibroblasts. The protocol is also applicable to the study of DNA damage accompanied by strand scission, such as by UV or dimethyl sulfate as well as their repair. The technique is sensitive enough to detect even single-strand breaks induced by 0.1 J/m2 UV and sufficiently reproducible that breaks induced by increasing UV dosages were dose dependent. Thus, this technique was proven to be very sensitive, reliable and simple to perform. Therefore, this improvement will be extremely useful to investigators studying DNA repair.
- Published
- 1996
- Full Text
- View/download PDF
229. Immobilized metal affinity chromatography of DNA.
- Author
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Min C and Verdine GL
- Subjects
- Metals, Polymerase Chain Reaction, Chromatography, Affinity methods, DNA, Single-Stranded isolation & purification
- Abstract
Many of the most widely employed operations in molecular biology hinge upon the use of single-stranded DNA as a probe or template. Here we report a straightforward method by which to produce long single-stranded DNA molecules using the polymerase chain reaction (PCR) in combination with immobilized metal affinity chromatography (IMAC). We demonstrate that a tag consisting of six successive 6-histaminylpurine (H) residues (H6-tag) endows a DNA strand with selective retentivity onto a Ni2+-NTA-agarose chromatography matrix. The H6-tagged strand can then be eluted from the resin using 200 mM imidazole. Quantitative phosphorimaging analysis revealed that the PCR/IMAC procedure typically yields unmodified strands comprising >90% of the unbound DNA and H6-tagged strands comprising >95% of the bound fractions. DNA strands generated in this manner are shown to be excellent substrates for template-directed polymerization. The chemistry reported herein should facilitate a wide variety of operations in molecular biology, including automated DNA sequencing, hybridization screening of DNA libraries, assembly of gene cassettes, run-off transcription, site-directed mutagenesis and footprinting of protein-DNA complexes by template-directed interference footprinting.
- Published
- 1996
- Full Text
- View/download PDF
230. Rapid preparation of single stranded DNA from PCR products by streptavidin induced electrophoretic mobility shift.
- Author
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Pagratis NC
- Subjects
- Base Sequence, Electrophoresis, Polyacrylamide Gel, Molecular Sequence Data, Streptavidin, Bacterial Proteins, DNA, Single-Stranded isolation & purification, Polymerase Chain Reaction methods
- Abstract
Streptavidin induced electrophoretic mobility shift was used to prepare single stranded (ss) DNA amplified with the polymerase chain reaction in the presence of a biotinylated and a non-biotinylated primer. A variety of denaturing conditions, including incubation at 95 degrees C in 50% formamide can be used without disrupting the streptavidin-biotinylated-ssDNA complex. Following electrophoresis, pure non-biotinylated DNA can be efficiently recovered from 7 M urea gels because it is well separated from the severely retarded streptavidin-biotinylated-ssDNA complex. Quantitative complexing of biotinylated ssDNA can occur at a streptavidin to DNA molar ratio of 1 or more.
- Published
- 1996
- Full Text
- View/download PDF
231. Characteristics of single-stranded DNA separation by capillary gel electrophoresis.
- Author
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Kamahori M and Kambara H
- Subjects
- Acrylic Resins chemistry, Electrochemistry, Models, Chemical, Particle Size, Time Factors, DNA, Single-Stranded isolation & purification, Electrophoresis, Capillary methods, Electrophoresis, Polyacrylamide Gel methods
- Abstract
The optimization of electrophoretic conditions for fast separation of single stranded DNA by capillary gel electrophoresis was investigated. Mobilities and band broadening of the DNA fragments were measured for a 4% T, 5% C gel at 100-300 V/cm. The resolution of DNA peaks was evaluated for band spacing and band width. Band spacing decreased with increasing electric field strengths as well as DNA fragment size; resolution for large DNA fragments also decreased at higher electric field strengths. The measured mobilities in a 4% T, 5% C gel were analyzed by the reptation theory and compared with those in a 9% T, 0% C matrix. The reptation plots indicate that the onset of reptation in a 4% T, 5% C gel occurs at a DNA size of about 200 bases and about 150 bases in a 9% T, 0% C matrix. The 4% T, 5% C gel proved superior to the 9% T, 0% C matrix with respect to resolution and analysis time. The optimum electric field strength and migration distance for specific DNA fragment length (200, 400 and 700 bases) in the shortest time were calculated from band spacing and the measured band width for the 4% T, 5% C gel at Rs = 0.5. Under optimized conditions, analysis of DNA fragments up to 200, 400 and 700 bases could be carried out in about 5, 50, and 230 min, respectively.
- Published
- 1996
- Full Text
- View/download PDF
232. Single-strand conformation polymorphism (SSCP) can be explained by semistable conformation dynamics of single-stranded DNA.
- Author
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Nakabayashi Y and Nishigaki K
- Subjects
- Base Sequence, Binding Sites genetics, DNA Primers genetics, DNA, Single-Stranded isolation & purification, Electrophoresis, Polyacrylamide Gel, Models, Chemical, Molecular Sequence Data, Nucleic Acid Conformation, Point Mutation, Thermodynamics, DNA, Single-Stranded chemistry, DNA, Single-Stranded genetics, Polymorphism, Single-Stranded Conformational
- Abstract
The cause of separation in gel electrophoresis between highly homologous ss (single-stranded) DNAs as observed in SSCP (single-strand conformation polymorphism) was pursued. Advancing our previous explanation [J. Biochem. 99, 663-671 (1986)], the mobility difference of ssDNAs was correlated to differences in their dynamic conformation (not to differences in their most stable structure), focusing on point-substituted sites. The contribution of semistable conformation dynamics was considered to be critical. Putative factors which may influence mobility (i.e., length of ssDNAs, location of substituted sites, and types of substitutions) were experimentally examined and critically discussed. Understanding of these phenomena should yield improvements in various techniques, such as SSCP, and in evaluation of the solution structures of DNA.
- Published
- 1996
- Full Text
- View/download PDF
233. Preparation of single-stranded phagemid DNA without chromosomal DNA contamination.
- Author
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Tang W, Liu JN, and Gurewich V
- Subjects
- Chemical Precipitation, Escherichia coli genetics, Nucleic Acid Denaturation, Nucleic Acid Renaturation, Bacteriophages genetics, DNA, Bacterial, DNA, Single-Stranded isolation & purification, DNA, Viral isolation & purification
- Published
- 1996
- Full Text
- View/download PDF
234. The telomeric GGGTTA repeats of Trypanosoma brucei contain the hypermodified base J in both strands.
- Author
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van Leeuwen F, Wijsman ER, Kuyl-Yeheskiely E, van der Marel GA, van Boom JH, and Borst P
- Subjects
- Animals, Base Sequence, DNA, Single-Stranded isolation & purification, Genome, Protozoan, Molecular Sequence Data, Nucleic Acid Hybridization, Telomere chemistry, Trypanosoma brucei brucei chemistry, Uracil analysis, DNA, Protozoan chemistry, Glucosides analysis, Repetitive Sequences, Nucleic Acid, Telomere genetics, Trypanosoma brucei brucei genetics, Uracil analogs & derivatives
- Abstract
We have previously shown that nuclear DNA of bloodstream from Trypanosoma brucei contains a novel base beta-glucosyl-hydroxymethyluracil, called J. Base J is enriched in minichromosome fractions but not in the minichromosome internal repeats, suggesting the association of J with telomeric DNA. To test whether J is present in the long telomeric (GGGTTA)n repeat arrays, which are 2-26 kb in T.brucei, we have purified these arrays both by hybrid selection and by isolating 2-26 kb fragments from DNA digested with multiple restriction enzymes. We find that in purified telomeric repeats approximately 13% of T is replaced by J, compared to 0.8% in total DNA, and we estimate that approximately 50% of the total J is in these repeats. Highly purified complementary strands of the repeats were obtained by alkaline CsCl equilibrium centrifugation. In the (TAACCC)n strand 14% of T was replaced by J. In the (GGGTTA)n strand approximately 36% of the second T was replaced by J; the first T was not detectably replaced. Modified bases have not been found in telomeric repeats before. How the bulky base J affects telomere function and structure in bloodstream form trypanosomes remains to be determined.
- Published
- 1996
- Full Text
- View/download PDF
235. Fractionation of nucleic acids into single-stranded and double-stranded forms.
- Author
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Beld M, Sol C, Goudsmit J, and Boom R
- Subjects
- DNA, Bacterial chemistry, DNA, Single-Stranded isolation & purification, DNA, Viral chemistry, RNA, Double-Stranded isolation & purification, Silicon Dioxide, DNA isolation & purification, Nucleic Acid Conformation, Nucleic Acid Heteroduplexes isolation & purification, RNA isolation & purification
- Published
- 1996
- Full Text
- View/download PDF
236. Improved method for the production of M13 phage and single-stranded DNA for DNA sequencing.
- Author
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Reddy P and McKenney K
- Subjects
- Bacteriophage M13 genetics, Base Sequence, Culture Media pharmacology, DNA, Single-Stranded isolation & purification, DNA, Viral isolation & purification, Escherichia coli virology, Molecular Sequence Data, Bacteriophage M13 physiology, DNA, Single-Stranded chemistry, Sequence Analysis, DNA methods
- Abstract
An improved method is described for the efficient production of M13 phage and M13 single-stranded (ss)DNA in a relatively short time period. Infection of E. coli (F') cells with as few as 5 phage particles can yield 10(12) phage particles/mL in 3 hours if the cells are grown in LB broth or SOB broth supplemented with about 5 mM Mg2+. The method tolerates large variations in the initial multiplicity of infection (5-5000 phage per 5 x 10(7) cells) and still yields about 10(12) phage particles/mL. These amounts are sufficient to purify 10-15 micrograms of ssDNA and to carry out at least 10-15 DNA sequencing reactions.
- Published
- 1996
- Full Text
- View/download PDF
237. Detection of the single-stranded DNA of Streptomyces plasmid pSA1.1 and a binding histone-like protein.
- Author
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Yokoyama E, Doi K, Kimura M, and Ogata S
- Subjects
- Amino Acid Sequence, Bacterial Proteins genetics, Bacterial Proteins metabolism, DNA Replication, DNA, Single-Stranded metabolism, DNA-Binding Proteins metabolism, Electrophoresis, Agar Gel, Histones isolation & purification, Histones metabolism, Molecular Sequence Data, Plasmids metabolism, Protein Binding, Streptomyces genetics, Streptomyces metabolism, Bacterial Proteins isolation & purification, DNA, Single-Stranded isolation & purification, DNA-Binding Proteins isolation & purification, Plasmids isolation & purification, Streptomyces chemistry
- Abstract
Streptomyces plasmid pSA1.1 accumulated single-stranded DNA as replication intermediates in S. lividans; therefore, this plasmid was considered to replicate by a rolling-circle mechanism. A DNA-binding protein (pI > 9.7 and about 10 kDa) was purified on a denatured DNA-Cellulose column, then on a native DNA-Cellulose column. The N-terminal amino acid sequence of this protein has a high homology with bacterial histone-like proteins. In the gel retardation assay, this protein bound with the single-stranded DNA of pSA1.1. We propose that this protein may participate in the replication of pSA1.1.
- Published
- 1996
- Full Text
- View/download PDF
238. Preparation of pure plasmid or cosmid DNA using single-strand affinity matrix and gel-filtration spin columns.
- Author
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Pham TT, Chillapagari S, and Suarez AR
- Subjects
- Chromatography, Affinity instrumentation, Chromatography, Gel instrumentation, Cloning, Molecular methods, Cosmids chemistry, Cosmids genetics, DNA Restriction Enzymes, DNA, Recombinant genetics, DNA, Single-Stranded genetics, Escherichia coli genetics, Genetic Vectors chemistry, Genetic Vectors genetics, Plasmids genetics, Chromatography, Affinity methods, Chromatography, Gel methods, DNA, Recombinant isolation & purification, DNA, Single-Stranded isolation & purification, Plasmids chemistry
- Abstract
A rapid method has been developed for ultrapure plasmid or cosmid DNA isolation from ten-mL to several hundred-mL cultures of Escherichia coli (midi to maxi prep). A cleared lysate is prepared by alkaline lysis, followed by a quick alcohol precipitation step. Denatured bacterial DNA and RNA having at least 20 nucleotides of single-stranded regions are removed from the supercoiled plasmid by binding strongly to the single-strand affinity matrix (SSAMTM). Plasmid DNA is then effectively purified on a gel-filtration spin column to remove SSAM, proteins, small RNA and salts. This method produces consistent yields of high-quality plasmids that are suitable for use in many molecular biology applications. In addition, recombinant cosmids of approximately 46 kb can be purified intact, free of chromosomal DNA.
- Published
- 1996
- Full Text
- View/download PDF
239. Simplified probe preparation facilitates S1 nuclease analysis.
- Author
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Noti JD and Reinemann BC
- Subjects
- DNA isolation & purification, DNA Probes genetics, Electrophoresis, Agar Gel, Integrins genetics, Leukocytes chemistry, Nucleic Acid Hybridization, Polymerase Chain Reaction, Promoter Regions, Genetic genetics, Single-Strand Specific DNA and RNA Endonucleases genetics, Transcription, Genetic genetics, DNA Probes isolation & purification, DNA, Single-Stranded isolation & purification, Single-Strand Specific DNA and RNA Endonucleases metabolism
- Published
- 1996
- Full Text
- View/download PDF
240. Purification of single-stranded M13 DNA by cooperative triple-helix-mediated affinity capture.
- Author
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Johnson AF, Wang R, Ji H, Chen D, Guilfoyle RA, and Smith LM
- Subjects
- Base Sequence, Binding Sites, Biotin, Chromatography, High Pressure Liquid methods, DNA, Single-Stranded chemistry, DNA, Viral chemistry, Genetic Vectors, Indicators and Reagents, Molecular Sequence Data, Oligodeoxyribonucleotides chemical synthesis, Sensitivity and Specificity, Templates, Genetic, Bacteriophage M13, DNA, Single-Stranded isolation & purification, DNA, Viral isolation & purification, Nucleic Acid Conformation
- Abstract
A solid-phase triple-helix-mediated affinity capture method is described for the purification of single-stranded M13 DNA for use as template in fluorescence-based DNA sequencing reactions. In this method, a biotinylated polypyrimidine oligonucleotide "loop" bound to streptavidin-coated magnetic beads is used to selectively capture single-stranded M13 DNA from high-titer phage supernatant through the formation of a cooperative triple helix (CTH) complex between the oligonucleotide and a polypurine site previously cloned into the M13 vector. Capture is accomplished at acidic pH to encourage triple-helix formation, while elution is performed at alkaline pH with heating to destroy the CTH complex. The beads can be reused up to three times without probe replenishment. Yields of M13 ssDNA in excess of 1 microgram per milliliter of culture are obtained, sufficient for use as template in fluorescence-based DNA sequencing reactions.
- Published
- 1996
- Full Text
- View/download PDF
241. M13 phage growth and single-stranded DNA preparation.
- Author
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Tomley FM
- Subjects
- DNA, Single-Stranded isolation & purification, DNA, Viral genetics, Escherichia coli genetics, Bacteriophage M13 genetics, Bacteriophage M13 growth & development, Cloning, Molecular methods, DNA, Single-Stranded genetics
- Published
- 1996
- Full Text
- View/download PDF
242. ErbB-2 expression is correlated with poor prognosis for patients with osteosarcoma.
- Author
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Onda M, Matsuda S, Higaki S, Iijima T, Fukushima J, Yokokura A, Kojima T, Horiuchi H, Kurokawa T, and Yamamoto T
- Subjects
- Adolescent, Adult, Aged, Base Sequence, Blotting, Southern, Bone Neoplasms mortality, Child, DNA, Single-Stranded isolation & purification, Female, Gene Amplification, Gene Expression Regulation, Neoplastic, Humans, Male, Middle Aged, Molecular Sequence Data, Osteosarcoma mortality, Polymerase Chain Reaction, Prognosis, Survival Analysis, Bone Neoplasms genetics, Osteosarcoma genetics, Receptor, ErbB-2 isolation & purification
- Abstract
Background: It has been reported that the c-erbB-2 protooncogene is frequently amplified and overexpressed in many types of cancers, except sarcomas and hematological malignancies., Methods: Expression of ErbB-2 in the tumors of 26 patients with conventional osteosarcoma was evaluated by immunoblotting. DNA from osteosarcoma tissues that expressed ErbB-2 were analyzed by Southern blot hybridization to examine gross rearrangement of the gene. The DNA was also surveyed for the presence of genetic mutation in the transmembrane domain of ErbB-2 by polymerase chain reaction-single-stranded DNA conformation polymorphism analysis. In addition, possible correlation of ErbB-2 expression with gender, age, histopathologic subtype, and response to chemotherapy was analyzed. Survival analysis was performed by the Kaplan-Meier test using the approximate chi-square statistic for the log-rank test., Results: The ErbB-2 protein was detected in 11 of 26 osteosarcoma tissues (42%) by immunoblot analysis. Expression of ErbB-2 was confirmed by immunohistochemical studies using specific anti-ErbB-2 monoclonal antibody. However, neither amplification of the c-erbB-2 gene nor evidence of significant genetic mutation was found in these osteosarcomas. Expression of ErbB-2 examined by immunoblotting was most strongly correlated with early pulmonary metastases (P < 0.05). Among the entire group of 26 patients in this study, Kaplan-Meier life table survival of the patients with apparent ErbB-2 expression was significantly worse than that of the patients with little ErbB-2 expression (P < 0.01)., Conclusions: In 42% of the osteosarcomas, the tumor cells expressed ErbB-2. Expression of ErbB-2 was strongly correlated with early pulmonary metastasis and poor survival rate for the patient. These data suggest that ErbB-2 plays a significant role in aggressive tumor growth and in the promotion of metastatic potential in osteosarcomas. ErbB-2 in the osteosarcoma tissues would be a useful prognostic marker for patients.
- Published
- 1996
- Full Text
- View/download PDF
243. Factors affecting DNA damage caused by lipid hydroperoxides and aldehydes.
- Author
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Yang MH and Schaich KM
- Subjects
- Cations pharmacology, DNA, Single-Stranded drug effects, DNA, Single-Stranded isolation & purification, Electrophoresis, Agar Gel, Iron pharmacology, Kinetics, Pentetic Acid pharmacology, Solvents, Spectrophotometry, Atomic, Structure-Activity Relationship, Aldehydes, Antioxidants pharmacology, DNA Damage, Free Radical Scavengers pharmacology, Linoleic Acids pharmacology, Lipid Peroxidation, Lipid Peroxides pharmacology, Plasmids drug effects, Plasmids isolation & purification
- Abstract
Single (SSB) and double strand breaks (DSB) in supercoiled plasmid DNA pBR322 reacted with linoleic acid hydroperoxides (LOOH) were followed by agarose gel electrophoresis to obtain definitive information about factors affecting LOOH interaction with DNA. In water, LOOH induced extensive DSB, which were metal mediated and increased with incubation time. Adventitious metal bound to DNA was sufficient to decompose LOOH to reactive radicals, activity that was not readily inhibited by chelators DTPA and desferrioxamine. Added Fe2+ and Fe3+ increased SSB and DSB, although the effects of Fe2+ were more extensive. Above 100 microM both valences inhibited DNA damage. Strand breakage by LOOH proceeded via lipid alkoxyl and peroxyl radicals. Aldehydic lipid peroxidation products induced strand breaks via oxidation of double bonds, not by reactions of the carbonyl groups. Lipophilic antioxidants BHA, BHT, and alpha-tocopherol were about 20 times more effective than hydrophilic free radical scavengers sodium benzoate, inositol, DMSO, and mannitol in preventing LOOH-induced strand breaks, supporting lipid phase localization of the damage.
- Published
- 1996
- Full Text
- View/download PDF
244. Preparation of ssDNA from phagemid vectors.
- Author
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Trower MK
- Subjects
- DNA, Viral genetics, Escherichia coli genetics, Plasmids genetics, Bacteriophages genetics, Cloning, Molecular methods, DNA, Single-Stranded isolation & purification, Genetic Vectors
- Published
- 1996
- Full Text
- View/download PDF
245. Solid phase purification and SSCP analysis of amplified genomic DNA by capillary electrophoresis.
- Author
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Debernardi S, Luzzana M, and De Bellis G
- Subjects
- DNA, Single-Stranded isolation & purification, Genome, Human, Globins genetics, Humans, Polymerase Chain Reaction, Polymorphism, Single-Stranded Conformational, DNA Mutational Analysis methods, Electrophoresis, Capillary methods, Point Mutation
- Abstract
Detection and identification of point mutations in genomic DNA has proven increasingly important in biomedical research. A variety of methods for the analysis of single base substitutions have been proposed among which Single Strand Conformational Polymorphism (SSCP) quickly gained success due to its simplicity. In this work we present an analytical on-line tool which combines the ease of solid phase purification of amplified genomic DNA, the simplicity of SSCP and the significant potential advantages offered by capillary electrophoresis (CE).
- Published
- 1996
- Full Text
- View/download PDF
246. Efficient PCR production of single-stranded DNA sequencing templates.
- Author
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Kaltenboeck B and Kousoulas KG
- Subjects
- Templates, Genetic, DNA, Single-Stranded isolation & purification, Polymerase Chain Reaction methods, Sequence Analysis, DNA methods
- Published
- 1996
- Full Text
- View/download PDF
247. PCR product with strands of unequal length.
- Author
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Williams KP and Bartel DP
- Subjects
- Base Sequence, DNA Primers, DNA, Single-Stranded genetics, Molecular Sequence Data, DNA, Single-Stranded isolation & purification, Polymerase Chain Reaction methods
- Published
- 1995
- Full Text
- View/download PDF
248. Spectroscopic demonstration of a linkage between the kinetics of NTP hydrolysis and the conformational state of the recA-single-stranded DNA complex.
- Author
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Stole E and Bryant FR
- Subjects
- Binding Sites, DNA, Single-Stranded isolation & purification, DNA, Single-Stranded metabolism, Histidine, Hydrogen-Ion Concentration, Kinetics, Point Mutation, Rec A Recombinases isolation & purification, Rec A Recombinases metabolism, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Spectrometry, Fluorescence, Structure-Activity Relationship, Tryptophan, DNA, Single-Stranded chemistry, Purine Nucleotides metabolism, Rec A Recombinases chemistry
- Abstract
We recently constructed a mutant recA protein in which His-163 was replaced by a tryptophan residue; the [H163W]recA protein is functionally identical to the wild-type protein, and the Trp-163 side chain serves as a reporter group for the conformational transitions of the [H163W]recA-single-stranded DNA (ssDNA) complex. We have now examined the fluorescence properties of the [H163W]recA-ssDNA complex in the presence of a series of alternate nucleoside triphosphate cofactors. Under standard conditions (pH 7.5), ATP (S0.5 = 70 microM) and purine riboside triphosphate (PTP) (S0.5 = 110 microM) effect a 44% decrease in Trp-163 fluorescence and are active as cofactors for the DNA strand exchange reaction. In contrast, ITP (S0.5 = 400 microM) elicits only a 20% decrease in Trp-163 fluorescence (a level identical to that observed with the nucleoside diphosphates ADP, PDP, and IDP) and is inactive as a strand exchange cofactor. If the S0.5 (PTP) is increased to 130 microM (by increasing the pH of the reaction solution), the PTP-mediated quenching of Trp-163 fluorescence decreases to 20%, and PTP becomes inactive as a strand exchange cofactor. These results provide direct evidence for a linkage between the S0.5 value of a nucleoside triphosphate and the conformational state of the recA-ssDNA complex, with an S0.5 of 100-120 microM or lower required for stabilization of the strand exchange-active conformation.
- Published
- 1995
- Full Text
- View/download PDF
249. Specific single-stranded breaks in mature bacteriophage T7 DNA.
- Author
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Khan SA, Hayes SJ, Wright ET, Watson RH, and Serwer P
- Subjects
- Bacteriophage T7 genetics, Base Sequence, Centrifugation, Zonal, DNA, Single-Stranded isolation & purification, DNA, Viral isolation & purification, Electrophoresis, Agar Gel, Molecular Sequence Data, Nucleic Acid Denaturation, Oligonucleotide Probes, Bacteriophage T7 metabolism, DNA, Single-Stranded chemistry, DNA, Viral chemistry
- Abstract
Both rate zonal centrifugation and gel electrophoresis have revealed that the mature double-stranded DNA of bacteriophage T5 has single-stranded breaks (nicks) at specific sites. Neither of these procedures has previously revealed site-specific nicks in the double-stranded DNA of other bacteriophages, including T7. In the present study, denaturing gel electrophoresis, followed by specific DNA detection, reveals that a small fraction of mature T7 DNA molecules, like most T5 DNA molecules, has site-specific nicks. The procedure of specific detection is to probe with an oligonucleotide specific for one of the ends of T7 DNA. If position 0.0 is the left genetic end and position 100.0 is the right genetic end of T7 DNA, the nicks on the 5' left-oriented strand are at 11.3, 12.4, 65.7, 79.2, and 86.0; the nicks on the 5' right-oriented strand are at 23.3 and 26.5 (+/- 0.5). The positions of the three rightmost nicks are indistinguishable from those of double-stranded breaks that produce previously demonstrated shorter than mature length DNAs packaged in vivo. We propose that the T7 nicks are produced by premature activity of the T7 terminase during DNA packaging.
- Published
- 1995
- Full Text
- View/download PDF
250. A novel and simple methodology to generate subtracted cDNA libraries.
- Author
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Rivolta MN and Wilcox ER
- Subjects
- Actins genetics, Animals, Base Sequence, Brain Chemistry, DNA metabolism, DNA Restriction Enzymes metabolism, DNA, Complementary isolation & purification, DNA, Single-Stranded chemistry, DNA, Single-Stranded genetics, DNA, Single-Stranded isolation & purification, Guinea Pigs, Molecular Sequence Data, Nucleic Acid Hybridization, Polymerase Chain Reaction, Receptors, Transferrin genetics, DNA, Complementary genetics, Gene Library
- Published
- 1995
- Full Text
- View/download PDF
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