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Sample records for plasmid dna extracted

  1. Dataset of plasmid DNA extraction using different magnetic nanoparticles (MNPs

    Directory of Open Access Journals (Sweden)

    H. Rahnama

    2016-12-01

    MNPs were characterized by energy dispersive spectroscopy (EDS and transmission electron microscopy (TEM. Finally, the overall efficiency of different MNPs (Fe3O4, Fe3O4/SiO2, Fe3O4/SiO2/TiO2 in plasmid DNA isolation was compared using gel electrophoresis analysis. The data supplied in this article supports the accompanying publication “Comparative study of three magnetic nano-particles (FeSO4, FeSO4/SiO2, FeSO4/SiO2/TiO2 in plasmid DNA extraction” (H. Rahnama, A. Sattarzadeh, F. Kazemi, N. Ahmadi, F. Sanjarian, Z. Zand, 2016 [1].

  2. Aqueous extract of Pinus caribaea inhibits the damage induced by ultraviolet radiations, in plasmid DNA

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    Marioly Vernhes Tamayo

    2017-08-01

    Full Text Available Context: The incidence of solar ultraviolet radiation (UV on Earth has increased due to diminish of the ozone layer. This enviromental agent is highly genotoxic causing numerous damage in DNA molecule. Nowadays there is a growing interest in the search of compounds capable to minimize these effects. In particular, phytocompounds have been tested as excelent candidates for their antigenotoxic properties. Aims: To evaluate the protective effect of the aqueous extract of Pinus caribaea (EPC against the damage induced by the UVB and UVC radiation. Methods: The cell-free plasmid DNA assay was employed. The forms of plasmid were separated electrophoretically in agarose gel. For genotoxic and photoprotective evaluation of P. caribaea, different concentrations of the extract (0.1 – 2.0 mg/mL and exposure times were evaluated. The CPD lesions were detected enzymatically. Additionally, the transmittance of the aqueous extract against 254 nm and 312 nm was measured. Results: None of the concentrations were genotoxic in 30 min of treatment, for superior times a clastogenic effect was observed. The EPC despite inhibiting the activity of the enzyme T4 endo V, impedes photolesions formation in DNA at concentrations ≥ 0.1 mg/mL. Conclusions: The EPC has photoprotective properties, this effect could be related with its antioxidants and absorptives capacities.

  3. Influence of an extract of Juglans regia on the growth of Escherichia coli, on the electrophoretic profile of plasmid DNA and on the radiolabeling of blood constituents

    Energy Technology Data Exchange (ETDEWEB)

    Santos-Filho, Sebastiao David; Diniz, Claudia Leite; Carmo, Fernanda Santos do; Fonseca, Adenilson de Souza da; Bernardo-Filho, Mario [Universidade do Estado do Rio de Janeiro (UERJ), RJ (Brazil). Instituto de Biologia Roberto Alcantara Gomes. Dept. de Biofisica e Biometria]. E-mail: santos-filho@uer.br

    2008-12-15

    The aim of this work was to study the influence of a walnut (Juglans regia) extract on the growth of Escherichia coli (E. coli) AB1157, on the plasmid DNA topology and on the labeling of blood constituents with technetium-99m ({sup 99m}Tc). An E. coli AB1157 culture, in stationary phase, was incubated with walnut and the growth of the culture was evaluated by optical density at 600 nm for 7 hours. Plasmid DNA samples were incubated with SnCl{sub 2} in presence or absence of walnut for 40 minutes, 0.8% agarose gel electrophoresis was performed, the gel was stained and the plasmid topological forms were visualized. Blood samples from Wistar rats were incubated with walnut extract and an assay of labeling of blood constituents with technetium-99m ({sup 99m}Tc) was performed. Blood cells and plasma were separated. The radioactivity in each fraction was counted and percentage of incorporated radioactivity (%ATI) was determined. The results presented an inhibitory action of the growth of the E. coli AB1157 culture, no protective action of the walnut extract in plasmid DNA treated with SnCl{sub 2}. Moreover, walnut was also not capable to induce modifications in the DNA mobility in agarose gel but walnut was capable to decrease the distribution of {sup 99}'mTc on the blood cell compartment. In conclusion, our experimental data suggest that in the walnut extract has substances with an effect on the growth of E. coli culture, a potential action to increase the SnCl{sub 2} effect on plasmid DNA and also is capable to alter the distribution of {sup 99m}Tc on the blood cell compartment probably due to redoxi properties. (author)

  4. Influence of an extract of Juglans regia on the growth of Escherichia coli, on the electrophoretic profile of plasmid DNA and on the radiolabeling of blood constituents

    Directory of Open Access Journals (Sweden)

    Sebastião David Santos-Filho

    2008-12-01

    Full Text Available The aim of this work was to study the influence of a walnut (Juglans regia extract on the growth of Escherichia coli (E. coli AB1157, on the plasmid DNA topology and on the labeling of blood constituents with technetium-99m (99mTc. An E. coli AB1157 culture, in stationary phase, was incubated with walnut and the growth of the culture was evaluated by optical density at 600 nm for 7 hours. Plasmid DNA samples were incubated with SnCl2 in presence or absence of walnut for 40 minutes, 0.8% agarose gel electrophoresis was performed, the gel was stained and the plasmid topological forms were visualized. Blood samples from Wistar rats were incubated with walnut extract and an assay of labeling of blood constituents with technetium-99m (99mTc was performed. Blood cells and plasma were separated. The radioactivity in each fraction was counted and percentage of incorporated radioactivity (%ATI was determined. The results presented an inhibitory action of the growth of the E. coli AB1157 culture, no protective action of the walnut extract in plasmid DNA treated with SnCl2. Moreover, walnut was also not capable to induce modifications in the DNA mobility in agarose gel but walnut was capable to decrease the distribution of 99mTc on the blood cell compartment. In conclusion, our experimental data suggest that in the walnut extract has substances with an effect on the growth of E. coli culture, a potential action to increase the SnCl2 effect on plasmid DNA and also is capable to alter the distribution of 99mTc on the blood cell compartment probably due to redoxi properties.O objetivo desse trabalho foi estudar a influência de um extrato de nogueira (Juglans regia no crescimento de Escherichia coli (E. coli AB1157, na topologia do DNA plasmidial e na marcação de constituintes sanguíneos com tecnécio-99m (99mTc. Uma cultura de E. coli AB1157, em fase estacionária, foi incubada com nogueira e o crescimento da cultura foi avaliado por densidade óptica a

  5. A Chrysobalanus icaco extract alters the plasmid topology and the effects of stannous chloride on the DNA of plasmids Um extrato de Chrysobalanus icaco altera a topologia de plasmídios e os efeitos do cloreto estanoso sobre o DNA de plasmídios

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    Giuseppe A. Presta

    2007-09-01

    Full Text Available Chrysobalanus icaco (C. icaco leaves are used in folk medicine (known as Abajeru in Brazil to control the glycaemia in diabetic patients. Stannous chloride (SnCl2 is a powerful reducing agent used for different purposes and presents cytotoxic and genotoxic effects. The aim of this work was to investigate the effect of an aqueous C. icaco extract on the plasmid DNA topology and on the effects of the stannous chloride on DNA plasmid. Plasmid pBSK was incubated with a C. icaco extract in the presence or absence of SnCl2 (200 mg/mL, after that, the agarose gel electrophoresis procedure was carried out. Plasmid incubated only SnCl2 was used as positive control and, as negative control, plasmid incubated with Tris buffer. The gels were stained with ethidium bromide, DNA bands were semiquantified by densitometry. The data showed that C. icaco extract alters the electrophoretic profile and decreases significantly (p Folhas de Chrysobalanus icaco (C. icaco são usadas na medicina popular (conhecido como Abajeru no Brasil para controlar a glicemia em pacientes diabéticos. Cloreto estanoso (SnCl2 é um agente redutor potente usado para diferentes propostas e apresenta efeitos citotóxico e genotóxico. O objetivo deste trabalho foi investigar os efeitos de um extrato aquoso de C. icaco na topologia de DNA plasmidial e nos efeitos do cloreto estanoso sobre o DNA plasmidial. Plasmídios pBSK foram incubados com um extrato de C. icaco na presença ou ausência do SnCl2 (200 mg/mL, em seguida, o procedimento de eletroforese em gel de agarose foi realizado. Plasmídios incubados somente com SnCl2 foram usados como controle positivo e, como controle negativo, plasmídios incubados com tampão Tris. Os géis foram corados com brometo de etídio e as bandas de DNA foram semiquantificadas por densitometria. Os dados mostraram que o extrato de C. icaco altera o perfil eletroforético e diminui significativamente (p < 0,05 os efeitos do SnCl2 sobre DNA plasmidial

  6. Plasmid DNA entry into postmitotic nuclei of primary rat myotubes.

    OpenAIRE

    Dowty, M E; Williams, P.; G. Zhang; Hagstrom, J E; Wolff, J A

    1995-01-01

    These studies were initiated to elucidate the mechanism of DNA nuclear transport in mammalian cells. Biotin- or gold-labeled plasmid and plasmid DNA expression vectors for Escherichia coli beta-galactosidase or firefly luciferase were microinjected into the cytoplasm of primary rat myotubes in culture. Plasmid DNA was expressed in up to 70% of the injected myotubes, which indicates that it entered intact, postmitotic nuclei. The nuclear transport of plasmid DNA occurred through the nuclear po...

  7. Persistence of plasmid DNA in different soils

    African Journals Online (AJOL)

    STORAGESEVER

    2008-08-04

    Aug 4, 2008 ... ... transformation in bacteria (Davison, 1999), binding of DNA from Bacillus subtilis on clay mineral montmorillonite, and the ability of ... soil was taken and soil extract was prepared with sterile water the DNA was isolated and.

  8. Sample displacement chromatography of plasmid DNA isoforms.

    Science.gov (United States)

    Černigoj, Urh; Martinuč, Urška; Cardoso, Sara; Sekirnik, Rok; Krajnc, Nika Lendero; Štrancar, Aleš

    2015-10-02

    Sample displacement chromatography (SDC) is a chromatographic technique that utilises different relative binding affinities of components in a sample mixture and has been widely studied in the context of peptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) or linear isoform. Since displacement is more efficient when mass transfer between stationary and mobile chromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobicities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoform separation was shown to be dependent on column selectivity for individual isoform, column efficiency and on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative mode elution often operate in parallel, therefore negative mode elution additionally influences the efficiency of the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNA homogeneity and a dynamic binding capacity of over 1mg/mL at a relatively low concentration of AS. SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes, and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used. This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, which is compatible with continuous, multicolumn chromatography systems, and could therefore be used to increase productivity of pDNA production in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Production and pharmaceutical formulation of plasmid DNA vaccines

    NARCIS (Netherlands)

    van der Heijden, I.

    2013-01-01

    Research leading to the thesis ‘Production and pharmaceutical formulation of plasmid DNA vaccines‘ can be divided into two parts. The first part describes the development of a Good Manufacturing Practice (GMP) compliant plasmid DNA production process of pDNA vaccines for the treatment of Human papil

  10. Production and pharmaceutical formulation of plasmid DNA vaccines

    NARCIS (Netherlands)

    van der Heijden, I.

    2013-01-01

    Research leading to the thesis ‘Production and pharmaceutical formulation of plasmid DNA vaccines‘ can be divided into two parts. The first part describes the development of a Good Manufacturing Practice (GMP) compliant plasmid DNA production process of pDNA vaccines for the treatment of Human papil

  11. Influenza Plasmid DNA Vaccines: Progress and Prospects.

    Science.gov (United States)

    Bicho, Diana; Queiroz, João António; Tomaz, Cândida Teixeira

    2015-01-01

    Current influenza vaccines have long been used to fight flu infectious; however, recent advances highlight the importance of produce new alternatives. Even though traditional influenza vaccines are safe and usually effective, they need to be uploaded every year to anticipate circulating flu viruses. This limitation together with the use of embryonated chicken eggs as the substrate for vaccine production, is time-consuming and could involve potential biohazards in growth of new virus strains. Plasmid DNA produced by prokaryote microorganisms and encoding foreign proteins had emerged as a promising therapeutic tool. This technology allows the expression of a gene of interest by eukaryotic cells in order to induce protective immune responses against the pathogen of interest. In this review, we discuss the strategies to choose the best DNA vaccine to be applied in the treatment and prevention of influenza. Specifically, we give an update of influenza DNA vaccines developments, all involved techniques, their main characteristics, applicability and technical features to obtain the best option against influenza infections.

  12. Human papillomavirus DNA from warts for typing by endonuclease restriction patterns: purification by alkaline plasmid methods.

    Science.gov (United States)

    Chinami, M; Tanikawa, E; Hachisuka, H; Sasai, Y; Shingu, M

    1990-01-01

    The alkaline plasmid DNA extraction method of Birnboim and Doly was applied for the isolation of human papillomavirus (HPV) from warts. Tissue from common and plantar warts was digested with proteinase K, and the extrachromosomal circular covalently-closed form of HPV-DNA was rapidly extracted by alkaline sodium dodecyl sulphate and phenol-chloroform treatment. Recovery of HPV-DNA from the tissue was sufficient for determination of endonuclease restriction patterns by agarose gel electrophoresis.

  13. DNA is a co-factor for its own replication in Xenopus egg extracts

    NARCIS (Netherlands)

    Lebofsky, Ronald; van Oijen, Antoine M.; Walter, Johannes C.

    Soluble Xenopus egg extracts efficiently replicate added plasmids using a physiological mechanism, and thus represent a powerful system to understand vertebrate DNA replication. Surprisingly, DNA replication in this system is highly sensitive to plasmid concentration, being undetectable below

  14. DNA is a co-factor for its own replication in Xenopus egg extracts

    NARCIS (Netherlands)

    Lebofsky, Ronald; van Oijen, Antoine M.; Walter, Johannes C.

    2011-01-01

    Soluble Xenopus egg extracts efficiently replicate added plasmids using a physiological mechanism, and thus represent a powerful system to understand vertebrate DNA replication. Surprisingly, DNA replication in this system is highly sensitive to plasmid concentration, being undetectable below simila

  15. DNA is a co-factor for its own replication in Xenopus egg extracts

    NARCIS (Netherlands)

    Lebofsky, Ronald; van Oijen, Antoine M.; Walter, Johannes C.

    2011-01-01

    Soluble Xenopus egg extracts efficiently replicate added plasmids using a physiological mechanism, and thus represent a powerful system to understand vertebrate DNA replication. Surprisingly, DNA replication in this system is highly sensitive to plasmid concentration, being undetectable below simila

  16. The heat-shock DnaK protein is required for plasmid R1 replication and it is dispensable for plasmid ColE1 replication.

    Science.gov (United States)

    Giraldo-Suárez, R; Fernández-Tresguerres, E; Díaz-Orejas, R; Malki, A; Kohiyama, M

    1993-01-01

    Plasmid R1 replication in vitro is inactive in extracts prepared from a dnaK756 strain but is restored to normal levels upon addition of purified DnaK protein. Replication of R1 in extracts of a dnaKwt strain can be specifically inhibited with polyclonal antibodies against DnaK. RepA-dependent replication of R1 in dnaK756 extracts supplemented with DnaKwt protein at maximum concentration is partially inhibited by rifampicin and it is severely inhibited at sub-optimal concentrations of DnaK protein. The copy number of a run-away R1 vector is reduced in a dnaK756 background at 30 degrees C and at 42 degrees C the amplification of the run-away R1 vector is prevented. However a runaway R1 vector containing dnaK gene allows the amplification of the plasmid at high temperature. These data indicate that DnaK is required for both in vitro and in vivo replication of plasmid R1 and show a partial compensation for the low level of DnaK by RNA polymerase. In contrast ColE1 replication is not affected by DnaK as indicated by the fact that ColE1 replicates with the same efficiency in extracts from dnaKwt and dnaK756 strains. Images PMID:8265367

  17. Plasmid P1 replication: negative control by repeated DNA sequences.

    OpenAIRE

    Chattoraj, D; Cordes, K.; Abeles, A

    1984-01-01

    The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pi...

  18. DNA polymerase beta can substitute for DNA polymerase I in the initiation of plasmid DNA replication.

    OpenAIRE

    1995-01-01

    We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant ...

  19. Separation of plasmid DNA topoisomers by multimodal chromatography.

    Science.gov (United States)

    Silva-Santos, A Rita; Alves, Cláudia P A; Prazeres, Duarte Miguel F; Azevedo, Ana M

    2016-06-15

    The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases. Here, we describe the separation of supercoiled (sc) and open circular (oc) topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid. The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Purification of transfection-grade plasmid DNA from bacterial cells with superparamagnetic nanoparticles

    Science.gov (United States)

    Chiang, Chen-Li; Sung, Ching-Shan

    2006-07-01

    The functionalized magnetic nanobeads were used to develop a rapid protocol for extracting and purifying transfection-grade plasmid DNA from bacterial culture. Nanosized superparamagnetic nanoparticles (Fe 3O 4) were prepared by chemical coprecipitation method using Fe 2+, Fe 3+ salt, and ammonium hydroxide under a nitrogen atmosphere. The surface of Fe 3O 4 nanoparticles was modified by coating with the multivalent cationic agent, polyethylenimine (PEI). The PEI-modified magnetic nanobeads were employed to simplify the purification of plasmid DNA from bacterial cells. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of Escherichia coli. The loaded nanobeads are recovered by magnetically driven separation and regenerated by exposure to the elution buffer with optimal ionic strength (1.25 M) and pH (9.0). Up to approximately 819 μg of high-purity (A 260/A 280 ratio=1.86) plasmid DNA was isolated from 100 ml of overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and animal cell transfection applications with success. The PEI-modified magnetic nanobead delivers significant time-savings, overall higher yields and better transfection efficiencies compared to anion-exchange and other methods. The results presented in this report show that PEI-modified magnetic nanobeads are suitable for isolation and purification of transfection-grade plasmid DNA.

  1. Plasmid DNA Manufacturing for Indirect and Direct Clinical Applications.

    Science.gov (United States)

    Schmeer, Marco; Buchholz, Tatjana; Schleef, Martin

    2017-10-01

    Plasmid DNA is currently gaining increasing importance for clinical research applications in gene therapy and genetic vaccination. For direct gene transfer into humans, good manufacturing practice (GMP)-grade plasmid DNA is mandatory. The same holds true if the drug substance contains a genetically modified cell, for example chimeric antigen receptor (CAR) T cells, where these cells as well as the contained plasmids are used. According to the responsible regulatory agencies, they have to be produced under full GMP. On the other hand, for GMP production of, for example, mRNA or viral vectors (lentiviral vectors, adeno-associated virus vectors, etc.), in many cases, High Quality Grade plasmid DNA is accepted as a starting material. The manufacturing process passes through different production steps. To ensure the right conditions are used for the plasmid, a pilot run must be conducted at the beginning. In this step, a followed upscaling with respect to reproducibility and influences on product quality is performed. Subsequently, a cell bank of the transformed productions strain is established and characterized. This cell bank is used for the cultivation process. After cell harvesting and lysis, several chromatography steps are conducted to receive a pure plasmid product. Depending on the respective required quality grade, the plasmid product is subject to several quality controls. The last step consists of formulation and filling of the product.

  2. Functional amyloids as inhibitors of plasmid DNA replication

    Science.gov (United States)

    Molina-García, Laura; Gasset-Rosa, Fátima; Moreno-del Álamo, María; Fernández-Tresguerres, M. Elena; Moreno-Díaz de la Espina, Susana; Lurz, Rudi; Giraldo, Rafael

    2016-01-01

    DNA replication is tightly regulated to constrain the genetic material within strict spatiotemporal boundaries and copy numbers. Bacterial plasmids are autonomously replicating DNA molecules of much clinical, environmental and biotechnological interest. A mechanism used by plasmids to prevent over-replication is ‘handcuffing’, i.e. inactivating the replication origins in two DNA molecules by holding them together through a bridge built by a plasmid-encoded initiator protein (Rep). Besides being involved in handcuffing, the WH1 domain in the RepA protein assembles as amyloid fibres upon binding to DNA in vitro. The amyloid state in proteins is linked to specific human diseases, but determines selectable and epigenetically transmissible phenotypes in microorganisms. Here we have explored the connection between handcuffing and amyloidogenesis of full-length RepA. Using a monoclonal antibody specific for an amyloidogenic conformation of RepA-WH1, we have found that the handcuffed RepA assemblies, either reconstructed in vitro or in plasmids clustering at the bacterial nucleoid, are amyloidogenic. The replication-inhibitory RepA handcuff assembly is, to our knowledge, the first protein amyloid directly dealing with DNA. Built on an amyloid scaffold, bacterial plasmid handcuffs can bring a novel molecular solution to the universal problem of keeping control on DNA replication initiation. PMID:27147472

  3. Efficient transformation of Bacillus thuringiensis requires nonmethylated plasmid DNA.

    OpenAIRE

    Macaluso, A; Mettus, A M

    1991-01-01

    The transformation efficiency of Bacillus thuringiensis depends upon the source of plasmid DNA. DNA isolated from B. thuringiensis, Bacillus megaterium, or a Dam- Dcm- Escherichia coli strain efficiently transformed several B. thuringiensis strains, B. thuringiensis strains were grouped according to which B. thuringiensis backgrounds were suitable sources of DNA for transformation of other B. thuringiensis strains, suggesting that B. thuringiensis strains differ in DNA modification and restri...

  4. Multiple Pathways of Plasmid DNA Transfer in Helicobacter pylori

    Science.gov (United States)

    Rohrer, Stefanie; Holsten, Lea; Weiss, Evelyn; Benghezal, Mohammed; Fischer, Wolfgang; Haas, Rainer

    2012-01-01

    Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species. PMID:23029142

  5. Comparative study of three magnetic nano-particles (FeSO4, FeSO4/SiO2, FeSO4/SiO2/TiO2) in plasmid DNA extraction.

    Science.gov (United States)

    Rahnama, H; Sattarzadeh, A; Kazemi, F; Ahmadi, N; Sanjarian, F; Zand, Z

    2016-11-15

    Recent updates on Magnetic Nano-Particles (MNPs) based separation of nucleic acids have received more attention due to their easy manipulation, simplicity, ease of automation and cost-effectiveness. It has been indicated that DNA molecules absorb on solid surfaces via hydrogen-bonding, and hydrophobic and electrostatic interactions. These properties highly depend on the surface condition of the solid support. Therefore, surface modification of MNPs may enhance their functionality and specification. In the present study, we functionalized Fe3O4 nano-particle surface utilizing SiO2 and TiO2 layer as Fe3O4/SiO2 and Fe3O4/SiO2/TiO2 and then compare their functionality in the adsorption of plasmid DNA molecules with the naked Fe3O4 nano-particles. The result obtained showed that the purity and amount of DNA extracted by Fe3O4 coated by SiO2 or SiO2/TiO2 were higher than the naked Fe3O4 nano-particles. Furthermore, we obtained pH 8 and 1.5 M NaCl as an optimal condition for desorption of DNA from MNPs. The result further showed that, 0.2 mg nano-particle and 10 min at 55 °C are the optimal conditions for DNA desorption from nano-particles. In conclusion, we recommended Fe3O4/SiO2/TiO2 as a new MNP for separation of DNA molecules from biological sources.

  6. Cationic lipids delay the transfer of plasmid DNA to lysosomes.

    Science.gov (United States)

    Wattiaux, R; Jadot, M; Laurent, N; Dubois, F; Wattiaux-De Coninck, S

    1996-10-14

    Plasmid 35S DNA, naked or associated with different cationic lipid preparations was injected to rats. Subcellular distribution of radioactivity in the liver one hour after injection, was established by centrifugation methods. Results show that at that time, 35S DNA has reached lysosomes. On the contrary, when 35S DNA was complexed with lipids, radioactivity remains located in organelles whose distribution after differential and isopycnic centrifugation, is clearly distinct from that of arylsulfatase, lysosome marker enzyme. Injection of Triton WR 1339, a specific density perturbant of lysosomes, four days before 35S DNA injection causes a density decrease of radioactivity bearing structures, apparent one hour after naked 35S DNA injection but visible only after more than five hours, when 35S DNA associated with a cationic lipid is injected. These observations show that cationic lipids delay the transfer to lysosomes, of plasmid DNA taken up by the liver.

  7. Liquid-Crystalline Mesophases of Plasmid DNA in Bacteria

    Science.gov (United States)

    Reich, Ziv; Wachtel, Ellen J.; Minsky, Abraham

    1994-06-01

    Bacterial plasmids may often reach a copy number larger than 1000 per cell, corresponding to a total amount of DNA that may exceed the amount of DNA within the bacterial chromosome. This observation highlights the problem of cellular accommodation of large amounts of closed-circular nucleic acids, whose interwound conformation offers negligible DNA compaction. As determined by x-ray scattering experiments conducted on intact bacteria, supercoiled plasmids segregate within the cells into dense clusters characterized by a long-range order. In vitro studies performed at physiological DNA concentrations indicated that interwound DNA spontaneously forms liquid crystalline phases whose macroscopic structural properties are determined by the features of the molecular supercoiling. Because these features respond to cellular factors, DNA supercoiling may provide a sensitive regulatory link between cellular parameters and the packaging modes of interwound DNA in vivo.

  8. Evaluation of plasmid and genomic DNA calibrants used for the quantification of genetically modified organisms.

    Science.gov (United States)

    Caprioara-Buda, M; Meyer, W; Jeynov, B; Corbisier, P; Trapmann, S; Emons, H

    2012-07-01

    The reliable quantification of genetically modified organisms (GMOs) by real-time PCR requires, besides thoroughly validated quantitative detection methods, sustainable calibration systems. The latter establishes the anchor points for the measured value and the measurement unit, respectively. In this paper, the suitability of two types of DNA calibrants, i.e. plasmid DNA and genomic DNA extracted from plant leaves, for the certification of the GMO content in reference materials as copy number ratio between two targeted DNA sequences was investigated. The PCR efficiencies and coefficients of determination of the calibration curves as well as the measured copy number ratios for three powder certified reference materials (CRMs), namely ERM-BF415e (NK603 maize), ERM-BF425c (356043 soya), and ERM-BF427c (98140 maize), originally certified for their mass fraction of GMO, were compared for both types of calibrants. In all three systems investigated, the PCR efficiencies of plasmid DNA were slightly closer to the PCR efficiencies observed for the genomic DNA extracted from seed powders rather than those of the genomic DNA extracted from leaves. Although the mean DNA copy number ratios for each CRM overlapped within their uncertainties, the DNA copy number ratios were significantly different using the two types of calibrants. Based on these observations, both plasmid and leaf genomic DNA calibrants would be technically suitable as anchor points for the calibration of the real-time PCR methods applied in this study. However, the most suitable approach to establish a sustainable traceability chain is to fix a reference system based on plasmid DNA.

  9. [Chromatographic separation of plasmid DNA by anion-exchange cryogel].

    Science.gov (United States)

    Guo, Yantao; Shen, Shaochuan; Yun, Junxian; Yao, Kejian

    2012-08-01

    Plasmid DNA (pDNA) is used as an important vector for gene therapy, and its wide application is restricted by the purity and yield. To obtain high-purity pDNA, a chromatographic method based on anion-exchange supermacroporous cryogel was explored. The anion-exchange cryogel was prepared by grafting diethylaminoethyl-dextran to the epoxide groups of polyacrylamide-based matrix and pUC19 plasmid was used as a target to test the method. The plasmid was transferred into Escherichia coli DH5alpha, cultivated, harvested and lysed. The obtained culture was centrifuged and the supernatant was used as the plasmid feedstock, which was loaded into the anion-exchange cryogel bed for chromatographic separation. By optimizing the pH of running buffer and the elution conditions, high-purity pDNA was obtained by elution with 0.5 mol/L sodium chloride solution at pH 6.6. Compared to the traditional methods for purification of pDNA, animal source enzymes and toxic reagents were not involved in the present separation process, ensuring the safety of both the purification operations and the obtained pDNA.

  10. Molecular mechanism of immune response induced by foreign plasmid DNA after oral administration in mice

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To study immune response induced by foreign plasmid DNA after oral administration in mice.METHODS: Mice were orally administered with 200 μg of plasmid pcDNA3 once and spleen was isolated 4 h and 18 h after administration. Total RNA was extracted from spleen and gene expression profile of BALB/c mice spleen was analyzed by using Affymetrix oligonucleotide GeneChip. Functional cluster analysis was conducted by GenMAPP software.RESULTS: At 4 h and 18 h after oral plasmid pcDNA3 administration, a number of immune-related genes,including cytokine and cytokine receptors, chemokines and chemokine receptor, complement molecule,proteasome, histocompatibility molecule, lymphocyte antigen complex and apoptotic genes, were up-regulated. Moreover, MAPPFinder results also showed that numerous immune response processes were up-regulated. In contrast, the immunoglobulin genes were down-regulated.CONCLUSION: Foreign plasmid DNA can modulate the genes expression related to immune system via the gastrointestinal tract, and further analysis of the related immune process may help understand the molecular mechanisms of immune response induced by foreign plasmid via the gastrointestinal tract.

  11. Pharmaceutical development of the plasmid DNA vaccine pDERMATT

    NARCIS (Netherlands)

    Quaak, S.G.L.

    2009-01-01

    The discovery of tumor specific antigens and self tolerance mechanisms against these antigens led to the assumption that antigens circulating at sufficient concentration levels could break this self tolerance mechanism and evoke immunological antitumor effects. pDERMATT (plasmid DNA encoding recombi

  12. Pharmaceutical development of the plasmid DNA vaccine pDERMATT

    NARCIS (Netherlands)

    Quaak, S.G.L.

    2009-01-01

    The discovery of tumor specific antigens and self tolerance mechanisms against these antigens led to the assumption that antigens circulating at sufficient concentration levels could break this self tolerance mechanism and evoke immunological antitumor effects. pDERMATT (plasmid DNA encoding

  13. Use of plasmid DNA for induction of protective immunity

    DEFF Research Database (Denmark)

    Lorenzen, Niels

    2004-01-01

    Vaccines based on plasmid DNA have been tested for a number of fish pathogens but so far it is only in case of the rhabdoviruses, where the technology has been a real break through in vaccine research. Aspects of dose, time-course and mechanisms of protection, as well as practical use are discussed....

  14. Current trends in separation of plasmid DNA vaccines: a review.

    Science.gov (United States)

    Ghanem, Ashraf; Healey, Robert; Adly, Frady G

    2013-01-14

    Plasmid DNA (pDNA)-based vaccines offer more rapid avenues for development and production if compared to those of conventional virus-based vaccines. They do not rely on time- or labour-intensive cell culture processes and allow greater flexibility in shipping and storage. Stimulating antibodies and cell-mediated components of the immune system are considered as some of the major advantages associated with the use of pDNA vaccines. This review summarizes the current trends in the purification of pDNA vaccines for practical and analytical applications. Special attention is paid to chromatographic techniques aimed at reducing the steps of final purification, post primary isolation and intermediate recovery, in order to reduce the number of steps necessary to reach a purified end product from the crude plasmid.

  15. Enhanced brain targeting efficiency of intranasally administered plasmid DNA: an alternative route for brain gene therapy.

    Science.gov (United States)

    Han, In-Kwon; Kim, Mi Young; Byun, Hyang-Min; Hwang, Tae Sun; Kim, Jung Mogg; Hwang, Kwang Woo; Park, Tae Gwan; Jung, Woon-Won; Chun, Taehoon; Jeong, Gil-Jae; Oh, Yu-Kyoung

    2007-01-01

    Recently, nasal administration has been studied as a noninvasive route for delivery of plasmid DNA encoding therapeutic or antigenic genes. Here, we examined the brain targeting efficiency and transport pathways of intranasally administered plasmid DNA. Quantitative polymerase chain reaction (PCR) measurements of plasmid DNA in blood and brain tissues revealed that intranasally administered pCMVbeta (7.2 kb) and pN2/CMVbeta (14.1 kb) showed systemic absorption and brain distribution. Following intranasal administration, the beta-galactosidase protein encoded by these plasmids was significantly expressed in brain tissues. Kinetic studies showed that intranasally administered plasmid DNA reached the brain with a 2,595-fold higher efficiency than intravenously administered plasmid DNA did, 10 min post-dose. Over 1 h post-dose, the brain targeting efficiencies were consistently higher for intranasally administered plasmid DNA than for intravenously administered DNA. To examine how plasmid DNA enters the brain and moves to the various regions, we examined tissues from nine brain regions, at 5 and 10 min after intranasal or intravenous administration of plasmid DNA. Intravenously administered plasmid DNA displayed similar levels of plasmid DNA in the nine different regions, whereas, intranasally administered plasmid DNA exhibited different levels of distribution among the regions, with the highest plasmid DNA levels in the olfactory bulb. Moreover, plasmid DNA was mainly detected in the endothelial cells, but not in glial cells. Our results suggest that intranasally applied plasmid DNA may reach the brain through a direct route, possibly via the olfactory bulb, and that the nasal route might be an alternative method for efficiently delivering plasmid DNA to the brain.

  16. Anion exchange purification of plasmid DNA using expanded bed adsorption.

    Science.gov (United States)

    Ferreira, G N; Cabral, J M; Prazeres, D M

    2000-01-01

    Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity of process streams is of major concern in the purification of plasmids, since it can cause high back pressures in column operations, thus limiting the throughput. In order to avoid these high back pressures, expanded bed anion exchange chromatography was evaluated as an alternative to fixed bed chromatography. A Streamline 25 column filled with 100 ml of Streamline QXL media, was equilibrated with 0.5 M NaCl in TE (10 mM Tris, 1 mM EDTA, pH = 8.0) buffer at an upward flow of 300 cmh-1, E. coli lysates (obtained from up to 3 liters of fermentation broth) were injected in the column. After washing out the unbound material, the media was allowed to sediment and the plasmid was eluted with 1 M NaCl in TE buffer at a downward flow of 120 cmh-1. Purification factors of 36 +/- 1 fold, 26 +/- 0.4 plasmid purity, and close to 100% yields were obtained when less than one settled column volume of plasmid feed was injected. However, both recovery yield and purity abruptly decreased when larger amounts were processed-values of 35 +/- 2 and 5 +/- 0.7 were obtained for the recovery yield and purity, respectively, when 250 ml of feedstock were processed. In these cases, gel clogging and expansion collapse were observed. The processing of larger volumes, thus larger plasmid quantities, was only possible by performing an isopropanol precipitation step prior to the chromatographic step. This step led to an enhancement of the purification step.

  17. Using Plasmids as DNA Vaccines for Infectious Diseases.

    Science.gov (United States)

    Tregoning, John S; Kinnear, Ekaterina

    2014-12-01

    DNA plasmids can be used to induce a protective (or therapeutic) immune response by delivering genes encoding vaccine antigens. That naked DNA (without the refinement of coat proteins or host evasion systems) can cross from outside the cell into the nucleus and be expressed is particularly remarkable given the sophistication of the immune system in preventing infection by pathogens. As a result of the ease, low cost, and speed of custom gene synthesis, DNA vaccines dangle a tantalizing prospect of the next wave of vaccine technology, promising individual designer vaccines for cancer or mass vaccines with a rapid response time to emerging pandemics. There is considerable enthusiasm for the use of DNA vaccination as an approach, but this enthusiasm should be tempered by the successive failures in clinical trials to induce a potent immune response. The technology is evolving with the development of improved delivery systems that increase expression levels, particularly electroporation and the incorporation of genetically encoded adjuvants. This review will introduce some key concepts in the use of DNA plasmids as vaccines, including how the DNA enters the cell and is expressed, how it induces an immune response, and a summary of clinical trials with DNA vaccines. The review also explores the advances being made in vector design, delivery, formulation, and adjuvants to try to realize the promise of this technology for new vaccines. If the immunogenicity and expression barriers can be cracked, then DNA vaccines may offer a step change in mass vaccination.

  18. Persistence of Free Plasmid DNA in Soil Monitored by Various Methods, Including a Transformation Assay

    Science.gov (United States)

    Romanowski, Gerd; Lorenz, Michael G.; Sayler, Gary; Wackernagel, Wilfried

    1992-01-01

    The persistence and stability of free plasmid pUC8-ISP DNA introduced into 10-g samples of various soils and kept at 23°C were monitored over a period of 60 days. The soils were sampled at a plant science farm and included a loamy sand soil (no. 1), a clay soil (no. 2), and a silty clay soil (no. 3). Four different methods allowed monitoring of (i) the production of acid-soluble radioactive material from [3H]thymidine-labeled plasmid DNA, (ii) the decrease of hybridizing nucleotide sequences in slot blot analysis, (iii) the loss of plasmid integrity measured by Southern hybridization, and (iv) the decay of the biological activity as determined by transformation of Ca2+-treated Escherichia coli cells with the DNA extracted from soil. Acid-soluble material was not produced within the first 24 h but then increased to 45% (soil no. 1), 27% (soil no. 2), and 77% (soil no. 3) until the end of incubation. A quite parallel loss of material giving a slot blot hybridization signal was observed. Southern hybridization indicated that after 1 h in the soils, plasmid DNA was mostly in the form of circular and full-length linear molecules but that, depending on the soil type, after 2 to 5 days full-length plasmid molecules were hardly detectable. The transforming activity of plasmid DNA reextracted from the soils followed inactivation curves over 2 to 4 orders of magnitude and dropped below the detection limit after 10 days. The inactivation was slower in soil no. 2 (28.2-h half-life time of the transforming activity of a plasmid molecule) than in soils no. 3 (15.1 h) and no. 1 (9.1 h). The studies provide data on the persistence of free DNA molecules in natural bacterial soil habitats. The data suggest that plasmid DNA may persist long enough to be available for uptake by competent recipient cells in situ. Images PMID:16348772

  19. Bacterial Mitosis: ParM of Plasmid R1 Moves Plasmid DNA by an Actin-like Insertional Polymerization Mechanism

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette

    2003-01-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system...

  20. DNA repair in bacterial cultures and plasmid DNA exposed to infrared laser for treatment of pain

    Science.gov (United States)

    Canuto, K. S.; Sergio, L. P. S.; Marciano, R. S.; Guimarães, O. R.; Polignano, G. A. C.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2013-06-01

    Biostimulation of tissues by low intensity lasers has been described on a photobiological basis and clinical protocols are recommended for treatment of various diseases, but their effects on DNA are controversial. The objective of this work was to evaluate effects of low intensity infrared laser exposure on survival and bacterial filamentation in Escherichia coli cultures, and induction of DNA lesions in bacterial plasmids. In E. coli cultures and plasmids exposed to an infrared laser at fluences used to treat pain, bacterial survival and filamentation and DNA lesions in plasmids were evaluated by electrophoretic profile. Data indicate that the infrared laser (i) increases survival of E. coli wild type in 24 h of stationary growth phase, (ii) induces bacterial filamentation, (iii) does not alter topological forms of plasmids and (iv) does not alter the electrophoretic profile of plasmids incubated with exonuclease III or formamidopyrimidine DNA glycosylase. A low intensity infrared laser at the therapeutic fluences used to treat pain can alter survival of E. coli wild type, induce filamentation in bacterial cells, depending on physiologic conditions and DNA repair, and induce DNA lesions other than single or double DNA strand breaks or alkali-labile sites, which are not targeted by exonuclease III or formamidopyrimidine DNA glycosylase.

  1. Complexation Between Cationic Diblock Copolymers and Plasmid DNA

    Science.gov (United States)

    Jung, Seyoung; Reineke, Theresa; Lodge, Timothy

    Deoxyribonucleic acids (DNA), as polyanions, can spontaneously bind with polycations to form polyelectrolyte complexes. When the polycation is a diblock copolymer with one cationic block and one uncharged hydrophilic block, the polyelectrolyte complexes formed with plasmid DNA (pDNA) are often colloidally stable, and show great promise in the field of polymeric gene therapy. While the resulting properties (size, stability, and toxicity to biological systems) of the complexes have been studied for numerous cationic diblocks, the fundamentals of the pDNA-diblock binding process have not been extensively investigated. Herein, we report how the cationic block content of a diblock influences the pDNA-diblock interactions. pDNA with 7164 base pairs and poly(2-deoxy-2-methacrylamido glucopyranose)-block-poly(N-(2-aminoethyl) methacrylamide) (PMAG-b-PAEMA) are used as the model pDNA and cationic diblock, respectively. To vary the cationic block content, two PMAG-b-PAEMA copolymers with similar PMAG block lengths but distinct PAEMA block lengths and a PAEMA homopolymer are utilized. We show that the enthalpy change from pDNA-diblock interactions is dependent on the cationic diblock composition, and is closely associated with both the binding strength and the pDNA tertiary structure.

  2. DNA extraction from crayfish exoskeleton

    National Research Council Canada - National Science Library

    Li, Yanhe; Wang, Weimin; Liu, Xiaolian; Luo, Wei; Zhang, Jie; Gul, Yasmeen

    2011-01-01

    .... However, it is difficult to extract DNA from them. This study was intended to investigate CE as a DNA source and design an easy and efficient DNA extraction protocol for polymerase chain reactions...

  3. The Cloning of the Human Tumor Supressor Gene INGI: DNA Cloning into Plasmid Vector and DNA Analysis by Restriction Enzymes

    Directory of Open Access Journals (Sweden)

    Elza Ibrahim Auerkari

    2015-11-01

    Full Text Available DNA cloning is one of the most important techniques In the field of molecular biology, with a critical role in analyzing the structure and function of genes and their adjacent regulatory regions. DNA cloning is helpful in learning fundamental molecular biological techniques, since DNA cloning involves a series of them, such as polymerase chain reaction (PCR, DNA ligation, bacterial transformation, bacterial culture, plasmid DNA extraction, DNA digestion with restriction enzymes and agarose gel electrophoresis. In this paper the cloning of the human tumor suppressor gene INGI has been used to illustrate the methodology. The gene was amplified by PCR, cloned into a TA-cloning vectore, and restriction enzyme mapping was used to distinguish the sense INGI construct from the antisense INGI construct.

  4. Influence of an aqueous extract of Hypericum perforatum (Hypericin on the survival of Escherichia coli AB1157 and on the electrophoretic mobility of pBSK plasmid DNA Influência de um extrato aquoso de Hypericum perforatum (Hipericina na sobreviência de Escherichia coli AB1157 e na mobilidade eletroforética de DNA plasmidial pBSK

    Directory of Open Access Journals (Sweden)

    Sebastião D. Santos-Filho

    2008-09-01

    Full Text Available Hiperico (Hypericum perforatum or St John's worth has been widely used as an herbal medicine to treat depression. Hypericin is the main chemical compound of hiperico. Stannous chloride (SnCl2 is the most used reducing agent in nuclear medicine. The aim of this work was to verify the effect of a hiperico extract on the survival of Escherichia coli AB1157 and on the plasmid DNA topology. Exponentially E. coli AB1157 cultures were incubated with SnCl2 in the presence or absence of hypericin. Aliquots were spread onto Petri dishes containing solidified rich medium, the colonies units were counted after overnight and the survival fraction was calculated. Plasmid DNA samples were incubated with SnCl2 in presence or absence of hypericin extract during 40 minutes, 0.8% agarose gel electrophoresis was performed, the gel was stained with ethidium bromide and the plasmid topological forms (bands were visualized. The results revealed that hiperico extract is neither capable of altering the survival of E. coli cells nor the plasmid DNA topology but it may have protected these cells against the SnCl2 action. The data suggest absence of cytotoxic and genotoxic effects of the aqueous hiperico extract and a protective effect on E. coli cells against the action of SnCl2.Hipérico (Hypericum perforatum or St John's worth tem sido usado como uma planta medicinal para tratar a depressão. Hipericina é o principal componente do hipérico. O cloreto estanoso (SnCl2 é o agente redutor mais utilizado em medicina nuclear. O objetivo desse trabalho foi verificar o efeito de um extrato de hipérico na sobrevivência de Escherichia coli AB1157 e na topologia do DNA plasmidial. Culturas de E. coli AB1157, em fase exponencial, foram incubadas com SnCl2 na presença ou ausência de hipericina. Alíquotas foram espalhadas em placas de Petri contendo meio sólido, as unidades formadoras de colônias foram contadas após incubação e as frações de sobrevivência calculadas

  5. Scaling-up recombinant plasmid DNA for clinical trial: current concern, solution and status.

    Science.gov (United States)

    Ismail, Ruzila; Allaudin, Zeenathul Nazariah; Lila, Mohd-Azmi Mohd

    2012-09-07

    Gene therapy and vaccines are rapidly developing field in which recombinant nucleic acids are introduced in mammalian cells for enhancement, restoration, initiation or silencing biochemical function. Beside simplicity in manipulation and rapid manufacture process, plasmid DNA-based vaccines have inherent features that make them promising vaccine candidates in a variety of diseases. This present review focuses on the safety concern of the genetic elements of plasmid such as propagation and expression units as well as their host genome for the production of recombinant plasmid DNA. The highlighted issues will be beneficial in characterizing and manufacturing plasmid DNA for save clinical use. Manipulation of regulatory units of plasmid will have impact towards addressing the safety concerns raised in human vaccine applications. The gene revolution with plasmid DNA by alteration of their plasmid and production host genetics will be promising for safe delivery and obtaining efficient outcomes.

  6. Microneedle-mediated transcutaneous immunization with plasmid DNA coated on cationic PLGA nanoparticles

    Science.gov (United States)

    Kumar, Amit; Wonganan, Piyanuch; Sandoval, Michael A.; Li, Xinran; Zhu, Saijie; Cui, Zhengrong

    2012-01-01

    Previously, it was shown that microneedle-mediated transcutaneous immunization with plasmid DNA can potentially induce a stronger immune response than intramuscular injection of the same plasmid DNA. In the present study, we showed that the immune responses induced by transcutaneous immunization by applying plasmid DNA onto a skin area pretreated with solid microneedles were significantly enhanced by coating the plasmid DNA on the surface of cationic nanoparticles. In addition, the net surface charge of the DNA-coated nanoparticles significantly affected their in vitro skin permeation and their ability to induce immune responses in vivo. Transcutaneous immunization with plasmid DNA-coated net positively charged anoparticles elicited a stronger immune response than with plasmid DNA-coated net negatively charged nanoparticles or by intramuscular immunization with plasmid DNA alone. Transcutaneous immunization with plasmid DNA-coated net positively charged nanoparticles induced comparable immune responses as intramuscular injection of them, but transcutaneous immunization was able to induce specific mucosal immunity and a more balanced T helper type 1 and type 2 response. The ability of the net positively charged DNA-coated nanoparticles to induce a strong immune response through microneedle-mediated transcutaneous immunization may be attributed to their ability to increase the expression of the antigen gene encoded by the plasmid and to more effectively stimulate the maturation of antigen-presenting cells. PMID:22921518

  7. Plasmid DNA gene therapy by electroporation: principles and recent advances.

    Science.gov (United States)

    Murakami, Tatsufumi; Sunada, Yoshihide

    2011-12-01

    Simple plasmid DNA injection is a safe and feasible gene transfer method, but it confers low transfection efficiency and transgene expression. This non-viral gene transfer method is enhanced by physical delivery methods, such as electroporation and the use of a gene gun. In vivo electroporation has been rapidly developed over the last two decades to deliver DNA to various tissues or organs. It is generally considered that membrane permeabilization and DNA electrophoresis play important roles in electro-gene transfer. Skeletal muscle is a well characterized target tissue for electroporation, because it is accessible and allows for long-lasting gene expression ( > one year). Skin is also a target tissue because of its accessibility and immunogenicity. Numerous studies have been performed using in vivo electroporation in animal models of disease. Clinical trials of DNA vaccines and immunotherapy for cancer treatment using in vivo electroporation have been initiated in patients with melanoma and prostate cancer. Furthermore, electroporation has been applied to DNA vaccines for infectious diseases to enhance immunogenicity, and the relevant clinical trials have been initiated. The gene gun approach is also being applied for the delivery of DNA vaccines against infectious diseases to the skin. Here, we review recent advances in the mechanism of in vivo electroporation, and summarize the findings of recent preclinical and clinical studies using this technology.

  8. Bacterial mitosis: ParM of plasmid R1 moves plasmid DNA by an actin-like insertional polymerization mechanism.

    Science.gov (United States)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette; Jensen, Rasmus B; Roepstorff, Peter; Gerdes, Kenn

    2003-12-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system act together to generate the force required for plasmid movement during segregation. ParR protein binds cooperatively to the centromeric parC DNA region, thereby forming a complex that interacts with the filament-forming actin-like ParM protein in an ATP-dependent manner, suggesting that plasmid movement is powered by insertional polymerization of ParM. Consistently, we find that segregating plasmids are positioned at the ends of extending ParM filaments. Thus, the process of R1 plasmid segregation in E. coli appears to be mechanistically analogous to the actin-based motility operating in eukaryotic cells. In addition, we find evidence suggesting that plasmid pairing is required for ParM polymerization.

  9. Determination of plasmid copy number reveals the total plasmid DNA amount is greater than the chromosomal DNA amount in Bacillus thuringiensis YBT-1520.

    Directory of Open Access Journals (Sweden)

    Chunying Zhong

    Full Text Available Bacillus thuringiensis is the most widely used bacterial bio-insecticide, and most insecticidal crystal protein-coding genes are located on plasmids. Most strains of B. thuringiensis harbor numerous diverse plasmids, although the plasmid copy numbers (PCNs of all native plasmids in this host and the corresponding total plasmid DNA amount remains unknown. In this study, we determined the PCNs of 11 plasmids (ranging from 2 kb to 416 kb in a sequenced B. thuringiensis subsp. kurstaki strain YBT-1520 using real-time qPCR. PCNs were found to range from 1.38 to 172, and were negatively correlated to plasmid size. The amount of total plasmid DNA (∼8.7 Mbp was 1.62-fold greater than the amount of chromosomal DNA (∼5.4 Mbp at the mid-exponential growth stage (OD(600 = 2.0 of the organism. Furthermore, we selected three plasmids with different sizes and replication mechanisms to determine the PCNs over the entire life cycle. We found that the PCNs dynamically shifted at different stages, reaching their maximum during the mid-exponential growth or stationary phases and remaining stable and close to their minimum after the prespore formation stage. The PCN of pBMB2062, which is the smallest plasmid (2062 bp and has the highest PCN of those tested, varied in strain YBT-1520, HD-1, and HD-136 (172, 115, and 94, respectively. These findings provide insight into both the total plasmid DNA amount of B. thuringiensis and the strong ability of the species to harbor plasmids.

  10. Adsorption behavior of plasmid DNA onto perfusion chromatographic matrix

    Institute of Scientific and Technical Information of China (English)

    Miladys LIMONTA; Lourdes ZUMALACARREGUI; Dayana SOLER

    2012-01-01

    Anion exchange chromatography is the most popular chromatographic method for plasmid separa-tion.POROS RI 50 is a perfusion chromatographic support which is a reversed phase matrix and is an alterna-tive to conventional ones due to its mass transfer properties.The adsorption and elution of the pIDKE2 plasmidonto reversed phase POROS RI 50 was studied.Langmuir isotherm model was adjusted in order to get the max-imum adsorption capacity and the dissociation constant for POROS RI 50-plasmid DNA (pDNA) system.Break-through curves were obtained for volumetric flows between 0.69-3.33mL/min,given dynamic capacity up to2.3 times higher than those reported for ionic exchange matrix used during the purification process of plasmidswith similar size to that of pIDKE2.The efficiency was less than 45% for the flow conditions and initial concen-tration studied,which means that the support will not be operated under saturation circumstances.

  11. Variety of molecular conformation of plasmid pUC18 DNA and solenoidally supercoiled DNA

    Institute of Scientific and Technical Information of China (English)

    黄熙泰; 王照清; 吴永文; 樊廷玉; 王树荣; 王勖焜

    1996-01-01

    The plasmid pUC18 DNA isolated from Escherichia coli HB101 were analyzed by two-dimensional agarose gel electrophoresis and hybridization. The results show that the DNA sample can be separated into six groups of different structural components. The plectonemically and solenoidally supercoiled pUC18 DNA coexist in it. These two different conformations of supercoiled DNA are interchangeable with the circumstances (ionic strength and type, etc.). The amount of solenoidally supercoiled pUC18 DNA in the samples can be changed by treatment of DNA topoisome rases. Under an electron microscope, the solenoidal supercoiling DNA has a round shape with an average diameter of 45 nm. The facts suggest that solenoidaUy supercoiled DNA be a structural entity independent of histones. The polymorphism of DNA structure may be important to packing of DNA in vivo.

  12. Rapid isolation of plasmid DNA by LiCl-ethidium bromide treatment and gel filtration.

    Science.gov (United States)

    Kondo, T; Mukai, M; Kondo, Y

    1991-10-01

    We established a simple and rapid plasmid DNA purification method. Crude plasmid DNA preparations are treated with 4 M LiCl in the presence of 0.6 mg/ml ethidium bromide to precipitate RNA and proteins contained in the DNA preparations. After removal of RNA and protein precipitates, the supernatant is filtered through a Sepharose CL6B column to remove low-molecular-weight contaminants. This procedure takes only 30 min and provides pure plasmid DNA preparations that consist mainly of covalently closed circular plasmid DNA but have no detectable RNA and protein. The purified DNA preparations are susceptible to various six- and four-base-recognition restriction endonucleases, T4 DNA ligase, the Klenow fragment of DNA polymerase I, and T7 and Taq DNA polymerase. Since no special equipment is needed for this purification method, 20 or more samples of microgram to milligram levels can be treated in parallel.

  13. RK2 plasmid dynamics in Caulobacter crescentus cells--two modes of DNA replication initiation.

    Science.gov (United States)

    Wegrzyn, Katarzyna; Witosinska, Monika; Schweiger, Pawel; Bury, Katarzyna; Jenal, Urs; Konieczny, Igor

    2013-06-01

    Undisturbed plasmid dynamics is required for the stable maintenance of plasmid DNA in bacterial cells. In this work, we analysed subcellular localization, DNA synthesis and nucleoprotein complex formation of plasmid RK2 during the cell cycle of Caulobacter crescentus. Our microscopic observations showed asymmetrical distribution of plasmid RK2 foci between the two compartments of Caulobacter predivisional cells, resulting in asymmetrical allocation of plasmids to progeny cells. Moreover, using a quantitative PCR (qPCR) method, we estimated that multiple plasmid particles form a single fluorescent focus and that the number of plasmids per focus is approximately equal in both swarmer and predivisional Caulobacter cells. Analysis of the dynamics of TrfA-oriV complex formation during the Caulobacter cell cycle revealed that TrfA binds oriV primarily during the G1 phase, however, plasmid DNA synthesis occurs during the S and G2 phases of the Caulobacter cell cycle. Both in vitro and in vivo analysis of RK2 replication initiation in C. crescentus cells demonstrated that it is independent of the Caulobacter DnaA protein in the presence of the longer version of TrfA protein, TrfA-44. However, in vivo stability tests of plasmid RK2 derivatives suggested that a DnaA-dependent mode of plasmid replication initiation is also possible.

  14. A Simple and Inexpensive Method for Sending Binary Vector Plasmid DNA by Mail

    Science.gov (United States)

    We describe a simple cost-effective technique for the transport of plasmid DNA by mail. Our results demonstrate that common multipurpose printing paper is a satisfactory substrate and superior to the more absorbent 3MM chromatography paper for the transport of plasmid DNA through the U.S. first clas...

  15. INVESTIGATION OF POLYDL-LACTIDE-b-POLY(ETHYLENE GLYCOL)-b-POLYDL-LACTIDE MICROSPHERES CONTAINING PLASMID DNA

    Institute of Scientific and Technical Information of China (English)

    Yu Liu; Long Huang; Xiao-rong Qiao; Xian-mo Deng; Wen-xiang Jia; Xiao-hong Li

    2004-01-01

    PolyDL-lactide (PDLLA) and the block copolymer, polyDL-lactide-b-poly(ethylene glycol)-b-polyDL-lactide (PELA) were used as the microsphere matrix to encapsulate plasmid DNA. The PDLLA, PELA, pBR322-1oaded PDLLA and pBR322-1oaded PELA microspheres were prepared by solvent extraction method based on the formation of multiple w1/o/w2 emulsion. The microspheres were characterized by surface morphology, mean particle size, particle size distribution and loading efficiency. The integrity of DNA molecules after being extracted from microspheres was determined by agarose gel electrophoresis. The result suggested that plasmid DNA molecules could retain their integrity after being encapsulated by PELA. The PELA microspheres could prevent plasmid DNA from being digested by DNase. The in vitro degradation and release profiles of plasmid DNA-loaded microspheres were measured in pH = 7.4 buffer solution at 37℃. The in vitro degradation profiles of the microspheres were evaluated by the deterioration in microspheres surface morphology, the molecular weight reduction of polymer, the mass loss of microspheres, the changes of pH values of degradation medium, and the changes of particle size. The in vitro release profiles of the microspheres were assessed by measurement of the amount of DNA presented in the release medium at determined intervals. The release profiles were correlation with the degradation profiles. The release of plasmid DNA from PELA microspheres showed a similar biphasic trend, that is, an initial burst release was followed by a slow, but sustained release.

  16. Linear forms of plasmid DNA are superior to supercoiled structures as active templates for gene expression in plant protoplasts.

    Science.gov (United States)

    Ballas, N; Zakai, N; Friedberg, D; Loyter, A

    1988-07-01

    Introduction of the plasmids pUC8CaMVCAT and pNOSCAT into plant protoplasts is known to result in transient expression of the chloramphenicol acetyl transferase (CAT) gene. Also, transfection with the plasmid pDO432 results in transient appearance of the luciferase enzyme. In the present work we have used these systems to study the effect of DNA topology on the expression of the above recombinant genes. Linear forms of the above plasmids exhibited much higher activity in supporting gene expression than their corresponding super-coiled structures. CAT activity in protoplasts transfected with the linear forms of pUC8CaMVCAT and pNOSCAT was up to ten-fold higher than that observed in protoplasts transfected by the supercoiled template of these plasmids. This effect was observed in protoplasts derived from two different lines of Petunia hybrida and from a Nicotiana tabacum cell line. Transfection with the relaxed form of pUC8CaMVCAT resulted in very low expression of the CAT gene.Northern blot analysis revealed that the amount of poly(A)(+) RNA extracted from protoplasts transformed with the linear forms of the DNA was about 10-fold higher than that found in protoplasts transformed with supercoiled DNA.Southern blot analysis revealed that about the same amounts of supercoiled and linear DNA molecules were present in nuclei of transfected protoplasts. No significant quantitative differences have been observed between the degradation rates of the various DNA templates used.

  17. Separation of topological forms of plasmid DNA by anion-exchange HPLC: shifts in elution order of linear DNA.

    Science.gov (United States)

    Smith, Clara R; DePrince, Randolph B; Dackor, Jennifer; Weigl, Debra; Griffith, Jack; Persmark, Magnus

    2007-07-01

    We sought to establish a single anion-exchange HPLC method for the separation of linear, open circular and supercoiled plasmid topoisomers using purified topoisomeric forms of three plasmids (3.0, 5.5 and 7.6 kb). However, finding one condition proved elusive as the topoisomer elution order was determined to depend on salt gradient slope. The observed change in selectivity increased with plasmid size and was most pronounced for the linear form. Indeed, the elution order of the linear 7.6 kb plasmid was reversed relative to the supercoiled form. This observation may have implications for methods used in quality control of plasmid DNA.

  18. Optimizing hyaluronidase dose and plasmid DNA delivery greatly improves gene electrotransfer efficiency in rat skeletal muscle

    DEFF Research Database (Denmark)

    Åkerström, Thorbjörn; Vedel, Kenneth; Needham Andersen, Josefine

    2015-01-01

    Transfection of rat skeletal muscle in vivo is a widely used research model. However, gene electrotransfer protocols have been developed for mice and yield variable results in rats. We investigated whether changes in hyaluronidase pre-treatment and plasmid DNA delivery can improve transfection...... efficiency in rat skeletal muscle. We found that pre-treating the muscle with a hyaluronidase dose suitable for rats (0.56. U/g b.w.) prior to plasmid DNA injection increased transfection efficiency by >200% whereas timing of the pre-treatment did not affect efficiency. Uniformly distributing plasmid DNA...... delivery across the muscle by increasing the number of plasmid DNA injections further enhanced transfection efficiency whereas increasing plasmid dose from 0.2 to 1.6. μg/g b.w. or vehicle volume had no effect. The optimized protocol resulted in ~80% (CI95%: 79-84%) transfected muscle fibers...

  19. Low-dose plasmid DNA treatment increases plasma vasopressin and regulates blood pressure in experimental endotoxemia

    Directory of Open Access Journals (Sweden)

    Malardo Thiago

    2012-11-01

    Full Text Available Abstract Background Although plasmid DNA encoding an antigen from pathogens or tumor cells has been widely studied as vaccine, the use of plasmid vector (without insert as therapeutic agent requires further investigation. Results Here, we showed that plasmid DNA (pcDNA3 at low doses inhibits the production of IL-6 and TNF-α by lipopolysaccharide (LPS-stimulated macrophage cell line J774. These findings led us to evaluate whether plasmid DNA could act as an anti-inflammatory agent in a Wistar rat endotoxemia model. Rats injected simultaneously with 1.5 mg/kg of LPS and 10 or 20 μg of plasmid DNA had a remarkable attenuation of mean arterial blood pressure (MAP drop at 2 hours after treatment when compared with rats injected with LPS only. The beneficial effect of the plasmid DNA on MAP was associated with decreased expression of IL-6 in liver and increased concentration of plasma vasopressin (AVP, a known vasoconstrictor that has been investigated in hemorrhagic shock management. No difference was observed in relation to nitric oxide (NO production. Conclusion Our results demonstrate for the first time that plasmid DNA vector at low doses presents anti-inflammatory property and constitutes a novel approach with therapeutic potential in inflammatory diseases.

  20. Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology

    Directory of Open Access Journals (Sweden)

    Li Li

    2010-01-01

    Full Text Available Abstract Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1 DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2 Miniprep DNA from E. coli can be eluted with as little as 5 μl water, leading to high DNA concentrations (>1 μg/μl for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3 Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4 High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.

  1. Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology.

    Science.gov (United States)

    Li, Jian-Feng; Li, Li; Sheen, Jen

    2010-01-14

    Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2) Miniprep DNA from E. coli can be eluted with as little as 5 mul water, leading to high DNA concentrations (>1 mug/mul) for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG)-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3) Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4) High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.

  2. A DNA polymerase mutation that suppresses the segregation bias of an ARS plasmid in Saccharomyces cerevisiae.

    Science.gov (United States)

    Houtteman, S W; Elder, R T

    1993-03-01

    Yeast autonomously replicating sequence (ARS) plasmids exhibit an unusual segregation pattern during mitosis. While the nucleus divides equally into mother and daughter cells, all copies of the ARS plasmid will often remain in the mother cell. A screen was designed to isolate mutations that suppress this segregation bias. A plasmid with a weak ARS (wARS) that displayed an extremely high segregation bias was constructed. When cells were grown under selection for the wARS plasmid, the resulting colonies grew slowly and had abnormal morphology. A spontaneous recessive mutation that restored normal colony morphology was identified. This mutation suppressed plasmid segregation bias, as indicated by the increased stability of the wARS plasmid in the mutant cells even though the plasmid was present at a lower copy number. An ARS1 plasmid was also more stable in mutant cells than in wild-type cells. The wild-type allele for this mutant gene was cloned and identified as POL delta (CDC2). This gene encodes DNA polymerase delta, which is essential for DNA replication. These results indicate that DNA polymerase delta plays some role in causing the segregation bias of ARS plasmids.

  3. Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis: isolation of a spontaneous mutant of Bacillus subtilis with enhanced transformability for Escherichia coli-propagated chimeric plasmid DNA.

    OpenAIRE

    Ostroff, G. R.; Pène, J. J.

    1983-01-01

    Hybrid plasmid DNA cloned in Escherichia coli undergoes deletions when returned to competent Bacillus subtilis, even in defined restriction and modification mutants of strain 168. We have isolated a mutant of B. subtilis MI112 which is stably transformed at high frequency by chimeric plasmid DNA propagated in E. coli.

  4. Differential interactions of plasmid DNA, RNA and genomic DNA with amino acid-based affinity matrices.

    Science.gov (United States)

    Sousa, Angela; Sousa, Fani; Queiroz, João A

    2010-09-01

    The development of a strategy to plasmid DNA (pDNA) purification has become necessary for the development of gene therapy and DNA vaccine production processes in recent years, since this nucleic acid and most of contaminants, such as RNA, genomic DNA and endotoxins, are negatively charged. An ideal separation methodology may be achieved with the use of affinity interactions between immobilized amino acids and nucleic acids. In this study, the binding behaviour of nucleic acids under the influence of different environmental conditions, such as the composition and ionic strength of elution buffer, and the temperature, is compared with various amino acids immobilized on chromatography resins. Supercoiled (sc) plasmid isoform was isolated with all matrices used, but in some cases preferential interactions with other nucleic acids were found. Particularly, lysine chromatography showed to be an ideal technology mainly on RNA purification using low salt concentration. On the other hand, arginine ligands have shown a greater ability to retain the sc isoform comparatively to the other nucleic acids retention, becoming this support more adequate to sc pDNA purification. The temperature variation, competitive elution and oligonucleotides affinity studies also allowed to recognize the dominant interactions inherent to biorecognition of pDNA molecule and the affinity matrices.

  5. Homology and repair of UV-irradiated plasmid DNA in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Cabrea-Juarez, E.; Setlow, J.K.

    1983-02-01

    UV-irradiated plasmid pNov1 containing a cloned fragment of chromosomal DNA could be repaired by excision, but plasmid p2265 without homology to the chromosome could not. Establishment of pNov1 was more UV resistant in Rec/sup -/ than in Rec/sup +/ cells. 19 references, 2 figures.

  6. A one-step miniprep for the isolation of plasmid DNA and lambda phage particles.

    Directory of Open Access Journals (Sweden)

    George Lezin

    Full Text Available Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E. coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.

  7. Characterization of Plasmid DNA Location within Chitosan/PLGA/pDNA Nanoparticle Complexes Designed for Gene Delivery

    Directory of Open Access Journals (Sweden)

    Hali Bordelon

    2011-01-01

    Full Text Available Poly(D,L-lactide-co-glycolide- (PLGA-chitosan nanoparticles are becoming an increasingly common choice for the delivery of nucleic acids to cells for various genetic manipulation techniques. These particles are biocompatible, with tunable size and surface properties, possessing an overall positive charge that promotes complex formation with negatively charged nucleic acids. This study examines properties of the PLGA-chitosan nanoparticle/plasmid DNA complex after formation. Specifically, the study aims to determine the optimal ratio of plasmid DNA:nanoparticles for nucleic acid delivery purposes and to elucidate the location of the pDNA within these complexes. Such characterization will be necessary for the adoption of these formulations in a clinical setting. The ability of PLGA-chitosan nanoparticles to form complexes with pDNA was evaluated by using the fluorescent intercalating due OliGreen to label free plasmid DNA. By monitoring the fluorescence at different plasmid: nanoparticle ratios, the ideal plasmid:nanoparticle ration for complete complexation of plasmid was determined to be 1:50. Surface-Enhanced Raman Spectroscopy and gel digest studies suggested that even at these optimal complexation ratios, a portion of the plasmid DNA was located on the outer complex surface. This knowledge will facilitate future investigations into the functionality of the system in vitro and in vivo.

  8. Mechanistic basis of plasmid-specific DNA binding of the F plasmid regulatory protein, TraM.

    Science.gov (United States)

    Peng, Yun; Lu, Jun; Wong, Joyce J W; Edwards, Ross A; Frost, Laura S; Mark Glover, J N

    2014-11-11

    The conjugative transfer of bacterial F plasmids relies on TraM, a plasmid-encoded protein that recognizes multiple DNA sites to recruit the plasmid to the conjugative pore. In spite of the high degree of amino acid sequence conservation between TraM proteins, many of these proteins have markedly different DNA binding specificities that ensure the selective recruitment of a plasmid to its cognate pore. Here we present the structure of F TraM RHH (ribbon-helix-helix) domain bound to its sbmA site. The structure indicates that a pair of TraM tetramers cooperatively binds an underwound sbmA site containing 12 base pairs per turn. The sbmA is composed of 4 copies of a 5-base-pair motif, each of which is recognized by an RHH domain. The structure reveals that a single conservative amino acid difference in the RHH β-ribbon between F and pED208 TraM changes its specificity for its cognate 5-base-pair sequence motif. Specificity is also dictated by the positioning of 2-base-pair spacer elements within sbmA; in F sbmA, the spacers are positioned between motifs 1 and 2 and between motifs 3 and 4, whereas in pED208 sbmA, there is a single spacer between motifs 2 and 3. We also demonstrate that a pair of F TraM tetramers can cooperatively bind its sbmC site with an affinity similar to that of sbmA in spite of a lack of sequence similarity between these DNA elements. These results provide a basis for the prediction of the DNA binding properties of the family of TraM proteins.

  9. Effects of medium composition on the production of plasmid DNA vector potentially for human gene therapy

    Institute of Scientific and Technical Information of China (English)

    XU Zhi-nan; SHEN Wen-he; CHEN Hao; CEN Pei-lin

    2005-01-01

    Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The effects of different MMBL components on plasmid yield, cell mass and specific plasmid DNA productivity were evaluated on shake-flask scale. The results showed that glucose was the optimal carbon source. High plasmid yield (58.3 mg/L) was obtained when 5.0 g/L glucose was added to MMBL. Glycerol could be chosen as a complementary carbon source because of the highest specific plasmid productivity (37.9 mg DNA/g DCW). After tests of different levels of nitrogen source and inorganic phosphate, a modified MMBL medium was formulated for optimal plasmid production. Further study showed that the initial acetate addition (less than 4.0 g/L) in MMBL improved plasmid production significantly, although it inhibited cell growth. The results will be useful for large-scale plasmid production using recombinant E. coli system.

  10. PLASMID DNA DAMAGE CAUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    Science.gov (United States)

    PLASMID DNA DAMAGE CAOUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITINABSTRACT Both dimethylarsinic acid (DMA(V)) and dimethylarsinous acid (DMA(III)) release iron from human liver ferritin (HLF) with or without the presence of ascorbic acid. ...

  11. Event extraction for DNA methylation

    Directory of Open Access Journals (Sweden)

    Ohta Tomoko

    2011-10-01

    Full Text Available Abstract Background We consider the task of automatically extracting DNA methylation events from the biomedical domain literature. DNA methylation is a key mechanism of epigenetic control of gene expression and implicated in many cancers, but there has been little study of automatic information extraction for DNA methylation. Results We present an annotation scheme for DNA methylation following the representation of the BioNLP shared task on event extraction, select a set of 200 abstracts including a representative sample of all PubMed citations relevant to DNA methylation, and introduce manual annotation for this corpus marking nearly 3000 gene/protein mentions and 1500 DNA methylation and demethylation events. We retrain a state-of-the-art event extraction system on the corpus and find that automatic extraction of DNA methylation events, the methylated genes, and their methylation sites can be performed at 78% precision and 76% recall. Conclusions Our results demonstrate that reliable extraction methods for DNA methylation events can be created through corpus annotation and straightforward retraining of a general event extraction system. The introduced resources are freely available for use in research from the GENIA project homepage http://www-tsujii.is.s.u-tokyo.ac.jp/GENIA.

  12. Effect of Plasmid Incompatibility on DNA Transfer to Streptococcus cremoris

    OpenAIRE

    Van Der Lelie, Daniel; Vossen, Jos M.B.M. van der; Venema, Gerard

    1988-01-01

    Several Streptococcus cremoris strains were used in protoplast transformation and interspecific protoplast fusion experiments with Streptococcus lactis and Bacillus subtilis, with pGKV110, pGKV21, and ΔpAMβ1 as the marker plasmids. ΔpAMβ1 is a 15.9-kilobase nonconjugative, deletion derivative of pAMβ1, which is considerably larger than the pGKV plasmids (approximately 4.5 kilobases). In general, ΔpAMβ1 was transferred more efficiently than the pGKV plasmids. Using electroporation, we were abl...

  13. Plasmid loss and changes within the chromosomal DNA of Streptomyces reticuli.

    OpenAIRE

    Schrempf, H

    1982-01-01

    The sporulating wild-type strain of Streptomyces reticuli, which produces a melanin pigment and the macrolide leucomycin, contains plasmid DNA of 48 to 49 megadaltons. Plasmidless variants had an altered secondary metabolism and a changed antibiotic resistance pattern. By using a new colony hybridization technique developed for streptomycetes, it could be shown that plasmidless variants could be transformed with the wild-type plasmid DNA, which, however, is quickly lost from regenerated mycel...

  14. Efficient encapsulation of plasmid DNA in anionic liposomes by a freeze/thaw extrusion procedure

    NARCIS (Netherlands)

    Schoen, P; Bijl, L; Wilschut, J

    1998-01-01

    In this study we investigated whether intact plasmid DNA can be efficiently encapsulated in anionic liposomes prepared by freeze/thaw and extrusion techniques. There is controversy about this method of DNA encapsulation, especially as to whether DNA remains intact and retains its biological activity

  15. Inheritance of Mitochondrial DNA and Plasmids in the Ascomycetous Fungus, Epichloe Typhina

    OpenAIRE

    Chung, K. R.; Leuchtmann, A.; Schardl, C. L.

    1996-01-01

    We analyzed the inheritance of mitochondrial DNA (mtDNA) species in matings of the grass symbiont Epichloe typhina. Eighty progeny were analyzed from a cross in which the maternal (stromal) parent possessed three linear plasmids, designated Callan-a (7.5 kb), Aubonne-a (2.1 kb) and Bergell (2.0 kb), and the paternal parent had one plasmid, Aubonne-b (2.1 kb). Maternal transmission of all plasmids was observed in 76 progeny; two progeny possessed Bergell and Callan-a, but had the maternal Aubo...

  16. Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent.

    Science.gov (United States)

    Wooddell, Christine I; Subbotin, Vladimir M; Sebestyén, Magdolna G; Griffin, Jacob B; Zhang, Guofeng; Schleef, Martin; Braun, Serge; Huss, Thierry; Wolff, Jon A

    2011-02-01

    Various plasmids were delivered into rodent limb muscles by hydrodynamic limb vein (HLV) injection of naked plasmid DNA (pDNA). Some of the pDNA preparations caused significant muscle necrosis and associated muscle regeneration 3 to 4 days after the injection whereas others caused no muscle damage. Occurrence of muscle damage was independent of plasmid sequence, size, and encoded genes. It was batch dependent and correlated with the quantity of bacterial genomic DNA (gDNA) that copurified with the pDNA. To determine whether such an effect was due to bacterial DNA or simply to fragmented DNA, mice were treated by HLV injection with sheared bacterial or murine gDNA. As little as 20 μg of the large fragments of bacterial gDNA caused muscle damage that morphologically resembled damage caused by the toxic pDNA preparations, whereas murine gDNA caused no damage even at a 10-fold higher dose. Toxicity from the bacterial gDNA was not due to endotoxin and was eliminated by DNase digestion. We conclude that pDNA itself does not cause muscle damage and that purification methods for the preparation of therapeutic pDNA should be optimized for removal of bacterial gDNA.

  17. Targeted Collection of Plasmid DNA in Large and Growing Animal Muscles 6 Weeks after DNA Vaccination with and without Electroporation

    Directory of Open Access Journals (Sweden)

    Daniel Dory

    2015-01-01

    Full Text Available DNA vaccination has been developed in the last two decades in human and animal species as a promising alternative to conventional vaccination. It consists in the injection, in the muscle, for example, of plasmid DNA encoding the vaccinating polypeptide. Electroporation which forces the entrance of the plasmid DNA in cells at the injection point has been described as a powerful and promising strategy to enhance DNA vaccine efficacy. Due to the fact that the vaccine is composed of DNA, close attention on the fate of the plasmid DNA upon vaccination has to be taken into account, especially at the injection point. To perform such studies, the muscle injection point has to be precisely recovered and collected several weeks after injection. This is even more difficult for large and growing animals. A technique has been developed to localize precisely and collect efficiently the muscle injection points in growing piglets 6 weeks after DNA vaccination accompanied or not by electroporation. Electroporation did not significantly increase the level of remaining plasmids compared to nonelectroporated piglets, and, in all the cases, the levels were below the limit recommended by the FDA to research integration events of plasmid DNA into the host DNA.

  18. TOL plasmid carriage enhances biofilm formation and increases extracellular DNA content in Pseudomonas putida KT2440

    DEFF Research Database (Denmark)

    D'Alvise, Paul; Sjoholm, O.R.; Yankelevich, T.;

    2010-01-01

    : TOL carriage leads to more extracellular DNA (eDNA) in pellicles and biofilms. Pellicles were dissolved by DNase I treatment. Enhanced cell lysis due to plasmid carriage was ruled out as the mechanism for eDNA release. We report, for the first time, that carriage of a conjugative plasmid leads......Adherent growth of Pseudomonas putida KT2440 with and without the TOL plasmid (pWWO) at the solid-liquid and air-liquid interface was examined. We compared biofilm formation on glass in flow cells, and assayed pellicle (air-liquid interface biofilm) formation in stagnant liquid cultures by confocal...... laser scanning microscopy. The TOL-carrying strains formed pellicles and thick biofilms, whereas the same strains without the plasmid displayed little adherent growth. Microscopy using fluorescent nucleic acid-specific stains revealed differences in the production of extracellular polymeric substances...

  19. Optimizing hyaluronidase dose and plasmid DNA delivery greatly improves gene electrotransfer efficiency in rat skeletal muscle

    DEFF Research Database (Denmark)

    Åkerström, Thorbjörn; Vedel, Kenneth; Needham Andersen, Josefine;

    2015-01-01

    delivery across the muscle by increasing the number of plasmid DNA injections further enhanced transfection efficiency whereas increasing plasmid dose from 0.2 to 1.6. μg/g b.w. or vehicle volume had no effect. The optimized protocol resulted in ~80% (CI95%: 79-84%) transfected muscle fibers......Transfection of rat skeletal muscle in vivo is a widely used research model. However, gene electrotransfer protocols have been developed for mice and yield variable results in rats. We investigated whether changes in hyaluronidase pre-treatment and plasmid DNA delivery can improve transfection...... with a homogenous distribution. We also show that transfection was stable over five weeks of regular exercise or inactivity. Our findings show that species-specific plasmid DNA delivery and hyaluronidase pre-treatment greatly improves transfection efficiency in rat skeletal muscle....

  20. First report on vertical transmission of a plasmid DNA in freshwater prawn, Macrobrachium rosenbergii.

    Science.gov (United States)

    Chowdhury, Labrechai Mog; Gireesh-Babu, P; Pavan-Kumar, A; Suresh Babu, P P; Chaudhari, Aparna

    2014-09-01

    Outbreak of WSSV disease is one of the major stumbling blocks in shrimp aquaculture. DNA vaccines have shown potential for mass scale vaccination owing to their stability, cost effectiveness and easy maintenance. Development of economically feasible delivery strategies remains to be a major challenge. This study demonstrates vertical transmission of a plasmid DNA in a decapod Macrobrachium rosenbergii for the first time. Females at three different maturation stages (immature, matured and berried) and mature males were injected with a plasmid DNA and allowed to spawn with untreated counterparts. Using specific primers the plasmid DNA could be amplified from the offspring of all groups except that of berried females. For this confirmation genomic DNA was isolated from 3 pools of 10 post larvae in each group. This presents an ideal strategy to protect young ones at zero stress.

  1. Identification of putative DnaN-binding motifs in plasmid replication initiation proteins.

    Science.gov (United States)

    Dalrymple, Brian P; Kongsuwan, Kritaya; Wijffels, Gene

    2007-01-01

    Recently the plasmid RK2 replication initiation protein, TrfA, has been shown to bind to the beta subunit of DNA Polymerase III (DnaN) via a short pentapeptide with the consensus QL[S/D]LF. A second consensus peptide, the hexapeptide QLxLxL, has also been demonstrated to mediate binding to DnaN. Here we describe the results of a comprehensive survey of replication initiation proteins encoded by bacterial plasmids to identify putative DnaN-binding sites. Both pentapeptide and hexapeptide motifs have been identified in a number of families of replication initiation proteins. The distribution of sites is sporadic and closely related families of proteins may differ in the presence, location, or type of putative DnaN-binding motif. Neither motif has been identified in replication initiation proteins encoded by plasmids that replicate via rolling circles or strand displacement. The results suggest that the recruitment of DnaN to the origin of replication of a replisome by plasmid replication initiation proteins is not generally required for plasmid replication, but that in some cases it may be beneficial for efficiency of replication initiation.

  2. Plasmid containing a DNA ligase gene from Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.; Griffin, K.; Setlow, J.K.

    1984-05-01

    A ligase gene from Haemophilus influenzae was cloned into the shuttle vector pDM2. Although the plasmid did not affect X-ray sensitivity, it caused an increase in UV sensitivity of the wild-type but not excision-defective H. influenzae and a decrease in UV sensitivity of the rec-1 mutant. 14 references, 2 figures.

  3. TOL Plasmid Carriage Enhances Biofilm Formation and Increases Extracellular DNA Content in Pseudomonas Putida KT2440

    DEFF Research Database (Denmark)

    Smets, Barth F.; D'Alvise, Paul; Yankelovich, T.;

    of extracellular polymeric substances: TOL carriage leads to more extracellular DNA (eDNA) in pellicles and biofilms. Pellicles were dissolved by DNAse I treatment. eDNA was observed as ominous fibrous structures. Quantitative analysis of live and dead cells in static cultures was performed by flow cytometry......Adherent growth of Pseudomonas putida KT2440 with and without the TOL plasmid (pWWO) at the solid-liquid and air-liquid interface was examined. We compared biofilm formation on glass in flow cells, and assayed pellicle (air-liquid interface biofilm) formation in stagnant liquid cultures by confocal...... combined with specific cytostains; release of cytoplasmic material was assayed by a β-glucosidase assay. Enhanced cell lysis due to plasmid carriage was ruled out as the mechanism for eDNA release. We report, for the first time, that carriage of a conjugative plasmid leads to increased biofilm formation...

  4. The Chlamydophila felis plasmid is highly conserved.

    Science.gov (United States)

    Harley, Ross; Day, Sarinder; Di Rocco, Camillo; Helps, Chris

    2010-11-20

    The presence of a plasmid in the Chlamydiaceae is both species and strain specific. Knowledge of the prevalence of the plasmid in different Chlamydia species is important for future studies aiming to investigate the role of the plasmid in chlamydial biology and disease. Although strains of Chlamydophila felis with or without the plasmid have been identified, only a small number of laboratory-adapted strains have been analysed and the prevalence of the plasmid in field isolates has not been determined. This study aimed to determine the prevalence of the plasmid in C. felis-positive conjunctival and oropharyngeal clinical samples submitted for routine diagnosis of C. felis by real-time (Q)PCR. DNA extracts from four laboratory-adapted strains were also analysed. QPCR assays targeting regions of C. felis plasmid genes pCF01, pCF02 and pCF03 were developed for the detection of plasmid DNA. QPCR analysis of DNA extracts from C. felis-positive clinical samples found evidence of plasmid DNA in 591 of 595 samples representing 561 of 564 (99.5%) clinical cases. Plasmid DNA was also detected by QPCR in laboratory-adapted strains 1497V, K2487 and K2490, but not strain 905. We conclude that the plasmid is highly conserved in C. felis, and plasmid-deficient strains represent a rare but important population for future studies of chlamydial plasmid function.

  5. Fabrication of Size-Tunable Metallic Nanoparticles Using Plasmid DNA as a Biomolecular Reactor.

    Science.gov (United States)

    Samson, Jacopo; Piscopo, Irene; Yampolski, Alex; Nahirney, Patrick; Parpas, Andrea; Aggarwal, Amit; Saleh, Raihan; Drain, Charles Michael

    2011-10-21

    Plasmid DNA can be used as a template to yield gold, palladium, silver, and chromium nanoparticles of different sizes based on variations in incubation time at 70 °C with gold phosphine complexes, with the acetates of silver or palladium, or chromium acetylacetonate. The employment of mild synthetic conditions, minimal procedural steps, and aqueous solvents makes this method environmentally greener and ensures general feasibility. The use of plasmids exploits the capabilities of the biotechnology industry as a source of nanoreactor materials.

  6. Repeated extraction of DNA from FTA cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Ferrero, Laura; Børsting, Claus;

    2011-01-01

    Extraction of DNA using magnetic bead based techniques on automated DNA extraction instruments provides a fast, reliable and reproducible method for DNA extraction from various matrices. However, the yield of extracted DNA from FTA-cards is typically low. Here, we demonstrate that it is possible ...

  7. Electromobility of plasmid DNA in tumor tissues during electric field-mediated gene delivery.

    Science.gov (United States)

    Zaharoff, D A; Barr, R C; Li, C-Y; Yuan, F

    2002-10-01

    Interstitial transport is a crucial step in plasmid DNA-based gene therapy. However, interstitial diffusion of large nucleic acids is prohibitively slow. Therefore, we proposed to facilitate interstitial transport of DNA via pulsed electric fields. To test the feasibility of this approach to gene delivery, we developed an ex vivo technique to quantify the magnitude of DNA movement due to pulsed electric fields in two tumor tissues: B16.F10 (a mouse melanoma) and 4T1 (a mouse mammary carcinoma). When the pulse duration and strength were 50 ms and 233 V/cm, respectively, we found that the average plasmid DNA movements per 10 pulses were 1.47 microm and 0.35 microm in B16.F10 and 4T1 tumors, respectively. The average plasmid DNA movements could be approximately tripled, ie to reach 3.69 microm and 1.01 microm, respectively, when the pulse strength was increased to 465 V/cm. The plasmid DNA mobility was correlated with the tumor collagen content, which was approximately eight times greater in 4T1 than in B16.F10 tumors. These data suggest that electric field can be a powerful driving force for improving interstitial transport of DNA during gene delivery.

  8. Automated Extraction of DNA from clothing

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Hjort, Benjamin Benn; Nøhr Hansen, Thomas

    2011-01-01

    Presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. We have compared three automated DNA extraction methods based on magnetic beads with a manual method with the aim of reducing...... the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable DNA profiles....

  9. Bacterial spores as particulate carriers for gene gun delivery of plasmid DNA.

    Science.gov (United States)

    Aps, Luana R M M; Tavares, Milene B; Rozenfeld, Julio H K; Lamy, M Teresa; Ferreira, Luís C S; Diniz, Mariana O

    2016-06-20

    Bacillus subtilis spores represent a suitable platform for the adsorption of proteins, enzymes and viral particles at physiological conditions. In the present work, we demonstrate that purified spores can also adsorb DNA on their surface after treatment with cationic molecules. In addition, we demonstrate that DNA-coated B. subtilis spores can be used as particulate carriers and act as an alternative to gold microparticles for the biolistic (gene gun) administration of plasmid DNA in mice. Gene gun delivery of spores pre-treated with DODAB (dioctadecyldimethylammonium bromide) allowed efficient plasmid DNA absorption and induced protein expression levels similar to those obtained with gold microparticles. More importantly, we demonstrated that a DNA vaccine adsorbed on spores can be loaded into biolistic cartridges and efficiently delivered into mice, which induced specific cellular and antibody responses. Altogether, these data indicate that B. subtilis spores represent a simple and low cost alternative for the in vivo delivery of DNA vaccines by the gene gun technology.

  10. Immune Responses in Mice Injected with gD Plasmid DNA of Infectious Bovine Rhinotracheitis Virus

    Institute of Scientific and Technical Information of China (English)

    LI Ji-chang; TONG Guang-zhi; QIU Hua-ji

    2004-01-01

    The gene encoding gD of isolate Luojing of infectious bovine rhinotracheitis virus (IBRV)was amplified,sequenced, and cloned into plasmid pcDNA 3.1, resulting in a recombinant pcDNA-gD. Groups of BALB/c mice were injected with 100 μ g of plasmid only or together with liposome. After immunization, serum samples were collected from mice every 2 weeks for a 10-week period and tested for protein-specific antibody with enzyme-linked immunosorbent assay(ELISA). It was showed that the plasmid encoding IBRV glycopretein D developed gene-specific antibody. This report indicates the potential of DNA injection as a method of vaccination.

  11. Transduction of plasmid DNA in Streptomyces spp. and related genera by bacteriophage FP43.

    Science.gov (United States)

    McHenney, M A; Baltz, R H

    1988-05-01

    A segment (hft) of bacteriophage FP43 DNA cloned into plasmid pIJ702 mediated high-frequency transduction of the resulting plasmid (pRHB101) by FP43 in Streptomyces griseofuscus. The transducing particles contained linear concatemers of plasmid DNA. Lysates of FP43 prepared on S. griseofuscus containing pRHB101 also transduced many other Streptomyces species, including several that restrict plaque formation by FP43 and at least two that produce restriction endonucleases that cut pRHB101 DNA. Transduction efficiencies in different species were influenced by the addition of anti-FP43 antiserum to the transduction plates, the temperature for cell growth before transduction, the multiplicity of infection, and the host on which the transducing lysate was prepared. FP43 lysates prepared on S. griseofuscus(pRHB101) also transduced species of Streptoverticillium, Chainia, and Saccharopolyspora.

  12. Mechanism of DNA Segregation in Prokaryotes: Replicon Pairing by parC of Plasmid R1

    Science.gov (United States)

    Jensen, Rasmus Bugge; Lurz, Rudi; Gerdes, Kenn

    1998-07-01

    Prokaryotic chromosomes and plasmids encode partitioning systems that are required for DNA segregation at cell division. The systems are thought to be functionally analogous to eukaryotic centromeres and to play a general role in DNA segregation. The parA system of plasmid R1 encodes two proteins ParM and ParR, and a cis-acting centromere-like site denoted parC. The ParR protein binds to parC in vivo and in vitro. The ParM protein is an ATPase that interacts with ParR specifically bound to parC. Using electron microscopy, we show here that parC mediates efficient pairing of plasmid molecules. The pairing requires binding of ParR to parC and is stimulated by the ParM ATPase. The ParM mediated stimulation of plasmid pairing is dependent on ATP hydrolysis by ParM. Using a ligation kinetics assay, we find that ParR stimulates ligation of parC-containing DNA fragments. The rate-of-ligation was increased by wild type ParM protein but not by mutant ParM protein deficient in the ATPase activity. Thus, two independent assays show that parC mediates pairing of plasmid molecules in vitro. These results are consistent with the proposal that replicon pairing is part of the mechanism of DNA segregation in prokaryotes.

  13. The pPSU Plasmids for Generating DNA Molecular Weight Markers.

    Science.gov (United States)

    Henrici, Ryan C; Pecen, Turner J; Johnston, James L; Tan, Song

    2017-05-26

    Visualizing nucleic acids by gel electrophoresis is one of the most common techniques in molecular biology, and reference molecular weight markers or ladders are commonly used for size estimation. We have created the pPSU1 & pPSU2 pair of molecular weight marker plasmids which produce both 100 bp and 1 kb DNA ladders when digested with two common restriction enzymes. The 100 bp ladder fragments have been optimized to migrate appropriately on both agarose and native polyacrylamide, unlike many currently available DNA ladders. Sufficient plasmid DNA can be isolated from 100 ml E. coli cultures for the two plasmids to produce 100 bp or 1 kb ladders for 1000 gels. As such, the pPSU1 and pPSU2 plasmids provide reference fragments from 50 to 10000 bp at a fraction of the cost of commercial DNA ladders. The pPSU1 and pPSU2 plasmids are available without licensing restrictions to nonprofit academic users, affording freely available high-quality, low-cost molecular weight standards for molecular biology applications.

  14. The effects of a low-intensity red laser on bacterial growth, filamentation and plasmid DNA

    Science.gov (United States)

    Roos, C.; Santos, J. N.; Guimarães, O. R.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2013-07-01

    Exposure of nonphotosynthesizing microorganisms to light could increase cell division in cultures, a phenomenon denominated as biostimulation. However, data concerning the importance of the genetic characteristics of cells on this effect are as yet scarce. The aim of this work was to evaluate the effects of a low-intensity red laser on the growth, filamentation and plasmids in Escherichia coli cells proficient and deficient in DNA repair. E. coli cultures were exposed to a laser (658 nm, 10 mW, 1 and 8 J cm-2) to study bacterial growth and filamentation. Also, bacterial cultures hosting pBSK plasmids were exposed to the laser to study DNA topological forms from the electrophoretic profile in agarose gels. Data indicate the low-intensity red laser: (i) had no effect on the growth of E. coli wild type and exonuclease III deficient cells; (ii) induced bacterial filamentation, (iii) led to no alteration in the electrophoretic profile of plasmids from exonuclease III deficient cells, but plasmids from wild type cells were altered. A low-intensity red laser at the low fluences used in phototherapy has no effect on growth, but induces filamentation and alters the topological forms of plasmid DNA in E. coli cultures depending on the DNA repair mechanisms.

  15. Repeated extraction of DNA from FTA cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Ferrero, Laura; Børsting, Claus

    2011-01-01

    Extraction of DNA using magnetic bead based techniques on automated DNA extraction instruments provides a fast, reliable and reproducible method for DNA extraction from various matrices. However, the yield of extracted DNA from FTA-cards is typically low. Here, we demonstrate that it is possible...... to repeatedly extract DNA from the processed FTA-disk. The method increases the yield from the nanogram range to the microgram range....

  16. Repeated extraction of DNA from FTA cards

    OpenAIRE

    Stangegaard, Michael; Ferrero, Laura; Børsting, Claus; Frank-Hansen, Rune; Hansen, Anders Johannes; Morling, Niels

    2011-01-01

    Extraction of DNA using magnetic bead based techniques on automated DNA extraction instruments provides a fast, reliable and reproducible method for DNA extraction from various matrices. However, the yield of extracted DNA from FTA-cards is typically low. Here, we demonstrate that it is possible to repeatedly extract DNA from the processed FTA-disk. The method increases the yield from the nanogram range to the microgram range.

  17. Repeated extraction of DNA from FTA cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Ferrero, Laura; Børsting, Claus

    2011-01-01

    Extraction of DNA using magnetic bead based techniques on automated DNA extraction instruments provides a fast, reliable and reproducible method for DNA extraction from various matrices. However, the yield of extracted DNA from FTA-cards is typically low. Here, we demonstrate that it is possible...... to repeatedly extract DNA from the processed FTA-disk. The method increases the yield from the nanogram range to the microgram range....

  18. Advances in host and vector development for the production of plasmid DNA vaccines.

    Science.gov (United States)

    Mairhofer, Juergen; Lara, Alvaro R

    2014-01-01

    Recent developments in DNA vaccine research provide a new momentum for this rather young and potentially disruptive technology. Gene-based vaccines are capable of eliciting protective immunity in humans to persistent intracellular pathogens, such as HIV, malaria, and tuberculosis, for which the conventional vaccine technologies have failed so far. The recent identification and characterization of genes coding for tumor antigens has stimulated the development of DNA-based antigen-specific cancer vaccines. Although most academic researchers consider the production of reasonable amounts of plasmid DNA (pDNA) for immunological studies relatively easy to solve, problems often arise during this first phase of production. In this chapter we review the current state of the art of pDNA production at small (shake flasks) and mid-scales (lab-scale bioreactor fermentations) and address new trends in vector design and strain engineering. We will guide the reader through the different stages of process design starting from choosing the most appropriate plasmid backbone, choosing the right Escherichia coli (E. coli) strain for production, and cultivation media and scale-up issues. In addition, we will address some points concerning the safety and potency of the produced plasmids, with special focus on producing antibiotic resistance-free plasmids. The main goal of this chapter is to make immunologists aware of the fact that production of the pDNA vaccine has to be performed with as much as attention and care as the rest of their research.

  19. DNA extraction from formalin-fixed material.

    Science.gov (United States)

    Campos, Paula F; Gilbert, Thomas M P

    2012-01-01

    The principal challenges facing PCR-based analyses of DNA extracted from formalin-fixed materials are fragmentation of the DNA and cross-linked protein-DNA complexes. Here, we present an efficient protocol to extract DNA from formalin-fixed or paraffin-embedded tissues (FFPE). In this protocol, protein-DNA cross-links are reversed using heat and alkali treatment, yielding significantly longer fragments and larger amounts of PCR-amplifiable DNA than standard DNA extraction protocols.

  20. Design of expanded bed supports for the recovery of plasmid DNA by anion exchange adsorption

    DEFF Research Database (Denmark)

    Theodossiou, Irini; Søndergaard, M.; Thomas, Owen R. T.

    2001-01-01

    In this study we detail the rational design of new chromatographic adsorbents tailored for the capture of plasmid DNA. Features present on current chromatographic supports that can significantly enhance plasmid binding capacity have been identified in packed bed chromatography experiments...... and blueprints for improved expanded bed adsorbents have been put forward. The characterisation and testing of small (20-40 mum) high density (>3.7 g cm(-3)) pellicular expanded bed materials functionalised with various anion exchange structures is presented. In studies with calf thymus DNA, dynamic binding...

  1. Plasmid DNA Analysis of Pathogenic Escherichia coli in Musk Deer%麝致病性大肠杆菌的质粒DNA分析

    Institute of Scientific and Technical Information of China (English)

    罗燕; 程建国; 郑士华; 赵翠; 李蓓; 李敏

    2009-01-01

    [Objective] The pathogenic Escherichia coli in musk deer was classified at molecular level to provide basic materials for molecular epidemiology of pathogenic Escherichia coli in musk deer. [Method] Plasmids from 24 pathogenic Escherichia coli in musk deer were extracted by the Lysis Triton method, and then identified by single enzyme digestion with three endonucleases of Hind Ⅲ, EcoR Ⅰ and BamH Ⅰ. [Result] The yield rate of plasmids was 91.6%, and 24 pathogenic Escherichia coli in musk deer had the identical or similar plasmid profiles. [Conclusion] Plasmid DNA analysis offers scientific basis for molecular epidemiology of pathogenic Escherichia coli in musk deer in Sichuan Institute of Musk Deer Breeding.

  2. Automated Extraction of DNA from clothing

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Hjort, Benjamin Benn; Nøhr Hansen, Thomas;

    2011-01-01

    Presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. We have compared three automated DNA extraction methods based on magnetic beads with a manual method with the aim of reducing t...... the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable DNA profiles.......Presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. We have compared three automated DNA extraction methods based on magnetic beads with a manual method with the aim of reducing...

  3. Novel synthetic (S,S) and (R,R)-secoisolariciresinol diglucosides (SDGs) protect naked plasmid and genomic DNA From gamma radiation damage.

    Science.gov (United States)

    Mishra, Om P; Pietrofesa, Ralph; Christofidou-Solomidou, Melpo

    2014-07-01

    Secoisolariciresinol diglucoside (SDG) is the major lignan in wholegrain flaxseed. However, extraction methods are complex and are associated with low yield and high costs. Using a novel synthetic pathway, our group succeeded in chemically synthesizing SDG (S,S and R,R enantiomers), which faithfully recapitulates the properties of their natural counterparts, possessing strong antioxidant and free radical scavenging properties. This study further extends initial findings by now investigating the DNA-radioprotective properties of the synthetic SDG enantiomers compared to the commercial SDG. DNA radioprotection was assessed by cell-free systems such as: (a) plasmid relaxation assay to determine the extent of the supercoiled (SC) converted to open-circular (OC) plasmid DNA (pBR322) after exposure of the plasmid to gamma radiation; and (b) determining the extent of genomic DNA fragmentation. Exposure of plasmid DNA to 25 Gy of γ radiation resulted in decreased supercoiled form and increased open-circular form, indicating radiation-induced DNA damage. Synthetic SDG (S,S) and SDG (R,R), and commercial SDG at concentrations of 25-250 μM significantly and equipotently reduced the radiation-induced supercoiled to open-circular plasmid DNA in a dose-dependent conversion. In addition, exposure of calf thymus DNA to 50 Gy of gamma radiation resulted in DNA fragments of low-molecular weight (DNA-radioprotective properties. Such properties along with their antioxidant and free radical scavenging activity, reported earlier, suggest that SDGs are promising candidates for radioprotection for normal tissue damage as a result of accidental exposure during radiation therapy for cancer treatment.

  4. Large-scale purification of pharmaceutical-grade plasmid DNA using tangential flow filtration and multi-step chromatography.

    Science.gov (United States)

    Sun, Bo; Yu, XiangHui; Yin, Yuhe; Liu, Xintao; Wu, Yongge; Chen, Yan; Zhang, Xizhen; Jiang, Chunlai; Kong, Wei

    2013-09-01

    The demand for pharmaceutical-grade plasmid DNA in vaccine applications and gene therapy has been increasing in recent years. In the present study, a process consisting of alkaline lysis, tangential flow filtration, purification by anion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography was developed. The final product met the requirements for pharmaceutical-grade plasmid DNA. The chromosomal DNA content was <1 μg/mg plasmid DNA, and RNA was not detectable by agarose gel electrophoresis. Moreover, the protein content was <2 μg/mg plasmid DNA, and the endotoxin content was <10 EU/mg plasmid DNA. The process was scaled up to yield 800 mg of pharmaceutical-grade plasmid DNA from approximately 2 kg of bacterial cell paste. The overall yield of the final plasmid DNA reached 48%. Therefore, we have established a rapid and efficient production process for pharmaceutical-grade plasmid DNA. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. 99mTc-Labeled HYNIC-DAPI Causes Plasmid DNA Damage with High Efficiency

    OpenAIRE

    Joerg Kotzerke; Robert Punzet; Roswitha Runge; Sandra Ferl; Liane Oehme; Gerd Wunderlich; Robert Freudenberg

    2014-01-01

    (99m)Tc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, (99m)Tc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments ca...

  6. The immunogenicity of viral haemorragic septicaemia rhabdovirus (VHSV) DNA vaccines can depend on plasmid regulatory sequences.

    Science.gov (United States)

    Chico, V; Ortega-Villaizan, M; Falco, A; Tafalla, C; Perez, L; Coll, J M; Estepa, A

    2009-03-18

    A plasmid DNA encoding the viral hemorrhagic septicaemia virus (VHSV)-G glycoprotein under the control of 5' sequences (enhancer/promoter sequence plus both non-coding 1st exon and 1st intron sequences) from carp beta-actin gene (pAE6-G(VHSV)) was compared to the vaccine plasmid usually described the gene expression is regulated by the human cytomegalovirus (CMV) immediate-early promoter (pMCV1.4-G(VHSV)). We observed that these two plasmids produced a markedly different profile in the level and time of expression of the encoded-antigen, and this may have a direct effect upon the intensity and suitability of the in vivo immune response. Thus, fish genetic immunisation assays were carried out to study the immune response of both plasmids. A significantly enhanced specific-antibody response against the viral glycoprotein was found in the fish immunised with pAE6-G(VHSV). However, the protective efficacy against VHSV challenge conferred by both plasmids was similar. Later analysis of the transcription profile of a set of representative immune-related genes in the DNA immunized fish suggested that depending on the plasmid-related regulatory sequences controlling its expression, the plasmid might activate distinct patterns of the immune system. All together, the results from this study mainly point out that the selection of a determinate encoded-antigen/vector combination for genetic immunisation is of extraordinary importance in designing optimised DNA vaccines that, when required for inducing protective immune response, could elicit responses biased to antigen-specific antibodies or cytotoxic T cells generation.

  7. Automated DNA extraction from pollen in honey.

    Science.gov (United States)

    Guertler, Patrick; Eicheldinger, Adelina; Muschler, Paul; Goerlich, Ottmar; Busch, Ulrich

    2014-04-15

    In recent years, honey has become subject of DNA analysis due to potential risks evoked by microorganisms, allergens or genetically modified organisms. However, so far, only a few DNA extraction procedures are available, mostly time-consuming and laborious. Therefore, we developed an automated DNA extraction method from pollen in honey based on a CTAB buffer-based DNA extraction using the Maxwell 16 instrument and the Maxwell 16 FFS Nucleic Acid Extraction System, Custom-Kit. We altered several components and extraction parameters and compared the optimised method with a manual CTAB buffer-based DNA isolation method. The automated DNA extraction was faster and resulted in higher DNA yield and sufficient DNA purity. Real-time PCR results obtained after automated DNA extraction are comparable to results after manual DNA extraction. No PCR inhibition was observed. The applicability of this method was further successfully confirmed by analysis of different routine honey samples. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. DNA Extraction and Primer Selection

    DEFF Research Database (Denmark)

    Karst, Søren Michael; Nielsen, Per Halkjær; Albertsen, Mads

    Talk regarding pitfalls in DNA extraction and 16S amplicon primer choice when performing community analysis of complex microbial communities. The talk was a part of Workshop 2 "Principles, Potential, and Limitations of Novel Molecular Methods in Water Engineering; from Amplicon Sequencing to -omics...

  9. Extraction of DNA by magnetic ionic liquids: tunable solvents for rapid and selective DNA analysis.

    Science.gov (United States)

    Clark, Kevin D; Nacham, Omprakash; Yu, Honglian; Li, Tianhao; Yamsek, Melissa M; Ronning, Donald R; Anderson, Jared L

    2015-02-03

    DNA extraction represents a significant bottleneck in nucleic acid analysis. In this study, hydrophobic magnetic ionic liquids (MILs) were synthesized and employed as solvents for the rapid and efficient extraction of DNA from aqueous solution. The DNA-enriched microdroplets were manipulated by application of a magnetic field. The three MILs examined in this study exhibited unique DNA extraction capabilities when applied toward a variety of DNA samples and matrices. High extraction efficiencies were obtained for smaller single-stranded and double-stranded DNA using the benzyltrioctylammonium bromotrichloroferrate(III) ([(C8)3BnN(+)][FeCl3Br(-)]) MIL, while the dicationic 1,12-di(3-hexadecylbenzimidazolium)dodecane bis[(trifluoromethyl)sulfonyl]imide bromotrichloroferrate(III) ([(C16BnIM)2C12(2+)][NTf2(-), FeCl3Br(-)]) MIL produced higher extraction efficiencies for larger DNA molecules. The MIL-based method was also employed for the extraction of DNA from a complex matrix containing albumin, revealing a competitive extraction behavior for the trihexyl(tetradecyl)phosphonium tetrachloroferrate(III) ([P6,6,6,14(+)][FeCl4(-)]) MIL in contrast to the [(C8)3BnN(+)][FeCl3Br(-)] MIL, which resulted in significantly less coextraction of albumin. The MIL-DNA method was employed for the extraction of plasmid DNA from bacterial cell lysate. DNA of sufficient quality and quantity for polymerase chain reaction (PCR) amplification was recovered from the MIL extraction phase, demonstrating the feasibility of MIL-based DNA sample preparation prior to downstream analysis.

  10. Isolation of T—DNA flanking plant DNA from T—DNA insertional embryo—lethal mutants of Arabidopsis thaliana by plasmid rescue technique

    Institute of Scientific and Technical Information of China (English)

    YAOXIAOLI; JIANGESUN; 等

    1996-01-01

    Three T-DNA insertional embryonic lethal mutants from NASC(The Nottingham Arabidopsis Stock Center) were first checked with their segregation ratio of abortive and normal seeds and the copy number of T-DNA insertion.The N4081 mutant has a segregation ratio of 1:3.04 in average and one T-DNA insertion site according to our assay.It was therefore chosen for further analysis.To isolate the joint fragment of T-DNA and plant DNA,the plasmid rescue technique was used.pEL-7,one of plasmids from left border of T-DNA,which contained pBR322 was selected from ampicillin plate.The T-DNA fragment of pEL-7 was checked by restriction enzyme analysis and Southern Blot.Restriction analysis confirmed the presence of known sites of EcoRI,PstI and PvuII on it.For confirming the presence of flanking plant DNA in this plasmid,pEL-7 DNA was labeled and hybridized with wild type and mutant plant DNA.The Southern Blot indicated the hybridization band in both of them.Furthermore,the junction of T-DNA/plant DNA was subcloned into bluescript SK+ and sequenced by Applied Biosystem 373A sequencer.The results showed the 822 bp fragment contained a 274 bp sequence,which is 99.6%homolog(273bp/274bp) to Ti plasmid pTi 15955,DNA.The bp of left 25 bp border repeat were also found in the juction of T-DNA and Plant DNA. Taken together,pEL-7 should coutain a joint fragment of T-DNA and flanking plant DNA.This plasmid DNA could be used for the isolation of plant gene,which will be helpful to elucidate the relationship between gene function and plant embryo development.

  11. Development of new plasmid DNA vaccine vectors with R1-based replicons

    Directory of Open Access Journals (Sweden)

    Bower Diana M

    2012-08-01

    Full Text Available Abstract Background There has been renewed interest in biopharmaceuticals based on plasmid DNA (pDNA in recent years due to the approval of several veterinary DNA vaccines, on-going clinical trials of human pDNA-based therapies, and significant advances in adjuvants and delivery vehicles that have helped overcome earlier efficacy deficits. With this interest comes the need for high-yield, cost-effective manufacturing processes. To this end, vector engineering is one promising strategy to improve plasmid production. Results In this work, we have constructed a new DNA vaccine vector, pDMB02-GFP, containing the runaway R1 origin of replication. The runaway replication phenotype should result in plasmid copy number amplification after a temperature shift from 30°C to 42°C. However, using Escherichia coli DH5α as a host, we observed that the highest yields of pDMB02-GFP were achieved during constant-temperature culture at 30°C, with a maximum yield of approximately 19 mg pDNA/g DCW being observed. By measuring mRNA and protein levels of the R1 replication initiator protein, RepA, we determined that RepA may be limiting pDMB02-GFP yield at 42°C. A mutant plasmid, pDMB-ATG, was constructed by changing the repA start codon from the sub-optimal GTG to ATG. In cultures of DH5α[pDMB-ATG], temperature-induced plasmid amplification was more dramatic than that observed with pDMB02-GFP, and RepA protein was detectable for several hours longer than in cultures of pDMB02-GFP at 42°C. Conclusions Overall, we have demonstrated that R1-based plasmids can produce high yields of high-quality pDNA without the need for a temperature shift, and have laid the groundwork for further investigation of this class of vectors in the context of plasmid DNA production.

  12. Ca2+ promoted the low transformation efficiency of plasmid DNA exposed to PAH contaminants.

    Directory of Open Access Journals (Sweden)

    Fuxing Kang

    Full Text Available The effects of interactions between genetic materials and polycyclic aromatic hydrocarbons (PAHs on gene expression in the extracellular environment remain to be elucidated and little information is currently available on the effect of ionic strength on the transformation of plasmid DNA exposed to PAHs. Phenanthrene and pyrene were used as representative PAHs to evaluate the transformation of plasmid DNA after PAH exposure and to determine the role of Ca(2+ during the transformation. Plasmid DNA exposed to the test PAHs demonstrated low transformation efficiency. In the absence of PAHs, the transformation efficiency was 4.7 log units; however, the efficiency decreased to 3.72-3.14 log units with phenanthrene/pyrene exposures of 50 µg · L(-1. The addition of Ca(2+ enhanced the low transformation efficiency of DNA exposed to PAHs. Based on the co-sorption of Ca(2+ and phenanthrene/pyrene by DNA, we employed Fourier-transform infrared spectroscopy (FTIR, X-ray photoelectron spectroscopy (XPS, and mass spectrometry (MS to determine the mechanisms involved in PAH-induced DNA transformation. The observed low transformation efficiency of DNA exposed to either phenanthrene or pyrene can be attributed to a broken hydrogen bond in the double helix caused by planar PAHs. Added Ca(2+ formed strong electrovalent bonds with "-POO(--" groups in the DNA, weakening the interaction between PAHs and DNA based on weak molecular forces. This decreased the damage of PAHs to hydrogen bonds in double-stranded DNA by isolating DNA molecules from PAHs and consequently enhanced the transformation efficiency of DNA exposed to PAH contaminants. The findings provide insight into the effects of anthropogenic trace PAHs on DNA transfer in natural environments.

  13. Evaluation of different buffers on plasmid DNA encapsulation into PLGA microparticles.

    Science.gov (United States)

    Tse, Man Tsuey; Blatchford, Chris; Oya Alpar, H

    2009-03-31

    Double emulsion solvent evaporation is a widely used method to prepare poly(dl-lactide-co-glycolide) (PLGA) microparticles encapsulating plasmid DNA. There are inherent problems associated with preparing plasmid DNA in this form, in particular the DNA is liable to degrade during manufacture and the resulting powder has low encapsulation efficiencies. This study compares the use of two buffers, 0.1M NaHCO(3) and 0.07M Na(2)HPO(4) and the effect these have on the encapsulation efficiency and other critical parameters associated with these encapsulated DNA materials. Both buffers preserved the conformation of the original plasmid DNA during the homogenization process, but those made with 0.07M Na(2)HPO(4) had higher encapsulation efficiencies, as well as smaller diameters, compared with those made with 0.1M NaHCO(3) (encapsulation efficiencies of 40.72-45.65%, and mean volume diameters of 2.96-4.45microm). Buffers with a range of pH from 5 to 12 were investigated, and it was demonstrated that pH 9 was the point at which the highest amount of supercoiled DNA was balanced with the highest encapsulation efficiency. To simulate in vitro release, it was shown that microparticles made with 0.07M Na(2)HPO(4) had lower DNA release rates than those made with 0.1M NaHCO(3). These results demonstrate that the use of different buffers can aid in retaining the conformation of plasmid DNA, and can also modulate the amount of DNA encapsulated and the release profiles of microparticles.

  14. Encapsulation of plasmid DNA in calcium phosphate nanoparticles: stem cell uptake and gene transfer efficiency

    Directory of Open Access Journals (Sweden)

    Cao X

    2011-12-01

    Full Text Available Xia Cao*, Wenwen Deng*, Yuan Wei*, Weiyan Su, Yan Yang, Yawei Wei, Jiangnan Yu, Ximing XuDepartment of Pharmaceutics, School of Pharmacy, and Center for Nano Drug/Gene Delivery and Tissue Engineering, Jiangsu University, Jingkou District, Zhenjiang, People's Republic of China*These authors contributed equally to this workBackground: The purpose of this study was to develop calcium phosphate nanocomposite particles encapsulating plasmid DNA (CP-pDNA nanoparticles as a nonviral vector for gene delivery.Methods: CP-pDNA nanoparticles employing plasmid transforming growth factor beta 1 (TGF-β1 were prepared and characterized. The transfection efficiency and cell viability of the CP-pDNA nanoparticles were evaluated in mesenchymal stem cells, which were identified by immunofluorescence staining. Cytotoxicity of plasmid TGF-β1 and calcium phosphate to mesenchymal stem cells were evaluated by MTT assay.Results: The integrity of TGF-β1 encapsulated in the CP-pDNA nanoparticles was maintained. The well dispersed CP-pDNA nanoparticles exhibited an ultralow particle size (20–50 nm and significantly lower cytotoxicity than Lipofectamine™ 2000. Immunofluorescence staining revealed that the cultured cells in this study were probably mesenchymal stem cells. The cellular uptake and transfection efficiency of the CP-pDNA nanoparticles into the mesenchymal stem cells were higher than that of needle-like calcium phosphate nanoparticles and a standard calcium phosphate transfection kit. Furthermore, live cell imaging and confocal laser microscopy vividly showed the transportation process of the CP-pDNA nanoparticles in mesenchymal stem cells. The results of a cytotoxicity assay found that both plasmid TGF-β1 and calcium phosphate were not toxic to mesenchymal stem cells.Conclusion: CP-pDNA nanoparticles can be developed into an effective alternative as a nonviral gene delivery system that is highly efficient and has low cytotoxicity.Keywords: calcium

  15. DNA Extraction Techniques for Use in Education

    Science.gov (United States)

    Hearn, R. P.; Arblaster, K. E.

    2010-01-01

    DNA extraction provides a hands-on introduction to DNA and enables students to gain real life experience and practical knowledge of DNA. Students gain a sense of ownership and are more enthusiastic when they use their own DNA. A cost effective, simple protocol for DNA extraction and visualization was devised. Buccal mucosal epithelia provide a…

  16. DNA Extraction Techniques for Use in Education

    Science.gov (United States)

    Hearn, R. P.; Arblaster, K. E.

    2010-01-01

    DNA extraction provides a hands-on introduction to DNA and enables students to gain real life experience and practical knowledge of DNA. Students gain a sense of ownership and are more enthusiastic when they use their own DNA. A cost effective, simple protocol for DNA extraction and visualization was devised. Buccal mucosal epithelia provide a…

  17. Production and purification of plasmid DNA vaccines: is there scope for further innovation?

    Science.gov (United States)

    Xenopoulos, Alex; Pattnaik, Priyabrata

    2014-12-01

    The demand for plasmid DNA (pDNA) has vastly increased over the past decade in response to significant advances that have been made in its application for gene therapy and vaccine development. Plasmid DNA-based vaccines are experiencing a resurgence due to success with prime-boost immunization strategies. The challenge has always been poor productivity and delivery of pDNA. Plasmid DNA-based vaccines have traditionally required milligram scale of GMP-grade product for vaccination due to the relatively low efficacy and duration of gene expression. However, efforts to increase pDNA vaccine effectiveness are evolving in genetic manipulations of bacterial host, improvements in product recovery and innovative delivery methods. This review summarizes recent advances in large-scale pDNA vaccine manufacturing, ranging from upstream processing, downstream processing and formulation, as such information is usually not available to the scientific community. The article will highlight technology gaps and offer insight on further scope of innovation.

  18. Improvement of in vivo transfer of plasmid DNA in muscle : Comparison of electroporation versus ultrasound

    NARCIS (Netherlands)

    Kusumanto, Yoka H.; Mulder, Nanno H.; Dam, Wendy A.; Losen, Mario H.; Meijer, Coby; Hospers, Geke A. P.

    2007-01-01

    Plasmid-based gene delivery to muscle is a treatment strategy for many diseases with potential advantages above viral-based gene delivery methods, however, with a relative low transfection efficiency. We compared two physical methods-electroporation and ultrasound-that facilitate DNA uptake into cel

  19. TOL plasmid carriage enhances biofilm formation and increases extracellular DNA content in Pseudomonas putida KT2440.

    Science.gov (United States)

    D'Alvise, Paul W; Sjøholm, Ole R; Yankelevich, Tatiana; Jin, Yujie; Wuertz, Stefan; Smets, Barth F

    2010-11-01

    Adherent growth of Pseudomonas putida KT2440 with and without the TOL plasmid (pWWO) at the solid-liquid and air-liquid interface was examined. We compared biofilm formation on glass in flow cells, and assayed pellicle (air-liquid interface biofilm) formation in stagnant liquid cultures by confocal laser scanning microscopy. The TOL-carrying strains formed pellicles and thick biofilms, whereas the same strains without the plasmid displayed little adherent growth. Microscopy using fluorescent nucleic acid-specific stains revealed differences in the production of extracellular polymeric substances: TOL carriage leads to more extracellular DNA (eDNA) in pellicles and biofilms. Pellicles were dissolved by DNase I treatment. Enhanced cell lysis due to plasmid carriage was ruled out as the mechanism for eDNA release. We report, for the first time, that carriage of a conjugative plasmid leads to increased biofilm formation by production of eDNA. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  20. Hundreds of circular novel plasmids and DNA elements identified in a rat cecum metamobilome.

    Directory of Open Access Journals (Sweden)

    Tue Sparholt Jørgensen

    Full Text Available Metagenomic approaches are widespread in microbiological research, but so far, the knowledge on extrachromosomal DNA diversity and composition has largely remained dependant on cultivating host organisms. Even with the emergence of metagenomics, complete circular sequences are rarely identified, and have required manual curation. We propose a robust in silico procedure for identifying complete small plasmids in metagenomic datasets from whole genome shotgun sequencing. From one very pure and exhaustively sequenced metamobilome from rat cecum, we identified a total of 616 circular sequences, 160 of which were carrying a gene with plasmid replication domain. Further homology analyses indicated that the majority of these plasmid sequences are novel. We confirmed the circularity of the complete plasmid candidates using an inverse-type PCR approach on a subset of sequences with 95% success, confirming the existence and length of discrete sequences. The implication of these findings is a broadened understanding of the traits of circular elements in nature and the possibility of massive data mining in existing metagenomic datasets to discover novel pools of complete plasmids thus vastly expanding the current plasmid database.

  1. Differential behavior of plasmids containing chromosomal DNA insertions of various sizes during transformation and conjugation in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1985-01-01

    Plasmids with chromosomal insertions were constructed by removal of a 1.1-kilobase-pair piece from the 9.8-kilobase-pair vector plasmid pDM2 by EcoRI digestion and inserting in its place various lengths of chromosomal DNA (1.7, 3.4, and 9.0 kilobase pairs) coding for resistance to novobiocin. A fourth plasmid was constructed by insertion of the largest piece of chromosomal DNA into the SmaI site of pDM2. The plasmids without inserts were taken up poorly by competent cells and thus were considered not to contain specific DNA uptake sites. The presence of even the smallest insert of chromosomal DNA caused a large increase in transformation of Rec/sup +/ and Rec/sup -/ strains. The frequency of plasmid establishment in Rec/sup +/ cells by transformation increased exponentially with increasing insert size, but in Rec/sup -/ cells there was less transformation by the larger plasmids. Conjugal transfer of these plasmids was carried out with the 35-kilobase-pair mobilizing plasmid pHD147. The frequency of establishment of plasmids by this method not only was not markedly affected by the presence of the insertions, but also decreased somewhat with increase in insert size and was independent of rec-1 and rec-2 genes. Recombination between plasmid and chromosome was readily detected after transformation, but could not be detected after transconjugation even when the recipient cells were Rec/sup +/ and made competent. These data suggested that there is a special processing of plasmid DNA that enters the competent cells in transformation that makes possible recombination of homologous regions of the plasmid with the chromosome and pairing with the chromosome that aids plasmid establishment.

  2. High-Throughput Plasmid cDNA Library Screening

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Kenneth H.; Yu, Charles; George, Reed A.; Carlson, JosephW.; Hoskins, Roger A.; Svirskas, Robert; Stapleton, Mark; Celniker, SusanE.

    2006-05-24

    Libraries of cDNA clones are valuable resources foranalysing the expression, structure, and regulation of genes, as well asfor studying protein functions and interactions. Full-length cDNA clonesprovide information about intron and exon structures, splice junctionsand 5'- and 3'-untranslated regions (UTRs). Open reading frames (ORFs)derived from cDNA clones can be used to generate constructs allowingexpression of native proteins and N- or C-terminally tagged proteins.Thus, obtaining full-length cDNA clones and sequences for most or allgenes in an organism is critical for understanding genome functions.Expressed sequence tag (EST) sequencing samples cDNA libraries at random,which is most useful at the beginning of large-scale screening projects.However, as projects progress towards completion, the probability ofidentifying unique cDNAs via EST sequencing diminishes, resulting in poorrecovery of rare transcripts. We describe an adapted, high-throughputprotocol intended for recovery of specific, full-length clones fromplasmid cDNA libraries in five days.

  3. Method for the preparation of plasmid DNA suitable for physicochemical measurements.

    Science.gov (United States)

    Vojtísková, M; Lukásová, E; Palecek, E

    1985-01-01

    A method has been developed for the isolation of plasmid DNA suitable for physical and physicochemical measurements. The procedure is based on the deproteinization of the cleared lysate of bacterial cells (after amplification of plasmids by chloramphenicol) by phenol at pH 8.0 and subsequent removal of chromosomal DNA by means of phenol at pH 4.0 and separation of RNA on a hydroxyapatite column at higher temperature. ColE1 DNA sample was compared with samples of the same DNA prepared by three thus far used methods. Samples obtained by means of the latter methods were contaminated with chromosomal DNA, RNA, or ethidium bromide. The presence of ethidium bromide in the DNA sample was a factor interfering in the electrochemical analysis, chromosomal DNA and RNA were disturbing in the use of other methods. DNA separated by the method devised by us was free of any detectable contaminants and fulfilled the high requirements for sample purity of differential pulse polarography. Measurements performed by means of differential pulse polarography showed that the content of single-stranded segments in superhelical ColE1 DNA is less than 0.15% (i.e. less than 20 bases per molecule). This is in keeping with the notion that a cruciform is formed in this DNA (as a result of tension due to supercoiling) in the region of inverted repeat sequence, containing only 5 bases in the single-stranded loop region.

  4. Fabrication of Size-Tunable Metallic Nanoparticles Using Plasmid DNA as a Biomolecular Reactor

    Directory of Open Access Journals (Sweden)

    Charles Michael Drain

    2011-10-01

    Full Text Available Plasmid DNA can be used as a template to yield gold, palladium, silver, and chromium nanoparticles of different sizes based on variations in incubation time at 70 °C with gold phosphine complexes, with the acetates of silver or palladium, or chromium acetylacetonate. The employment of mild synthetic conditions, minimal procedural steps, and aqueous solvents makes this method environmentally greener and ensures general feasibility. The use of plasmids exploits the capabilities of the biotechnology industry as a source of nanoreactor materials.

  5. Putative DNA-dependent RNA polymerase in Mitochondrial Plasmid of Paramecium caudatum Stock GT704

    Directory of Open Access Journals (Sweden)

    Trina Ekawati Tallei

    2015-10-01

    Full Text Available Mitochondria of Paramecium caudatum stock GT704 has a set of four kinds of linear plasmids with sizes of 8.2, 4.1, 2.8 and 1.4 kb. The plasmids of 8.2 and 2.8 kb exist as dimers consisting of 4.1- and 1.4-kb monomers, respectively. The plasmid 2.8 kb, designated as pGT704-2.8, contains an open reading frame encodes for putative DNA-dependent RNA polymerase (RNAP. This study reveals that this RNAP belongs to superfamily of DNA/RNA polymerase and family of T7/T3 single chain RNA polymerase and those of mitochondrial plasmid of fungi belonging to Basidiomycota and Ascomycota. It is suggested that RNAP of pGT704-2.8 can perform transcription without transcription factor as promoter recognition. Given that only two motifs were found, it could not be ascertained whether this RNAP has a full function independently or integrated with mtDNA in carrying out its function.

  6. PEGylation enhances tumor targeting of plasmid DNA by an artificial cationized protein with repeated RGD sequences, Pronectin.

    Science.gov (United States)

    Hosseinkhani, Hossein; Tabata, Yasuhiko

    2004-05-31

    The objective of this study is to investigate feasibility of a non-viral gene carrier with repeated RGD sequences (Pronectin F+) in tumor targeting for gene expression. The Pronectin F+ was cationized by introducing spermine (Sm) to the hydroxyl groups to allow to polyionically complex with plasmid DNA. The cationized Pronectin F+ prepared was additionally modified with poly(ethylene glycol) (PEG) molecules which have active ester and methoxy groups at the terminal, to form various PEG-introduced cationized Pronectin F+. The cationized Pronectin F+ with or without PEGylation at different extents was mixed with a plasmid DNA of LacZ to form respective cationized Pronectin F+-plasmid DNA complexes. The plasmid DNA was electrophoretically complexed with cationized Pronectin F+ and PEG-introduced cationized Pronectin F+, irrespective of the PEGylation extent, although the higher N/P ratio of complexes was needed for complexation with the latter Pronectin F+. The molecular size and zeta potential measurements revealed that the plasmid DNA was reduced in size to about 250 nm and the charge was changed to be positive by the complexation with cationized Pronectin F+. For the complexation with PEG-introduced cationized Pronectin F+, the charge of complex became neutral being almost 0 mV with the increasing PEGylation extents, while the molecular size was similar to that of cationized Pronectin F+. When cationized Pronectin F+-plasmid DNA complexes with or without PEGylation were intravenously injected to mice carrying a subcutaneous Meth-AR-1 fibrosarcoma mass, the PEG-introduced cationized Pronectin F+-plasmid DNA complex specifically enhanced the level of gene expression in the tumor, to a significantly high extent compared with the cationized Pronectin F+-plasmid DNA complexes and free plasmid DNA. The enhanced level of gene expression depended on the percentage of PEG introduced, the N/P ratio, and the plasmid DNA dose. A fluorescent microscopic study revealed that the

  7. Insight into F plasmid DNA segregation revealed by structures of SopB and SopB–DNA complexes

    OpenAIRE

    2010-01-01

    Accurate DNA segregation is essential for genome transmission. Segregation of the prototypical F plasmid requires the centromere-binding protein SopB, the NTPase SopA and the sopC centromere. SopB displays an intriguing range of DNA-binding properties essential for partition; it binds sopC to form a partition complex, which recruits SopA, and it also coats DNA to prevent non-specific SopA–DNA interactions, which inhibits SopA polymerization. To understand the myriad functions of SopB, we dete...

  8. Binding and elution strategy for improved performance of arginine affinity chromatography in supercoiled plasmid DNA purification.

    Science.gov (United States)

    Sousa, F; Prazeres, D M F; Queiroz, J A

    2009-02-01

    New interesting strategies for plasmid DNA (pDNA) purification were designed, exploiting affinity interactions between amino acids and nucleic acids. The potential application of arginine-based chromatography to purify pDNA has been recently described in our work; however, to achieve higher efficiency and selectivity in arginine affinity chromatography, it is essential to characterize the behaviour of binding/elution of supercoiled (sc) isoforms. In this study, two different strategies based on increased sodium chloride (225-250 mm) or arginine (20-70 mm) stepwise gradients are described to purify sc isoforms. Thus, it was proved that well-defined binding/elution conditions are crucial to enhance the purification performance, resulting in an improvement of the final plasmids yields and transfection efficiency, as this could represent a significant impact on therapeutic applications of the purified sc isoform. Copyright (c) 2008 John Wiley & Sons, Ltd.

  9. Isothermal titration calorimetric analysis of the interaction between cationic lipids and plasmid DNA.

    Science.gov (United States)

    Lobo, B A; Davis, A; Koe, G; Smith, J G; Middaugh, C R

    2001-02-01

    The effects of buffer and ionic strength upon the enthalpy of binding between plasmid DNA and a variety of cationic lipids used to enhance cellular transfection were studied using isothermal titration calorimetry at 25.0 degrees C and pH 7.4. The cationic lipids DOTAP (1,2-dioleoyl-3-trimethyl ammonium propane), DDAB (dimethyl dioctadecyl ammonium bromide), DOTAP:cholesterol (1:1), and DDAB:cholesterol (1:1) bound endothermally to plasmid DNA with a negligible proton exchange with buffer. In contrast, DOTAP: DOPE (L-alpha-dioleoyl phosphatidyl ethanolamine) (1:1) and DDAB:DOPE (1:1) liposomes displayed a negative enthalpy and a significant uptake of protons upon binding to plasmid DNA at neutral pH. These findings are most easily explained by a change in the apparent pKa of the amino group of DOPE upon binding. Complexes formed by reverse addition methods (DNA into lipid) produced different thermograms, sizes, zeta potentials, and aggregation behavior, suggesting that structurally different complexes were formed in each titration direction. Titrations performed in both directions in the presence of increasing ionic strength revealed a progressive decrease in the heat of binding and an increase in the lipid to DNA charge ratio at which aggregation occurred. The unfavorable binding enthalpy for the cationic lipids alone and with cholesterol implies an entropy-driven interaction, while the negative enthalpies observed with DOPE-containing lipid mixtures suggest an additional contribution from changes in protonation of DOPE.

  10. Evaluation of DNA and RNA extraction methods.

    Science.gov (United States)

    Edwin Shiaw, C S; Shiran, M S; Cheah, Y K; Tan, G C; Sabariah, A R

    2010-06-01

    This study was done to evaluate various DNA and RNA extractions from archival FFPE tissues. A total of 30 FFPE blocks from the years of 2004 to 2006 were assessed with each modified and adapted method. Extraction protocols evaluated include the modified enzymatic extraction method (Method A), Chelex-100 extraction method (Method B), heat-induced retrieval in alkaline solution extraction method (Methods C and D) and one commercial FFPE DNA Extraction kit (Qiagen, Crawley, UK). For RNA extraction, 2 extraction protocols were evaluated including the enzymatic extraction method (Method 1), and Chelex-100 RNA extraction method (Method 2). Results show that the modified enzymatic extraction method (Method A) is an efficient DNA extraction protocol, while for RNA extraction, the enzymatic method (Method 1) and the Chelex-100 RNA extraction method (Method 2) are equally efficient RNA extraction protocols.

  11. 99mTc-labeled HYNIC-DAPI causes plasmid DNA damage with high efficiency.

    Science.gov (United States)

    Kotzerke, Joerg; Punzet, Robert; Runge, Roswitha; Ferl, Sandra; Oehme, Liane; Wunderlich, Gerd; Freudenberg, Robert

    2014-01-01

    (99m)Tc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, (99m)Tc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a (99m)Tc-labeled HYNIC-DAPI compound with that of (99m)Tc pertechnetate ((99m)TcO4(-)). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03) was twice that caused by (99m)TcO4(-) (0.51), and the number of DSBs increased fivefold in the (99m)Tc-HYNIC-DAPI-treated sample compared with the (99m)TcO4(-) treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the (99m)TcO4(-) treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the (99m)Tc-HYNIC-DAPI-treated samples. These results indicated that (99m)Tc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the (99m)Tc-labeled compound with DNA. In contrast to these results, (99m)TcO4(-) induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of (99m)Tc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by

  12. 99mTc-labeled HYNIC-DAPI causes plasmid DNA damage with high efficiency.

    Directory of Open Access Journals (Sweden)

    Joerg Kotzerke

    Full Text Available (99mTc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, (99mTc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs or double-strand breaks (DSBs; the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a (99mTc-labeled HYNIC-DAPI compound with that of (99mTc pertechnetate ((99mTcO4(-. pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03 was twice that caused by (99mTcO4(- (0.51, and the number of DSBs increased fivefold in the (99mTc-HYNIC-DAPI-treated sample compared with the (99mTcO4(- treated sample (0.02 to 0.10. In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the (99mTcO4(- treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the (99mTc-HYNIC-DAPI-treated samples. These results indicated that (99mTc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the (99mTc-labeled compound with DNA. In contrast to these results, (99mTcO4(- induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of (99mTc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by approximately

  13. Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

    Science.gov (United States)

    Fantoni, A; Bozzoni, I; Ullu, E; Farace, M G

    1979-08-10

    Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z.

  14. Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

    Energy Technology Data Exchange (ETDEWEB)

    Chowdhury, E.H., E-mail: md.ezharul.hoque@med.monash.edu.my [Jeffrey Cheah School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, Selangor Darul Ehsan (Malaysia)

    2011-06-17

    Highlights: {yields} Cytoplasmic stability of plasmid DNA is enhanced by fluoride incorporation into carbonate apatite carrier. {yields} Fluoridated carbonate apatite promotes a robust increase in transgene expression. {yields} Controlled dissolution of fluoridated carbonate apatite in endosomal acidic environment might buffer the endosomes and prevent degradation of the released DNA. -- Abstract: Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.

  15. Hypermutable ligation of plasmid DNA ends in cells from patients with Werner syndrome.

    Science.gov (United States)

    Rünger, T M; Bauer, C; Dekant, B; Möller, K; Sobotta, P; Czerny, C; Poot, M; Martin, G M

    1994-01-01

    Werner Syndrome is a rare autosomal recessive disorder characterized by an increased cancer risk and by symptoms suggestive of premature aging. Cells from these patients demonstrate a typical pattern of chromosomal instability and a spontaneous hypermutability with a high rate of unusually large deletions. We have studied the in vivo DNA ligation in three lymphoblast cell lines from Werner syndrome patients and three from normal donors. In our host cell ligation assay we transfected linearized plasmid pZ189 and measured the amount of plasmid DNA ends rejoined by these host cells as the ability of the recovered plasmid to transform bacteria. A mutagenesis marker gene close to the ligation site allowed screening for mutations. Subsequent mutation analysis provided information about the accuracy of the ligation process. The cells from Werner syndrome patients were as effective as normal cells in ligating DNA ends. However, mutation analysis revealed that the three Werner syndrome cell lines introduced 2.4-4.6 times more mutations (p < 0.001) than the normal cell lines during ligation of the DNA ends: the mutation rates were 69.4, 97.2, and 58.7%, as compared to 23.6, 21.7, and 24.4% in the normal cell lines. These increased mutation frequencies in plasmids ligated during passage through Werner syndrome cells were mainly due to a significant (p < 0.001) increase in deletions. This error-prone DNA ligation might be responsible for the spontaneous hypermutability and the genomic instability in Werner syndrome cells and related to the apparently accelerated aging and high cancer risk in affected patients.

  16. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation.

    Science.gov (United States)

    Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J; Fox, Catherine A

    2016-04-07

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

  17. Dichromatic laser radiation effects on DNA of Escherichia coli and plasmids

    Science.gov (United States)

    Martins, W. A.; Polignano, G. A. C.; Guimarães, O. R.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2015-04-01

    Dichromatic and consecutive laser radiations have attracted increased attention for clinical applications as offering new tools for the treatment of dysfunctional tissues in situations where monochromatic radiation is not effective. This work evaluated the survival, filamentation and morphology of Escherichia coli cells, and the induction of DNA lesions, in plasmid DNA exposed to low-intensity consecutive dichromatic laser radiation. Exponential and stationary wild type and formamidopyrimidine DNA glycosylase/MutM protein deficient E. coli cultures were exposed to consecutive low-intensity dichromatic laser radiation (infrared laser immediately after red laser) to study the survival, filamentation and morphology of bacterial cells. Plasmid DNA samples were exposed to dichromatic radiation to study DNA lesions by electrophoretic profile. Dichromatic laser radiation affects the survival, filamentation and morphology of E. coli cultures depending on the growth phase and the functional repair mechanism of oxidizing lesions in DNA, but does not induce single/double strands breaks or alkali-labile DNA lesions. Results show that low-intensity consecutive dichromatic laser radiation induces biological effects that differ from those induced by monochromatic laser radiation, suggesting that other therapeutic effects could be obtained using dichromatic radiation.

  18. Design of amphiphilic oligopeptides as models for fine tuning peptide assembly with plasmid DNA.

    Science.gov (United States)

    Goparaju, Geetha N; Gupta, Pardeep K

    2014-08-01

    We discuss the design of novel amphiphilic oligopeptides with hydrophobic and cationic amino acids to serve as models to understand peptide-DNA assembly. Biophysical and thermodynamic characterization of interaction of these amphiphilic peptides with plasmid DNA is presented. Peptides with at least +4 charges favor stable complex formation. Surface potential is dependent on the type of hydrophobic amino acid for a certain charge. Thermodynamically it is a spontaneous interaction between most of the peptides and plasmid DNA. Lys(7) and Tyr peptides with +4/+5 charges indicate cooperative binding with pDNA without saturation of interaction while Val(2)-Gly-Lys(4), Val-Gly-Lys(5), and Phe-Gly-Lys(5) lead to saturation of interaction indicating condensed pDNA within the range of N/Ps studied. We show that the biophysical properties of DNA-peptide complexes could be modulated by design and the peptides presented here could be used as building blocks for creating DNA-peptide complexes for various biomedical applications, mainly nucleic acid delivery.

  19. Enhanced transformation efficiency of recalcitrant Bacillus cereus and Bacillus weihenstephanensis isolates upon in vitro methylation of plasmid DNA

    NARCIS (Netherlands)

    Nierop Groot, M.N.; Nieboer, F.; Abee, T.

    2008-01-01

    Digestion patterns of chromosomal DNAs of Bacillus cereus and Bacillus weihenstephanensis strains suggest that Sau3AI-type restriction modification systems are widely present among the isolates tested. In vitro methylation of plasmid DNA was used to enhance poor plasmid transfer upon electroporation

  20. Effects of the presence of ColE1 plasmid DNA in Escherichia coli on the host cell metabolism

    Directory of Open Access Journals (Sweden)

    Węgrzyn Alicja

    2006-11-01

    Full Text Available Abstract Background Although understanding of physiological interactions between plasmid DNA and its host is important for vector design and host optimization in many biotechnological applications, to our knowledge, global studies on plasmid-host interactions have not been performed to date even for well-characterized plasmids. Results Escherichia coli cells, either devoid of plasmid DNA or bearing plasmid pOri1 (with a single ColE1 replication origin or plasmid pOri2 (with double ColE1 replication origins, were cultured in a chemostat. We used a combination of metabolic flux analysis, DNA microarray and enzyme activity analysis methods to explore differences in the metabolism between these strains. We found that the presence of plasmids significantly influenced various metabolic pathways in the host cells, e.g. glycolysis, the tricarboxylic acid (TCA cycle and the pentose phosphate (PP pathway. Expression of rpiA, a gene coding for ribose-5-phosphate isomerase A, was considerably decreased in E. coli carrying a high copy number plasmid relative to E. coli carrying a low copy number plasmid and plasmid-free E. coli. The rpiA gene was cloned into an expression vector to construct plasmid pETrpiA. Following induction of pETrpiA-bearing E. coli, which harbored either pOri1 or pOri2, with isopropyl-β-D-thiogalactopyranoside (IPTG, the copy number of pOri1 and pOri2 was sigificantly higher than that measured in a host devoid of pETrpiA. Conclusion The presence of plasmids can significantly influence some metabolic pathways in the host cell. We believe that the results of detailed metabolic analysis may be useful in optimizing host strains, vectors and cultivation conditions for various biotechnological purposes.

  1. Investigation Into the Effects of Nucleotide Content on the Electrical Characteristics of DNA Plasmid Molecular Wires.

    Science.gov (United States)

    Goshi, Noah; Narenji, Alaleh; Bui, Chris; Mokili, John L; Kassegne, Sam

    2016-09-01

    In this study, we investigate the effect of nucleotide content on the conductivity of plasmid length DNA molecular wires covalently bound to high aspect-ratio gold electrodes. The DNA wires were all between [Formula: see text] in length (>6000bp), and contained either 39%, 53%, or 64% GC base-pairs. We compared the current-voltage (I-V) and frequency-impedance characteristics of the DNA wires with varying GC content, and observed statistically significantly higher conductivity in DNA wires containing higher GC content in both AC and DC measurement methods. Additionally, we noted that the conductivity decreased as a function of time for all DNA wires, with the impedance at 100 Hz nearly doubling over a period of seven days. All readings were taken in humidity and temperature controlled environments on DNA wires suspended above an insulative substrate, thus minimizing the effect of experimental and environmental factors as well as potential for nonlinear alternate DNA confirmations. While other groups have studied the effect of GC content on the conductivity of nanoscale DNA molecules (DNA wires at scales that may be required during the fabrication of DNA-based electronics. Furthermore, our results provide further evidence that many of the charge transfer theories developed from experiments using nanoscale DNA molecules may still be applicable for DNA wires at the micro scale.

  2. Investigation of Effects of Nucleotide Content on Electrical Characteristics of DNA Plasmid Molecular Wires.

    Science.gov (United States)

    Goshi, Noah; Narenji, Alaleh; Bui, Chris; Mokili, John L; Kassegne, Sam

    2016-07-28

    In this study, we investigate the effect of nucleotide content on the conductivity of plasmid length DNA molecular wires covalently bound to high aspect-ratio gold electrodes. The DNA wires were all between 2.20-2.35μm in length (>6000bp), and contained either 39%, 53%, or 64% GC base-pairs. We compared the current-voltage (I-V) and frequency-impedance characteristics of the DNA wires with varying GC content, and observed statistically significantly higher conductivity in DNA wires containing higher GC content in both AC and DC measurement methods. Additionally, we noted that the conductivity decreased as a function of time for all DNA wires, with the impedance at 100Hz nearly doubling over a period of seven days. All readings were taken in humidity and temperature controlled environments on DNA wires suspended above an insulative substrate, thus minimizing the effect of experimental and environmental factors as well as potential for nonlinear alternate DNA confirmations. While other groups have studied the effect of GC content on the conductivity of nano-scale DNA molecules (DNA wires at scales that may be required during the fabrication of DNA-based electronics. Furthermore, our results provide further evidence that many of the charge transfer theories developed from experiments using nano-scale DNA molecules may still be applicable for DNA wires at the micro-scale.

  3. "Curing" of plasmid DNA in acetogen using microwave or applying an electric pulse improves cell growth and metabolite production as compared to the plasmid-harboring strain.

    Science.gov (United States)

    Berzin, Vel; Kiriukhin, Michael; Tyurin, Michael

    2013-03-01

    Plasmid-free acetogen Clostridium sp. MT962 electrotransformed with a small cryptic plasmid pMT351 was used to develop time- and cost-effective methods for plasmid elimination. Elimination of pMT351 restored production of acetate and ethanol to the levels of the plasmid-free strain with no dry cell weight changes. Destabilizing cell membrane via microwave at 2.45 GHz, or exposure to a single 12 ms square electric pulse at 35 kV cm⁻¹, eliminated pMT351 in 42-47 % of cells. Plasmid elimination with a single square electric pulse required 10 versus 0.1 J needed to introduce the same 3,202-bp plasmid into the cells as calculated per cell sample of Clostridium sp. MT962. Microwave caused visible changes in repPCR pattern and increased ethanol production at the expense of acetate. This is the first report on microwave of microwave ovens, wireless routers, and mobile devices causing chromosomal DNA aberrations in microbes along with carbon flux change.

  4. Exponential megapriming PCR (EMP) cloning--seamless DNA insertion into any target plasmid without sequence constraints.

    Science.gov (United States)

    Ulrich, Alexander; Andersen, Kasper R; Schwartz, Thomas U

    2012-01-01

    We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.

  5. Analysis of heavy-ion-induced DNA strand breaks in plasmid pUC18

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Plasmid DNA was irradiated or implanted by mixed particle field(CR) or lithium-ion-beam to detect strand breaks.The primary results showed that mixed particle field could induce single and double strand breaks with positive linear-dose-effects;most of sequence changes induced by CR were point mutant.Lithium-ion-beam could induce strand breaks also,but it was only at dose of 20Gy.

  6. Exponential megapriming PCR (EMP cloning--seamless DNA insertion into any target plasmid without sequence constraints.

    Directory of Open Access Journals (Sweden)

    Alexander Ulrich

    Full Text Available We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.

  7. Optimum range of plasmid supercoiled DNA for preparation of ccompetent Top 10 E. coli

    Directory of Open Access Journals (Sweden)

    Muhammad Tahir Majeed

    2011-05-01

    Full Text Available Objectives: In-house preparation of chemically competent andelectrocompetent Top 10 E. coli is not only economical butmeets the needs for most of the molecular cloning work. Forsuch transformations an optimum range of plasmidsupercoiled DNA is needed. Therefore, the present studydescribes the modification of two protocols for the preparationof such cells, and optimization of the amount of plasmidsupercoiled DNA required for better efficiency.Materials and methods: As most of the available protocols torender bacterial cells competent need special media orchemicals and are time consuming, the methods from HelenDonis-Keller Laboratory Manual of Washington University inSt. Louis and Goldberg Laboratory Standard Protocols of theUnited States Department of Agriculture have been used aftermeticulous selection and with few modifications for preparingchemically competent and electrocompetent Top 10 E. coli,respectively. The transformation was carried out using pUC19supercoiled plasmid DNA.Results: The transformation efficiencies of chemicallycompetent and electrocompetent Top 10 E. coli were found tobe 1.1 x 106 and 7.88 x 107 tranformants/μg of DNA,respectively. Such efficiencies are slightly higher than therequired (105-106 transformants/μg DNA for most of thecloning experimentation.Conclusion: The results of the present study indicatethat for sufficient transformation competence rates theoptimum range of plasmid supercoiled DNA is 10 ng forchemically competent and 0.1 ng for electrocompetentTop 10 E. coli.

  8. Encapsulation and delivery of plasmid DNA by virus-like nanoparticles engineered from Macrobrachium rosenbergii nodavirus.

    Science.gov (United States)

    Jariyapong, Pitchanee; Chotwiwatthanakun, Charoonroj; Somrit, Monsicha; Jitrapakdee, Sarawut; Xing, Li; Cheng, Holland R; Weerachatyanukul, Wattana

    2014-01-22

    Virus-like particles (VLPs) are potential candidates in developing biological containers for packaging therapeutic or biologically active agents. Here, we expressed Macrobrachium rosenbergii nodavirus (MrNv) capsid protein (encoding amino acids M1-N371 with 6 histidine residuals) in an Escherichia coli BL21(DE3). These easily purified capsid protein self-assembled into VLPs, and disassembly/reassembly could be controlled in a calcium-dependent manner. Physically, MrNv VLPs resisted to digestive enzymes, a property that should be advantageous for protection of active compounds against harsh conditions. We also proved that MrNv VLPs were capable of encapsulating plasmid DNA in the range of 0.035-0.042 mol ratio (DNA/protein) or 2-3 plasmids/VLP (assuming that MrNV VLPs is T=1, i made up of 60 capsid monomers). These VLPs interacted with cultured insect cells and delivered loaded plasmid DNA into the cells as shown by green fluorescent protein (GFP) reporter. With many advantageous properties including self-encapsulation, MrNv VLPs are good candidates for delivery of therapeutic agents.

  9. Process considerations related to the microencapsulation of plasmid DNA via ultrasonic atomization.

    Science.gov (United States)

    Ho, Jenny; Wang, Huanting; Forde, Gareth M

    2008-09-01

    An effective means of facilitating DNA vaccine delivery to antigen presenting cells is through biodegradable microspheres. Microspheres offer distinct advantages over other delivery technologies by providing release of DNA vaccine in its bioactive form in a controlled fashion. In this study, biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres containing polyethylenimine (PEI) condensed plasmid DNA (pDNA) were prepared using a 40 kHz ultrasonic atomization system. Process synthesis parameters, which are important to the scale-up of microspheres that are suitable for nasal delivery (i.e., less than 20 microm), were studied. These parameters include polymer concentration; feed flowrate; volumetric ratio of polymer and pDNA-PEI (plasmid DNA-polyethylenimine) complexes; and nitrogen to phosphorous (N/P) ratio. PDNA encapsulation efficiencies were predominantly in the range 82-96%, and the mean sizes of the particle were between 6 and 15 microm. The ultrasonic synthesis method was shown to have excellent reproducibility. PEI affected morphology of the microspheres, as it induced the formation of porous particles that accelerate the release rate of pDNA. The PLGA microspheres displayed an in vitro release of pDNA of 95-99% within 30 days and demonstrated zero order release kinetics without an initial spike of pDNA. Agarose electrophoresis confirmed conservation of the supercoiled form of pDNA throughout the synthesis and in vitro release stages. It was concluded that ultrasonic atomization is an efficient technique to overcome the key obstacles in scaling-up the manufacture of encapsulated vaccine for clinical trials and ultimately, commercial applications.

  10. Automated extraction of DNA from clothing

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Hjort, Benjamin Benn; Nøhr Hansen, Thomas

    2011-01-01

    Presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. We have compared three automated DNA extraction methods based on magnetic beads with a manual method with the aim of reducing t...

  11. Vaccination with Trypomastigote Surface Antigen 1-Encoding Plasmid DNA Confers Protection against Lethal Trypanosoma cruzi Infection

    OpenAIRE

    1998-01-01

    DNA vaccination was evaluated with the experimental murine model of Trypanosoma cruzi infection as a means to induce antiparasite protective immunity, and the trypomastigote surface antigen 1 (TSA-1), a target of anti-T. cruzi antibody and major histocompatibility complex (MHC) class I-restricted CD8+ cytotoxic T-lymphocyte (CTL) responses, was used as the model antigen. Following the intramuscular immunization of H-2b and H-2d mice with a plasmid DNA encoding an N-terminally truncated TSA-1 ...

  12. Implementation of an Automated High-Throughput Plasmid DNA Production Pipeline.

    Science.gov (United States)

    Billeci, Karen; Suh, Christopher; Di Ioia, Tina; Singh, Lovejit; Abraham, Ryan; Baldwin, Anne; Monteclaro, Stephen

    2016-12-01

    Biologics sample management facilities are often responsible for a diversity of large-molecule reagent types, such as DNA, RNAi, and protein libraries. Historically, the management of large molecules was dispersed into multiple laboratories. As methodologies to support pathway discovery, antibody discovery, and protein production have become high throughput, the implementation of automation and centralized inventory management tools has become important. To this end, to improve sample tracking, throughput, and accuracy, we have implemented a module-based automation system integrated into inventory management software using multiple platforms (Hamilton, Hudson, Dynamic Devices, and Brooks). Here we describe the implementation of these systems with a focus on high-throughput plasmid DNA production management.

  13. Local gene delivery via endovascular stents coated with dodecylated chitosan–plasmid DNA nanoparticles

    Directory of Open Access Journals (Sweden)

    Dunwan Zhu

    2010-12-01

    Full Text Available Dunwan Zhu1*, Xu Jin2*, Xigang Leng1, Hai Wang1, Junbo Bao1, Wenguang Liu3, Kangde Yao3, Cunxian Song11Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China; 2Department of Anesthesia and Pain Therapy, Capital Medical University Affiliated Beijing Tiantan Hospital, Beijing, China; 3Research Institute of Polymeric Materials, Tianjin University, Tianjin, China; *Both investigators contributed equally to this work and are senior authors.Abstract: Development of efficacious therapeutic strategies to prevent and inhibit the occurrences of restenosis after percutaneous transluminal coronary angioplasty is critical for the treatment of cardiovascular diseases. In this study, the feasibility and efficiency of stents coated with dodecylated chitosan–plasmid DNA nanoparticles (DCDNPs were evaluated as scaffolds for localized and prolonged delivery of reporter genes into the diseased blood vessel wall. Dodecylated chitosan–plasmid DNA complexes formed stable positive charged nanospheres with mean diameter of approximately 90–180 nm and zeta potential of +28 ± 3 mV. As prepared DCDNPs were spray-coated on stents, a thin layer of dense DCDNPs was successfully distributed onto the metal struts of the endovascular stents as demonstrated by scanning electron microscopy. The DCDNP stents were characterized for the release kinetics of plasmid DNA, and further evaluated for gene delivery and expression both in vitro and in vivo. In cell culture, DCDNP stents containing plasmid EGFP-C1 exhibited high level of GFP expression in cells grown on the stent surface and along the adjacent area. In animal studies, reporter gene activity was observed in the region of the artery in contact with the DCDNP stents, but not in adjacent arterial segments or distal organs. The DCDNP stent provides a very promising strategy for cardiovascular gene therapy

  14. Interleukin-12 plasmid DNA delivery using l-thyroxine-conjugated polyethylenimine nanocarriers

    Science.gov (United States)

    Dehshahri, Ali; Sadeghpour, Hossein; Kazemi Oskuee, Reza; Fadaei, Mahin; Sabahi, Zahra; Alhashemi, Samira Hossaini; Mohazabieh, Erfaneh

    2014-05-01

    In this study, l-thyroxine was covalently grafted on 25 kDa branched polyethylenimine (PEI), and the ability of the nano-sized polyplexes for transferring plasmid encoding interleukin-12 (IL-12) gene was evaluated. As there are several problems in systemic administration of recombinant IL-12 protein, local expression of the plasmid encoding IL-12 gene inside the tumor tissue has been considered as an effective alternative approach. The l-thyroxine-conjugated PEI polyplexes were prepared using pUMVC3-hIL12 plasmid, and their transfection activity was determined in HepG2 human liver carcinoma and Neuro2A neuroblastoma cell lines. The polyplexes characterized in terms of DNA condensation ability, particle size, zeta potential, and buffering capacity as well as cytotoxicity and resistance to enzyme digestion. The results revealed that l-thyroxine conjugation of PEI increased gene transfer ability by up to two fold relative to unmodified 25 kDa PEI, the gold standard for non-viral gene delivery, with the highest increase occurring at degrees of conjugation around 10 %. pDNA condensation tests and dynamic light scattering measurements exhibited the ability of PEI conjugates to optimally condense the plasmid DNA into polyplexes in the size range around 200 nm. The modified polymers showed remarkable buffering capacity and protection against enzymatic degradation comparable to that of unmodified PEI. These results suggest that l-thyroxine conjugation of PEI is a simple modification strategy for future investigations aimed at developing a targeting gene vehicle.

  15. Photoinduced interactions of supramolecular ruthenium(II) complexes with plasmid DNA: synthesis and spectroscopic, electrochemical, and DNA photocleavage studies.

    Science.gov (United States)

    Swavey, Shawn; DeBeer, Madeleine; Li, Kaiyu

    2015-04-06

    Two new bridging ligands have been synthesized by combining substituted benzaldehydes with phenanthrolinopyrrole (php), resulting in new polyazine bridging ligands. The ligands have been characterized by (1)H NMR, mass spectroscopy, and elemental analysis. These new ligands display π-π* transitions above 500 nm with modest molar absorptivities. Upon excitation at the ligand-centered charge-transfer transition, weak emission with a maximum wavelength of 612 nm is observed. When coordinated to two ruthenium(II) bis(bipyridyl) groups, the new bimetallic complexes generated give an overall 4+ charge. The electronic transitions of the bimetallic ruthenium(II) complexes display traditional π-π* transitions at 287 nm and metal-to-ligand charge-transfer transitions at 452 nm with molar absorptivities greater than 30000 M(-1) cm(-1). Oxidation of the ruthenium(II) metal centers to ruthenium(III) occurs at potentials above 1.4 V versus the Ag/AgCl reference electrode. Spectroscopic and electrochemical measurements indicate that the ruthenium(II) moieties behave independently. Both complexes are water-soluble and show the ability to photonick plasmid DNA when irradiated with low-energy light above 550 nm. In addition, one of the complexes, [Ru(bpy)2php]2Van(4+), shows the ability to linearize plasmid DNA and gives evidence, by gel electrophoresis, of photoinduced binding to plasmid DNA.

  16. Condensation of Plasmid DNA Enhances Mitochondrial Association in Skeletal Muscle Following Hydrodynamic Limb Vein Injection

    Directory of Open Access Journals (Sweden)

    Yukari Yasuzaki

    2014-08-01

    Full Text Available Mitochondrial gene therapy and diagnosis have the potential to provide substantial medical benefits. However, the utility of this approach has not yet been realized because the technology available for mitochondrial gene delivery continues to be a bottleneck. We previously reported on mitochondrial gene delivery in skeletal muscle using hydrodynamic limb vein (HLV injection. HLV injection, a useful method for nuclear transgene expression, involves the rapid injection of a large volume of naked plasmid DNA (pDNA. Moreover, the use of a condensed form of pDNA enhances the nuclear transgene expression by the HLV injection. The purpose of this study was to compare naked pDNA and condensed pDNA for mitochondrial association in skeletal muscle, when used in conjunction with HLV injection. PCR analysis showed that the use of condensed pDNA rather than naked pDNA resulted in a more effective mitochondrial association with pDNA, suggesting that the physicochemical state of pDNA plays a key role. Moreover, no mitochondrial toxicities in skeletal muscle following the HLV injection of condensed pDNA were confirmed, as evidenced by cytochrome c oxidase activity and mitochondrial membrane potential. These findings have the potential to contribute to the development for in vivo mitochondrial gene delivery system.

  17. High-Voltage Electroporation of Bacteria: Genetic Transformation of Campylobacter jejuni with Plasmid DNA

    Science.gov (United States)

    Miller, Jeff F.; Dower, William J.; Tompkins, Lucy S.

    1988-02-01

    Electroporation permits the uptake of DNA by mammalian cells and plant protoplasts because it induces transient permeability of the cell membrane. We investigated the utility of high-voltage electroporation as a method for genetic transformation of intact bacterial cells by using the enteric pathogen Campylobacter jejuni as a model system. This report demonstrates that the application of high-voltage discharges to bacterial cells permits genetic transformation. Our method involves exposure of a Campylobacter cell suspension to a high-voltage exponential decay discharge (5-13 kV/cm) for a brief period of time (resistance-capacitance time constant = 2.4-26 msec) in the presence of plasmid DNA. Electrical transformation of C. jejuni results in frequencies as high as 1.2 × 106 transformants per μ g of DNA. We have investigated the effects of pulse amplitude and duration, cell growth conditions, divalent cations, and DNA concentration on the efficiency of transformation. Transformants of C. jejuni obtained by electroporation contained structurally intact plasmid molecules. In addition, evidence is presented that indicates that C. jejuni possesses DNA restriction and modification systems. The use of electroporation as a method for transforming other bacterial species and guidelines for its implementation are also discussed.

  18. Antibacterial effect of cationic porphyrazines and anionic phthalocyanine and their interaction with plasmid DNA

    Science.gov (United States)

    Hassani, Leila; Hakimian, Fatemeh; Safaei, Elham; Fazeli, Zahra

    2013-11-01

    Resistance to antibiotics is a public health issue and identification of new antibacterial agents is one of the most important goals of pharmacological research. Among the novel developed antibacterial agents, porphyrin complexes and their derivatives are ideal candidates for use in medical applications. Phthalocyanines differ from porphyrins by having nitrogen atoms link the individual pyrrol units. The aza analogues of the phthalocyanines (azaPcs) such as tetramethylmetalloporphyrazines are heterocyclic Pc analogues. In this investigation, interaction of an anionic phthalocyanine (Cu(PcTs)) and two cationic tetrapyridinoporphyrazines including [Cu(2,3-tmtppa)]4+ and [Cu(3,4-tmtppa)]4+ complexes with plasmid DNA was studied using spectroscopic and gel electrophoresis methods. In addition, antibacterial effect of the complexes against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria was investigated using dilution test method. The results indicated that both porphyrazines have significant antibacterial properties, but Cu(PcTs) has weak antibacterial effect. Compairing the binding of the phthalocyanine and the porphyrazines to DNA demonstrated that the interaction of cationic porphyrazines is stronger than the anionic phthalocyanine remarkably. The extent of hypochromicity and red shift of absorption spectra indicated preferential intercalation of the two porphyrazine into the base pairs of DNA helix. Gel electrophoresis result implied Cu(2,3-tmtppa) and Cu(3,4-tmtppa) are able to perform cleavage of the plasmid DNA. Consequently, DNA binding and cleavage might be one of the antibacterial mechanisms of the complexes.

  19. Synthesis and Characterization of Chitosan-Saponin Nanoparticle for Application in Plasmid DNA Delivery

    Directory of Open Access Journals (Sweden)

    Faruku Bande

    2015-01-01

    Full Text Available Nonviral delivery system receives attention over the last decade. Chitosan (CS is a cationic polymer whereas saponin (SP is classified as glycoside. In this study, a spherically-shaped CS-SP nanoparticle was synthesized and characterized. The ability of the nanoparticle to protect DNA from enzymatic degradation, its thermostability and cytotoxicity were evaluated. The particle size was found below 100 nm as determined by Zetasizer, transmission electron microscopy (TEM, and field scanning electron microscopy (FSEM results. The surface charge ranges from 43.7 mV to 38.5 mV before and after encapsulation with DNA plasmid, respectively. In terms of thermostability, Thermal Gravimetric Analysis (TGA and Differential Scanning Calorimetry (DSC revealed that CS-SP nanoparticle had a melting temperature of 110°C, with rapid decomposition occurring at 120°C. Encapsulation of DNA with the synthesized nanoparticle was evidenced by changes in the FTIR spectra including characteristic peaks at 3267.39 and 1635.58 cm−1, wavenumbers. Additional peak was also observed at 1169.7 cm−1 following encapsulation. Electrophoretic mobility showed that CS-SP nanoparticle protected plasmid DNA from enzymatic degradation, while cell viability assays confirmed that the synthesized nanoparticle exhibited low cytotoxicity at different concentrations in avian cells. Taken together these, CS-SP nanoparticle showed potentials for applications as a DNA delivery system.

  20. Multiple factors affect immunogenicity of DNA plasmid HIV vaccines in human clinical trials.

    Science.gov (United States)

    Jin, Xia; Morgan, Cecilia; Yu, Xuesong; DeRosa, Stephen; Tomaras, Georgia D; Montefiori, David C; Kublin, James; Corey, Larry; Keefer, Michael C

    2015-05-11

    Plasmid DNA vaccines have been licensed for use in domesticated animals because of their excellent immunogenicity, but none have yet been licensed for use in humans. Here we report a retrospective analysis of 1218 healthy human volunteers enrolled in 10 phase I clinical trials in which DNA plasmids encoding HIV antigens were administered. Elicited T-cell immune responses were quantified by validated intracellular cytokine staining (ICS) stimulated with HIV peptide pools. HIV-specific binding and neutralizing antibody activities were also analyzed using validated assays. Results showed that, in the absence of adjuvants and boosting with alternative vaccines, DNA vaccines elicited CD8+ and CD4+ T-cell responses in an average of 13.3% (95% CI: 9.8-17.8%) and 37.7% (95% CI: 31.9-43.8%) of vaccine recipients, respectively. Three vaccinations (vs. 2) improved the proportion of subjects with antigen-specific CD8+ responses (p=0.02), as did increased DNA dosage (p=0.007). Furthermore, female gender and participants having a lower body mass index were independently associated with higher CD4+ T-cell response rate (p=0.001 and p=0.008, respectively). These vaccines elicited minimal neutralizing and binding antibody responses. These findings of the immunogenicity of HIV DNA vaccines in humans can provide guidance for future clinical trials. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Fed-batch microbioreactor platform for scale down and analysis of a plasmid DNA production process.

    Science.gov (United States)

    Bower, Diana M; Lee, Kevin S; Ram, Rajeev J; Prather, Kristala L J

    2012-08-01

    The rising costs of bioprocess research and development emphasize the need for high-throughput, low-cost alternatives to bench-scale bioreactors for process development. In particular, there is a need for platforms that can go beyond simple batch growth of the organism of interest to include more advanced monitoring, control, and operation schemes such as fed-batch or continuous. We have developed a 1-mL microbioreactor capable of monitoring and control of dissolved oxygen, pH, and temperature. Optical density can also be measured online for continuous monitoring of cell growth. To test our microbioreactor platform, we used production of a plasmid DNA vaccine vector (pVAX1-GFP) in Escherichia coli via a fed-batch temperature-inducible process as a model system. We demonstrated that our platform can accurately predict growth, glycerol and acetate concentrations, as well as plasmid copy number and quality obtained in a bench-scale bioreactor. The predictive abilities of the micro-scale system were robust over a range of feed rates as long as key process parameters, such as dissolved oxygen, were kept constant across scales. We have highlighted plasmid DNA production as a potential application for our microbioreactor, but the device has broad utility for microbial process development in other industries as well. Copyright © 2012 Wiley Periodicals, Inc.

  2. Gene Transfer into the Lung by Nanoparticle Dextran-Spermine/Plasmid DNA Complexes

    Directory of Open Access Journals (Sweden)

    Syahril Abdullah

    2010-01-01

    Full Text Available A novel cationic polymer, dextran-spermine (D-SPM, has been found to mediate gene expression in a wide variety of cell lines and in vivo through systemic delivery. Here, we extended the observations by determining the optimal conditions for gene expression of D-SPM/plasmid DNA (D-SPM/pDNA in cell lines and in the lungs of BALB/c mice via instillation delivery. In vitro studies showed that D-SPM could partially protect pDNA from degradation by nuclease and exhibited optimal gene transfer efficiency at D-SPM to pDNA weight-mixing ratio of 12. In the lungs of mice, the levels of gene expression generated by D-SPM/pDNA are highly dependent on the weight-mixing ratio of D-SPM to pDNA, amount of pDNA in the complex, and the assay time postdelivery. Readministration of the complex at day 1 following the first dosing showed no significant effect on the retention and duration of gene expression. The study also showed that there was a clear trend of increasing size of the complexes as the amount of pDNA was increased, where the sizes of the D-SPM/pDNA complexes were within the nanometer range.

  3. Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells.

    Science.gov (United States)

    Pimentel, Belén; Nair, Radhika; Bermejo-Rodríguez, Camino; Preston, Mark A; Agu, Chukwuma A; Wang, Xindan; Bernal, Juan A; Sherratt, David J; de la Cueva-Méndez, Guillermo

    2014-02-18

    Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid-bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.

  4. Low energy electrons and ultra-soft X-rays irradiation of plasmid DNA. Technical innovations

    Science.gov (United States)

    Fromm, Michel; Boulanouar, Omar

    2016-11-01

    In this paper we present in a first part the latest results of our group which are in relation with the study of DNA damages inflicted by low energy electrons (0-20 eV) in ultra-high vacuum as well as in air under atmospheric conditions. A short description of the drop-casting technique we developed to produce thin and nanometre-scaled DNA layers onto graphite sheets is given. We provide the absolute cross-section for loss of supercoiled topology of plasmid DNA complexed with 1,3-diaminopropane (Dap) in the vacuum under 10 eV electron impact and suggest a specific pathway for the dissociation of the transient negative ion formed by resonant capture of such a low energy electron (LEE) by the DNA's phosphate group when complexed to Dap. Well-gauged DNA-Dap layers with various nanometre-scaled thicknesses are used to evaluate the effective attenuation length of secondary photo-LEEs in the energy range (0-20 eV). The values of 11-16 nm for DNA kept under atmospheric conditions are in good agreement with the rare literature data available and which are stemming from computer simulations. In a second part, we describe the method we have developed in order to expose liquid samples of plasmid DNA to ultra-soft X-rays (Al Kα line at 1.5 keV) under hydroxyl radical scavenging conditions. We provide an experimentally determined percentage of indirect effects in aqueous medium kept under standard conditions of 94.7±2.1% indirect effects; in satisfactory agreement with the data published by others (i.e. 97.7%) relative to gamma irradiation of frozen solutions (Tomita et al., 1995).

  5. Vaccination with trypomastigote surface antigen 1-encoding plasmid DNA confers protection against lethal Trypanosoma cruzi infection.

    Science.gov (United States)

    Wizel, B; Garg, N; Tarleton, R L

    1998-11-01

    DNA vaccination was evaluated with the experimental murine model of Trypanosoma cruzi infection as a means to induce antiparasite protective immunity, and the trypomastigote surface antigen 1 (TSA-1), a target of anti-T. cruzi antibody and major histocompatibility complex (MHC) class I-restricted CD8(+) cytotoxic T-lymphocyte (CTL) responses, was used as the model antigen. Following the intramuscular immunization of H-2(b) and H-2(d) mice with a plasmid DNA encoding an N-terminally truncated TSA-1 lacking or containing the C-terminal nonapeptide tandem repeats, the antibody level, CTL response, and protection against challenge with T. cruzi were assessed. In H-2(b) mice, antiparasite antibodies were induced only by immunization with the DNA construct encoding TSA-1 containing the C-terminal repeats. However, both DNA constructs were efficient in eliciting long-lasting CTL responses against the protective H-2Kb-restricted TSA-1515-522 epitope. In H-2(d) mice, inoculation with either of the two TSA-1-expressing vectors effectively generated antiparasite antibodies and primed CTLs that lysed T. cruzi-infected cells in an antigen-specific, MHC class I-restricted, and CD8(+)-T-cell-dependent manner. When TSA-1 DNA-vaccinated animals were challenged with T. cruzi, 14 of 22 (64%) H-2(b) and 16 of 18 (89%) H-2(d) mice survived the infection. The ability to induce significant murine anti-T. cruzi protective immunity by immunization with plasmid DNA expressing TSA-1 provides the basis for the application of this technology in the design of optimal DNA multicomponent anti-T. cruzi vaccines which may ultimately be used for the prevention or treatment of Chagas' disease.

  6. Expression and humoral immune response to Hepatitis C virus using a plasmid DNA construct

    Directory of Open Access Journals (Sweden)

    Ray S

    2003-01-01

    Full Text Available PURPOSE: The objective of this study was to clone a c-DNA fragment of hepatitis C virus in a eukaryotic expression vector and to measure the efficacy of humoral immune responses in mice inoculated with this recombinant plasmid. This study was an attempt to lay a foundation for HCV nucleic acid vaccine development in the future. METHODS: A c-DNA fragment of BK146, a clone of HCV type 1b, was sub-cloned into an eukaryotic expression vector pMT3. HepG2 and COS cells were transfected with this construct, named pMT3-BK146. The expression of HCV mRNA and proteins was studied by reverse transcribed polymerase chain reaction, radio Immunoprecipitation (RIPA and immunofluorescence (IFA. The DNA of this construct was injected into the footpad of BALB/c mice and antibody response was tested by enzyme immunoassay and indirect immunofluorescence. RESULTS: COS and HepG2 cells transiently transfected with the recombinant plasmid pMT3-BK146 showed the expression of HCV proteins by RT-PCR, RIPA and immunofluorescence. This DNA clone when injected into Balb/c mice was able to generate specific antibody response to hepatitis C virus by ELISA and IFA. CONCLUSIONS: A c-DNA fragment of HCV cloned in an eukaryotic expression vector was able to express core protein. This DNA clone was also able to elicit antibody response in mice. This can be an initial step towards the development of a potential DNA vaccine for hepatitis C virus infection.

  7. Specific recognition of supercoiled plasmid DNA by affinity chromatography using the intercalator DAPP as ligand.

    Science.gov (United States)

    Caramelo-Nunes, C; Almeida, P; Marcos, J C; Tomaz, C T

    2013-06-01

    Small molecules that bind DNA with high specificity present a promising opportunity for application as chromatographic ligands for plasmid DNA (pDNA) purification. This research used the intercalator 3,8-diamino-6-phenylphenanthridine (DAPP) as an immobilized ligand for the specific separation of supercoiled (sc) pDNA by affinity chromatography. The results showed that the protonated DAPP-Sepharose support has a great affinity for sc pDNA isoform, separating it from the less active open circular and linear isoforms. All pDNA isoforms were retained in the column using 10mM acetate buffer pH 5. Selective elution of oc and linear isoforms was achieved with 0.22M of sodium chloride in the same buffer. Finally, increasing the concentration to 0.55M led to the elution of the sc isoform. The binding of pDNA to DAPP-Sepharose varies in function of pH, and the stability of the protonated DAPP-DNA complex decreases with increasing salt concentration. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Transformation of Azotobacter vinelandii OP with a broad host range plasmid containing a cloned chromosomal nif-DNA marker.

    Science.gov (United States)

    Bingle, W H

    1988-05-01

    The non-nitrogen-fixing (Nif-) strain UW10 of Azotobacter vinelandii OP (UW) was naturally induced to competence and transformed with broad host range plasmid pKT210 containing the cloned wild-type nif-10 locus from A. vinelandii UW (Nif+); this marker was unable to complement the nif-10 mutation in trans, but could through recombination with the chromosome. The most frequent type of transformation event observed was recombination between the homologous regions of the plasmid and chromosome (producing Nif+ transformants) with loss of the plasmid vector. At a substantially lower frequency, transformants expressing the plasmid-encoded antibiotic resistance determinants were isolated which were phenotypically Nif-. Agarose gel electrophoresis showed that these transformants contained a plasmid migrating with the same mobility as the original donor plasmid. During culture these transformants acquired a Nif+ phenotype without the loss of the plasmid, as judged by the use of a hybridization probe specific for the cloned nif-DNA fragment. These data indicate that plasmids carrying sequences homologous to chromosomal sequences could be maintained in recombination-proficient A. vinelandii UW. The introduction of plasmids containing sequences homologous to chromosomal sequences was facilitated by prelinearization of the plasmid using a restriction endonuclease generating cohesive ends. Because the site of linearization could be chosen outside the region of shared homology, it was unlikely that the route of plasmid establishment occurred via a homology-facilitated transformation mechanism. The data also indicated that A. vinelandii UW could harbor broad host range cloning vectors based on plasmid RSF1010 without significant impairment of its nitrogen-fixation ability.

  9. Scientific advice on the suitability of data for the assessment of DNA integration into the fish genome of a genetically modified DNA plasmid-based veterinary vaccine

    Directory of Open Access Journals (Sweden)

    European Food Safety Authority

    2013-05-01

    Full Text Available Pancreas disease caused by salmonid alphavirus in farmed Atlantic salmon (Salmo salar leads to high mortality rates post infection and histopathological lesions in several organs. As protection against pancreas disease, Novartis developed a prophylactic DNA plasmid-based vaccine to be administered to salmon as naked plasmid in a single intramuscular injection. In order to assess the legal status of the fish vaccinated with this new vaccine with regard to the legislation on genetically modified organisms, the European Commission suggested that the company carry out a scientific study on the integration/non-integration of the plasmid DNA into the fish genome. Subsequently, the European Commission requested EFSA to give scientific advice on the study design and the conclusions drawn by the company. PCR based analysis of genomic DNA from muscle samples, taken from at or around the injection site 436 days post vaccination, led the company to conclude that integration of plasmid DNA into the fish genome is extremely unlikely. After an assessment of the study, EFSA considers that the study presented by Novartis Animal Health on the integration/non-integration of DNA plasmid-based vaccine into the salmon genomic DNA provides insufficient information on the potential integration of plasmid DNA fragments into the fish genome due to a limited coverage of the plasmid DNA by the detection method provided, the limited number of samples analysed and an insufficient limit of detection and method validation. Therefore, EFSA is of the opinion that the results from the integration/non-integration study submitted by Novartis Animal Health are not sufficient to support the conclusion of non-integration of plasmid DNA into the fish genome drawn by the company.

  10. Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA.

    Science.gov (United States)

    Tullman, Jennifer; Guntas, Gurkan; Dumont, Matthew; Ostermeier, Marc

    2011-11-01

    We demonstrate that S1 nuclease converts supercoiled plasmid DNA to unit-length, linear dsDNA through the creation of a single, double-stranded break in a plasmid molecule. These double-stranded breaks occur not only in the origin of replication near inverted repeats but also at a wide variety of locations throughout the plasmid. S1 nuclease exhibits this activity under conditions typically employed for the nuclease's single-stranded nuclease activity. Thus, S1 nuclease digestion of plasmid DNA, unlike analogous digestion with DNaseI, effectively halts after the first double-stranded break. This property makes easier the construction of large domain insertion libraries in which the goal is to insert linear DNA at a variety of locations throughout a plasmid. We used this property to create a library in which a circularly permuted TEM1 β-lactamase gene was inserted throughout a plasmid containing the gene encoding Escherichia coli ribose binding protein. Gene fusions that encode allosteric switch proteins in which ribose modulates β-lactamase catalytic activity were isolated from this library using a combination of a genetic selection and a screen.

  11. Optimization of a lipitoid-based plasmid DNA transfection protocol for bovine trophectoderm CT-1 cells.

    Science.gov (United States)

    Schiffmacher, Andrew T; Keefer, Carol L

    2012-08-01

    Embryo-derived cell lines are important in vitro models for investigating the molecular mechanisms directing embryonic tissue lineage segregation and maintenance. The bovine trophectoderm-derived CT-1 cell line has been widely used to identify regulatory mechanisms of interferon tau gene expression, and it possesses potential as a model for characterizing the gene regulatory network controlling trophoblast lineage differentiation and development. This functional potential, however, is severely limited as CT-1 cells are very recalcitrant to standard transfection methods. The focus of this study was to test the cationic lipitoid reagent as an effective transfection reagent for DNA plasmid delivery. Optimization of liptoid-based transfection of plasmid DNA resulted in 9% transfection efficiency averaged across entire CT-1 colonies, with many subregions of CT-1 colonies achieving transfection rates of 15%. These rates are a substantial improvement over near-zero efficiencies achieved using other standard transfection techniques. CT-1 cells were also successfully adapted to substrate-free culture for over 20 passages, eliminating the need to culture CT-1 colonies on feeder cells or matrix-coated cultureware. Together, these results increase the utility of the CT-1 cell line as an in vitro bovine trophoblast model and provide insight into overcoming DNA delivery difficulties in other cell lines not amenable to genetic manipulation.

  12. A bimetallic nanocomposite electrode for direct and rapid biosensing of p53 DNA plasmid

    Indian Academy of Sciences (India)

    Ezat Hamidi-Asl; Jahan-Bakhsh Raoof; Nahid Naghizadeh; Simin Sharifi; Mohammad Saeid Hejazi

    2015-09-01

    A new label-free electrochemical DNA biosensor is presented based on carbon paste electrode (CPE) modified with gold (Au) and platinum (Pt) nanoparticles to prepare the bimetallic nanocomposite electrode. The proposed sensor was made by immobilization of 15-mer single stranded oligonucleotide probe related to p53 gene for detection of DNA plasmid samples. The hybridization detection relied on the alternation in the guanine oxidation signal following hybridization of the probe with complementary genomic DNA.The technique of differential pulse voltammetry (DPV) was used for monitoring guanine oxidation. To optimize the performance of the modified CPE, different electrodes were prepared in various percentages of Au and Pt nanoparticles. The modified electrode containing 15% Au/Pt bimetallic nanoparticles (15% Au/Pt-MCPE) was selected as the best working electrode. The selectivity of the sensor was investigated using plasmid samples containing non-complementary oligonucleotides. The detection limit of the biosensor was studied and calculated to be 53.10 pg L−1.

  13. Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography.

    Science.gov (United States)

    Hilbrig, Frank; Freitag, Ruth

    2012-01-01

    Hydroxyapatite and related stationary phases increasingly play a role in the downstream processing of high-value biological materials, such as recombinant proteins, therapeutic antibodies and pharmaceutical-grade plasmid DNA. Chromatographic hydroxyapatite is an inorganic, ceramic material identical in composition, if not in structure, to calcium phosphate found in human bones and teeth. The interaction of hydroxyapatite with biomacromolecules is complex and highly dynamic, which can make predicting performance difficult, but also allows the design of very selective isolation processes. This review discusses the currently commercially available chromatographic materials, different retention mechanisms supported by these materials and differential exploitation for the design of highly specific isolation procedures. The state of the art of antibody purification by hydroxy- and fluoroapatite is reviewed together with tested routines for method development and implementation. Finally, the isolation of plasmid DNA is discussed, since the purification of DNA therapeutics at a sufficiently large scale is an emerging need in bioprocess development and perhaps the area in bioseparation where apatite chromatography can make its most important contribution to date.

  14. Metal chelate affinity precipitation of RNA and purification of plasmid DNA

    Science.gov (United States)

    Balan, Sindhu; Murphy, Jason; Galaev, Igor; Kumar, Ashok; Fox, George E.; Mattiasson, Bo; Willson, Richard C.

    2003-01-01

    The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine 'tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.

  15. Metal chelate affinity precipitation of RNA and purification of plasmid DNA

    Science.gov (United States)

    Balan, Sindhu; Murphy, Jason; Galaev, Igor; Kumar, Ashok; Fox, George E.; Mattiasson, Bo; Willson, Richard C.

    2003-01-01

    The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine 'tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.

  16. [Localization of denitrification genes in plasmid DNA of bacteria Azospirillum brasilense].

    Science.gov (United States)

    Petrova, L P; Varshalomidze, O É; Shelud'ko, A V; Katsy, E I

    2010-07-01

    In 85-Mda plasmid (p85) of plant-associated bacteria Azospirillum brasilense Sp245 model strain, the genes encoding copper-containing nitrite reductase (nirK); heterodimeric NO-reductase (norCB); NorQ and NorD proteins affecting synthesis and (or) activation of NirK and (or) NO-reductase (norQD); catalytic subunit I ofcytochrom c oxidase (CccoN); presumable NO sensor carrying two hemeerythrine domains (orf181); and an enzyme required for synthesis of presumable NO antagonist, homocystein (metC) were identified. In the same region of p85, orf293 encoding transcriptional regulator of LysR type, orf208 whose protein product carries a formylmethanofuran dehydrogenase subunit E domain, and an orf164-encoding conservative secretory protein with unknown function were also found. Localization of a set of denitrification genes in the plasmid DNA A. brasilense Sp245 adjacent to IS elements ISAzba1 and ISAzba2 indicates potential mobility of these genes and high probability of their horizontal transfer among populations of rhizospheric bacteria. A site homologous to p85 nirK-orf208-orf181 genes was detected in the 115 kb plasmid of A. brasilense Sp7 type strain.

  17. CONSECUTIVE IMMUNIZATION WITH RECOMBINANT FOWLPOX VIRUS AND PLASMID DNA FOR ENHANCING CELLULAR AND HUMORAL IMMUNITY

    Institute of Scientific and Technical Information of China (English)

    罗坤; 金宁一; 郭志儒; 秦云龙; 郭炎; 方厚华; 安汝国; 殷震

    2001-01-01

    To investigate the influence of consecutive immunization on cellular and humoral immunity in mice. Methods: We evaluated a consecutive immunization strategy of priming with recombinant fowlpox virus vUTALG and boosting with plasmid DNA pcDNAG encoding HIV-1 capsid protein Gag. Results: In immunized mice, the number of CD4+ T cells from splenic lymphocytes increased significantly and the proliferation response of splenocytes to ConA and LPS elevated markedly and HIV-1-specific antibody response could be induced. Conclusion: Consecutive immunization could increase cellular and humoral immunity responses in mice.

  18. Nondestructive DNA extraction from museum specimens.

    Science.gov (United States)

    Hofreiter, Michael

    2012-01-01

    Natural history museums around the world hold millions of animal and plant specimens that are potentially amenable to genetic analyses. With more and more populations and species becoming extinct, the importance of these specimens for phylogenetic and phylogeographic analyses is rapidly increasing. However, as most DNA extraction methods damage the specimens, nondestructive extraction methods are useful to balance the demands of molecular biologists, morphologists, and museum curators. Here, I describe a method for nondestructive DNA extraction from bony specimens (i.e., bones and teeth). In this method, the specimens are soaked in extraction buffer, and DNA is then purified from the soaking solution using adsorption to silica. The method reliably yields mitochondrial and often also nuclear DNA. The method has been adapted to DNA extraction from other types of specimens such as arthropods.

  19. Plasmid DNA Initiates Replication of Yellow Fever Vaccine In Vitro and Elicits Virus-Specific Immune Response in Mice

    OpenAIRE

    Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat; Jokinen, Jenny; Lukashevich, Igor S.; Pushko, Peter

    2014-01-01

    Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10ng of iDNA plasmid was sufficient to s...

  20. Transgenic Crops by Direct Treatment of Exogenous DNA Without Agrobacterium tumefaciens Plasmid and Tissue Culture

    Institute of Scientific and Technical Information of China (English)

    ZhangGuodong

    1995-01-01

    Gene transfter methods are developing quickly recently,but each method has its limitations.We introduce a new gene transfer technique in this paper,which is simple,effective,and easy to operate,but does not get enough attention from scientists.This technique is used to transform plants by injecting exogenous DNA to stigma,style,ovary,young fruit or meristem of the recipient,or soaking the recipient's seeds in exogenous DNA solution.Los of heritable variations were found in many characters of many crops,It may be used to creaste new germplasms or realize gene exchange between different species,gerera,or families,even between animals and plants,A brief discussion was given to the mechanism of exogenous DNA introduction,integration into and expression in the recipient.We also discussed the merits and limitations of the technique.Currently there are two successful approaches that can be used to transform plants genetically,but each method has its limitations that are delaying the application of the techniques to certaincommercially important crops.The first tecnhique exploits a natural genetic engineer,Agrobacterium tumefaciens,which contains a tumor-inducing(Ti) plasmid that transfers a DNA segment(the T-DNA) from the plasmid to the nuclear genome of infected plants(or in vitro to plant tissue).The method is restricted to dicotyledenous plants;monocotyledenous plants are usually not susceptible to agrobacterial infection.The second technique involves direct transfter of DNA to plant protoplast ,prepared by enzymatic digestion of cell walls,for example by chemically stimulated uptake using polyethylene glycol or a high voltage pulse,generating transient'holes'in the protoplast membrane.This technique depends on a tissue culture system that allows regeneration of mature plants from protoplasts,But so far it is impossible to achieve plant regeneration from protoplasts in many crops.Both techniques use dominant selectable markers(for example,kanamycin resistance) to

  1. Poly(hydroxyethyl methacrylate) based magnetic nanoparticles for plasmid DNA purification from Escherichia coli lysate

    Energy Technology Data Exchange (ETDEWEB)

    Percin, Is Latin-Small-Letter-Dotless-I k [Department of Biology, Hacettepe University, Ankara (Turkey); Karakoc, Veyis [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey); Akgoel, Sinan [Department of Biochemistry, Ege University, Izmir (Turkey); Aksoez, Erol [Department of Biology, Hacettepe University, Ankara (Turkey); Denizli, Adil, E-mail: denizli@hacettepe.edu.tr [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey)

    2012-07-01

    The aim of this study is to prepare poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine) [PHEMAH] magnetic nanoparticles for plasmid DNA (pDNA) purification from Escherichia coli (E. coli) cell lysate. Magnetic nanoparticles were produced by surfactant free emulsion polymerization. mPHEMAH nanoparticles were characterized by elemental analysis, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), vibrating sample magnetometer (VSM), electron spin resonance (ESR), thermogravimetric analyses (TGA) and transmission electron microscopy (TEM). Surface area, average particle size and size distribution were also performed. Specific surface area of the mPHEMAH nanoparticles was found to be 1180 m{sup 2}/g. Elemental analysis of MAH for nitrogen was estimated as 0.18 mmol/g polymer. The amount of pDNA adsorbed onto the mPHEMAH nanoparticles first increased and then reached a saturation value at around 1.0 mg/mL of pDNA concentration. Compared with the mPHEMA nanoparticles (50 {mu}g/g polymer), the pDNA adsorption capacity of the mPHEMAH nanoparticles (154 mg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The maximum pDNA adsorption was achieved at 25 Degree-Sign C. The overall recovery of pDNA was calculated as 92%. The mPHEMAH nanoparticles could be used six times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH nanoparticles promise high selectivity for pDNA. - Highlights: Black-Right-Pointing-Pointer Magnetic nanoparticles have several advantages over conventional adsorbents. Black-Right-Pointing-Pointer MAH acted as the pseudospecific ligand, ligand immobilization step was eliminated. Black-Right-Pointing-Pointer pDNA adsorption amount was 154 mg/g. Black-Right-Pointing-Pointer Fifty-fold capacity increase was obtained when compared to conventional matrices.

  2. Efficient expression of human factor Ⅸ cDNA in livermediated by hydrodynamics-based plasmid administration

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Hydrodynamics-based administration via tail vein was used to deliver naked plasmid with human factor Ⅸ (hFⅨ) cDNA in 2.2 mL Ringer's solution into mice within 7 s. The peak level of expression of hFⅨ was 2921 ng/mL in mouse plasma. The hFⅨ cDNA expression increased with increasing the amount of plasmid DNA injected. The peak level of gene expression declined after repeated injection of plasmid (1459 ng/mL). The hFⅨ cDNA was detected in various organs, but the highest level of gene expression appeared in liver. Transaminase levels and liver histological results showed that rapid intravenous plasmid injection into mice induced transient focal acute liver damage, which was rapidly repaired within 3-10 d. These results suggested that high-level expression of hFⅨ cDNA can be achieved by hydrodynamics-based plasmid transfer and this method is now further used for gene therapy and gene function study in our lab.

  3. Ultrasound enhances the transfection of plasmid DNA by non-viral vectors.

    Science.gov (United States)

    Hosseinkhani, Hossein; Aoyama, Teruyoshi; Ogawa, Osamu; Tabata, Yasuhiko

    2003-04-01

    Increasing attention has been paid to technology used for the delivery of genetic materials into cells for gene therapy and the generation of genetically engineered cells. So far, viral vectors have been mainly used because of their inherently high transfection efficiency of gene. However, there are some problems to be resolved for the clinical applications, such as the pathogenicity and immunogenicity of viral vectors themselves. Therefore, many research trials with non-viral vectors have been performed to enhance their efficiency to a level comparable to the viral vector. Two directions of these trials exist: material improvement of non-viral vectors and their combination with various external physical stimuli. This paper reviews the latter research trials, with special attention paid to the enhancement of gene expression by ultrasound (US). The expression level of plasmid DNA by various cationized polymers and liposomes is promoted by US irradiation in vitro as well as in vivo. This US-enhanced expression of plasmid DNA will be discussed to emphasize the technical feasibility of US in gene therapy and biotechnology.

  4. Mapping of bionic array electric field focusing in plasmid DNA-based gene electrotransfer.

    Science.gov (United States)

    Browne, C J; Pinyon, J L; Housley, D M; Crawford, E N; Lovell, N H; Klugmann, M; Housley, G D

    2016-04-01

    Molecular medicine through gene therapy is challenged to achieve targeted action. This is now possible utilizing bionic electrode arrays for focal delivery of naked (plasmid) DNA via gene electrotransfer. Here, we establish the properties of array-based electroporation affecting targeted gene delivery. An array with eight 300 μm platinum ring electrodes configured as a cochlear implant bionic interface was used to transduce HEK293 cell monolayers with a plasmid-DNA green fluorescent protein (GFP) reporter gene construct. Electroporation parameters were pulse intensity, number, duration, separation and electrode configuration. The latter determined the shape of the electric fields, which were mapped using a voltage probe. Electrode array-based electroporation was found to require ~100 × lower applied voltages for cell transduction than conventional electroporation. This was found to be due to compression of the field lines orthogonal to the array. A circular area of GFP-positive cells was created when the electrodes were ganged together as four adjacent anodes and four cathodes, whereas alternating electrode polarity created a linear area of GFP-positive cells. The refinement of gene delivery parameters was validated in vivo in the guinea pig cochlea. These findings have significant clinical ramifications, where spatiotemporal control of gene expression can be predicted by manipulation of the electric field via current steering at a cellular level.

  5. Construction of a eukaryotic expression plasmid pcDNA3.1-HuR-FLAG and its transient expression in NIH3T3 cells

    Directory of Open Access Journals (Sweden)

    Tao LI

    2011-04-01

    Full Text Available Objective To construct a eukaryotic expression vector for HuR and analyze its expression and biological function in NIH3T3 cells.Methods The total RNA was extracted from NIH3T3 cells and reverse transcribed to cDNAs.The coding region sequence of mouse HuR was then amplified by PCR and subcloned into the pcDNA3.1-FLAG plasmid.The recombinant plasmid pcDNA3.1-HuR-FLAG was verified by PCR and restriction endonuclease analysis,confirmed by DNA sequence analysis,and then transiently transfected into NIH3T3 cells with Lipofectamine LTX.The expression of HuR protein was determined by Western blotting,and the mRNA level of HuR and DUSP1 were analyzed by using real-time PCR.Result The recombinant plasmid pcDNA3.1-HuR-FLAG was correctly constructed.Twenty-four hours after transfection of the recombinant plasmid into NIH3T3 cells,the fusion protein was found to have highly expressed in the cells as revealed by Western blotting.Real-time PCR results detected that the over-expression of HuR could up-regulate the expression of DUSP1.Conclusion The eukaryotic expression vector for HuR-FLAG fusion protein has been successfully constructed and transiently expressed in NIH3T3 cells.It can be used in further analysis of the posttranscriptional regulation of DUSP1 by HuR in cancer cells.

  6. Plasmid DNA initiates replication of yellow fever vaccine in vitro and elicits virus-specific immune response in mice

    Energy Technology Data Exchange (ETDEWEB)

    Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Jokinen, Jenny; Lukashevich, Igor S. [Department of Pharmacology and Toxicology, School of Medicine, Center for Predictive Medicine and Emerging Infectious Diseases, University of Louisville, Louisville, KY (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States)

    2014-11-15

    Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20 μg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF. - Highlights: • The iDNA{sup ®} platform combines advantages of DNA and live attenuated vaccines. • Yellow fever (YF) 17D vaccine was launched from iDNA plasmid in vitro and in vivo. • Safety of iDNA-generated 17D virus was confirmed in AG129 mice. • BALB/c mice seroconverted after a single-dose vaccination with iDNA. • YF virus-neutralizing response was elicited in iDNA-vaccinated mice.

  7. Plasmid DNA is internalized from the apical plasma membrane of the salivary gland epithelium in live animals.

    Science.gov (United States)

    Sramkova, Monika; Masedunskas, Andrius; Weigert, Roberto

    2012-08-01

    Non-viral-mediated gene delivery represents an alternative way to express the gene of interest without inducing immune responses or other adverse effects. Understanding the mechanisms by which plasmid DNAs are delivered to the proper target in vivo is a fundamental issue that needs to be addressed in order to design more effective strategies for gene therapy. As a model system, we have used the submandibular salivary glands in live rats and we have recently shown that reporter transgenes can be expressed in different cell populations of the glandular epithelium, depending on the modality of administration of plasmid DNA. Here, by using a combination of immunofluorescence and intravital microscopy, we have explored the relationship between the pattern of transgenes expression and the internalization of plasmid DNA. We found that plasmid DNA is internalized: (1) by all the cells in the salivary gland epithelium, when administered alone, (2) by large ducts, when mixed with empty adenoviral particles, and (3) by acinar cells upon stimulation of compensatory endocytosis. Moreover, we showed that plasmid DNA utilizes different routes of internalization, and evades both the lysosomal degradative pathway and the retrograde pathway towards the Golgi apparatus. This study clearly shows that in vivo approaches have the potential to address fundamental questions on the cellular mechanisms regulating gene delivery.

  8. Extracting DNA from submerged pine wood.

    Science.gov (United States)

    Reynolds, M Megan; Williams, Claire G

    2004-10-01

    A DNA extraction protocol for submerged pine logs was developed with the following properties: (i) high molecular weight DNA, (ii) PCR amplification of chloroplast and nuclear sequences, and (iii) high sequence homology to voucher pine specimens. The DNA extraction protocol was modified from a cetyltrimehtylammonium bromide (CTAB) protocol by adding stringent electrophoretic purification, proteinase K, RNAse, polyvinyl pyrrolidone (PVP), and Gene Releaser. Chloroplast rbcL (ribulose-1,5-bisphosphate carboxylase) could be amplified. Nuclear ribosomal sequences had >95% homology to Pinus taeda and Pinus palustris. Microsatellite polymorphism for PtTX2082 matched 2 of 14 known P. taeda alleles. Our results show DNA analysis for submerged conifer wood is feasible.

  9. Boldo prevents UV light and nitric oxide-mediated plasmid DNA damage and reduces the expression of Hsp70 protein in melanoma cancer cells.

    Science.gov (United States)

    Russo, Alessandra; Cardile, Venera; Caggia, Silvia; Gunther, Germán; Troncoso, Nicolas; Garbarino, Juan

    2011-09-01

    This study was designed to investigate the potential protective effect of a methanolic extract of Peumus boldus leaves on UV light and nitric oxide (NO)-mediated DNA damage. In addition, we investigated the growth inhibitory activity of this natural product against human melanoma cells (M14). Boldine, catechin, quercetin and rutin were identified using a HPLC method. The extract was incubated with plasmid DNA and, before irradiating the samples with UV-R, H(2) O(2) was added. For analysis of DNA single-strand breaks induced by NO, the experiments were performed by incubating the extract with Angeli's salt. In the study on M14 cell line, cell viability was measured using MTT assay. Release of lactate dehydrogenase, a marker of membrane breakdown, was also measured. For the detection of apoptosis, the evaluation of DNA fragmentation (COMET assay) and caspase-3 activity assay were employed. The expression of heat shock protein 70 (Hsp70) was detected by Western blot analysis. Generation of reactive oxygen species was measured by using a fluorescent probe. The extract (demonstrating the synergistic effect of the constituents boldine and flavonoids), showed a protective effect on plasmid DNA and selectively inhibited the growth of melanoma cells. But a novel finding was that apoptosis evoked by this natural product in M14 cells, appears to be mediated, at least in part, via the inhibition of Hsp70 expression, which may be correlated with a modulation of redox-sensitive mechanisms. These results confirm the promising biological properties of Peumus boldus and encourage in-vivo investigations into its potential anti-cancer activity. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

  10. Validation and scale-up of plasmid DNA purification by phenyl-boronic acid chromatography.

    Science.gov (United States)

    Gomes, A Gabriela; Azevedo, Ana M; Aires-Barros, M Raquel; Prazeres, D Miguel F

    2012-11-01

    This study addresses the feasibility of scaling-up the removal of host cell impurities from plasmid DNA (pDNA)-containing Escherichia coli lysates by phenyl-boronic (PB) acid chromatography using columns packed with 7.6 and 15.2 cm(3) of controlled porous glass beads (CPG) derivatized with PB ligands. Equilibration was performed with water at 10 cm(3) /min and no conditioning of the lysate feed was required. At a ratio of lysate feed to adsorbent volume of 1.3, 93-96% of pDNA was recovered in the flow through while 66-71% of impurities remained bound (~2.5-fold purification). The entire sequence of loading, washing, elution, and re-equilibration was completed in 20 min. Run-to-run consistency was observed in terms of chromatogram features and performance (yield, purification factor, agarose electrophoresis) across the different amounts of adsorbent (0.75-15.2 cm(3) ) by performing successive injections of lysates prepared independently and containing 3.7 or 6.1 kbp plasmids. The column productivity at large scale was 4 dm(3) of alkaline lysate per hour per dm(3) of PB-CPG resin. The method is rapid, reproducible, simple, and straightforward to scale-up. Furthermore, it is capable of handling heavily contaminated samples, constituting a good alternative to purification techniques such as isopropanol precipitation, aqueous two-phase systems, and tangential flow filtration. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Charge density and particle size effects on oligonucleotide and plasmid DNA binding to nanosized hydrotalcite.

    Science.gov (United States)

    Sanderson, Brian A; Sowersby, Drew S; Crosby, Sergio; Goss, Marcus; Lewis, L Kevin; Beall, Gary W

    2013-12-01

    Hydrotalcite (HT) and other layered double metal hydroxides are of great interest as gene delivery and timed release drug delivery systems and as enteric vehicles for biologically active molecules that are sensitive to gastric fluids. HT is a naturally occurring double metal hydroxide that can be synthesized as a nanomaterial consisting of a brucite structure with isomorphous substitution of aluminum ions. These positively charged nanoparticles exhibit plate-like morphology with very high aspect ratios. Biomolecules such as nucleic acids and proteins form strong associations with HT because they can associate with the positively charged layers. The binding of nucleic acids with HT and other nanomaterials is currently being investigated for potential use in gene therapy; however, the binding of specific nucleic acid forms, such as single- and double-stranded DNA, has been little explored. In addition, the effects of charge density and particle size on DNA adsorption has not been studied. In this paper, the binding of different forms of DNA to a series of HTs prepared at different temperatures and with different anion exchange capacities has been investigated. Experiments demonstrated that HTs synthesized at higher temperatures associate with both single- and double-stranded oligomers and circular plasmid DNA more tightly than HTs synthesized at room temperature, likely due to the hydrothermal conditions promoting larger particle sizes. HT with an anion exchange capacity of 300 meq/100 g demonstrated the highest binding of DNA, likely due to the closer match of charge densities between the HT and DNA. The details of the interaction of various forms of DNA with HT as a function of charge density, particle size, and concentration are discussed.

  12. Comparison of Methods of Extracting Salmonella enterica Serovar Enteritidis DNA from Environmental Substrates and Quantification of Organisms by Using a General Internal Procedural Control

    NARCIS (Netherlands)

    Klerks, M.M.; Bruggen, van A.H.C.; Zijlstra, C.; Donnikov, M.; Vos, de R.

    2006-01-01

    This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general

  13. Comparison of Methods of Extracting Salmonella enterica Serovar Enteritidis DNA from Environmental Substrates and Quantification of Organisms by Using a General Internal Procedural Control

    NARCIS (Netherlands)

    Klerks, M.M.; Bruggen, van A.H.C.; Zijlstra, C.; Donnikov, M.; Vos, de R.

    2006-01-01

    This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general interna

  14. Phototransfection of mouse embryonic stem cells with plasmid DNA using femtosecond laser pulses

    Science.gov (United States)

    Thobakgale, Lebogang; Manoto, Sello Lebohang; Ombinda Lemboumba, Saturnin; Maaza, Malik; Mthunzi-Kufa, Patience

    2017-02-01

    Cellular manipulation by delivery of molecules into cells has been applied extensively in tissue engineering research for medical applications . The different molecular delivery techniques used range from viral and chemical agents to physical and electrical methods. Although successful in most studies, these techniques have inherent difficulties such as toxicity, unwanted genetic mutations and low reproducibility respectively. Literature recognizes pulsed lasers at femtosecond level to be most efficient in photonic interactions with biological material. As of late, laser pulses have been used for drug and DNA delivery into cells via transient optical perforation of the cellular membrane. Thus in this study, we design and construct an optical system coupled to a femtosecond laser for the purpose of phototransfection or insertion of plasmid DNA (pDNA) into cells using lasers. We used fluorescent green protein (pGFP) to transfect mouse embryonic stem cells as our model. Secondly, we applied fluorescence imaging to view the extent of DNA delivery using this method. We also assessed the biocompatibility of our system by performing molecular assays of the cells post irradiation using adenosine triphosphate (ATP) and lactate dehydrogenase (LDH).

  15. Plasmid Biopharmaceuticals.

    Science.gov (United States)

    Prazeres, Duarte Miguel F; Monteiro, Gabriel A

    2014-12-01

    Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its mission to produce pharmaceutical proteins, antibodies, vaccines, industrial enzymes, and molecular diagnostics, to name a few key products. Furthermore, plasmids have gradually stepped up in the past 20 years as useful biopharmaceuticals in the context of gene therapy and DNA vaccination interventions. This review provides a concise coverage of the scientific progress that has been made since the emergence of what are called today plasmid biopharmaceuticals. The most relevant topics are discussed to provide researchers with an updated overview of the field. A brief outline of the initial breakthroughs and innovations is followed by a discussion of the motivation behind the medical uses of plasmids in the context of therapeutic and prophylactic interventions. The molecular characteristics and rationale underlying the design of plasmid vectors as gene transfer agents are described and a description of the most important methods used to deliver plasmid biopharmaceuticals in vivo (gene gun, electroporation, cationic lipids and polymers, and micro- and nanoparticles) is provided. The major safety issues (integration and autoimmunity) surrounding the use of plasmid biopharmaceuticals is discussed next. Aspects related to the large-scale manufacturing are also covered, and reference is made to the plasmid products that have received marketing authorization as of today.

  16. DNA extraction from keratin and chitin.

    Science.gov (United States)

    Campos, Paula F; Gilbert, Thomas M P

    2012-01-01

    DNA extracted from keratinous and chitinous materials can be a useful source of genetic information. To effectively liberate the DNA from these materials, buffers containing relatively high levels of DTT, proteinase K, and detergent are recommended, followed by purification using either silica-column or organic methods.

  17. [Application of the QIAamp DNA Investigator Kit and Prepfiler Forensic DNA Extraction Kit in genomic DNA extraction from skeletal remains].

    Science.gov (United States)

    Ludwikowska-Pawłowska, Małgorzata; Jacewicz, Renata; Jedrzejczyk, Maciej; Prośniak, Adam; Berent, Jarosław

    2009-01-01

    The report presents an application of the QIAamp DNA Investigator Kit and PrepFiler Forensic DNA Extraction Kit in genomic DNA extraction from post-mortem highly degraded skeletal remains. The analysis included 25 bone samples collected on autopsy. DNA extraction was performed in accordance with the QIAamp DNA Investigator Kit and PrepFiler Forensic DNA Extraction Kit manufacturer's isolation protocols. Amplification was performed on a Biometra termocycler using the AmpFISTR Identifiler PCR Amplification Kit according to the manufacturer's protocol. Typing of PCR products was carried out on an ABI Prism 377 DNA sequencer. The recommended parameters for GeneScan analysis and Genotyper software were followed. The authors demonstrated that the QIAamp DNA Investigator Kit was more effective, convenient and statistically significantly better method which may be employed in DNA extraction from bone specimens.

  18. Anti-oxidative effects of Phellinus linteus and red ginseng extracts on oxidative stress-induced DNA damage.

    Science.gov (United States)

    Park, Byung-Jae; Lim, Yeong-Seok; Lee, Hee-Jung; Eum, Won Sik; Park, Jinseu; Han, Kyu Hyung; Choi, Soo Young; Lee, Kil Soo

    2009-08-31

    Anti-oxidative effect of Phellinus linteus (P. linteus) and red ginseng extracts on DNA damage induced by reactive oxygen species (ROS) were investigated in this study. P. linteus (PLE) and red ginseng extracts (RGE) inhibited the breaking of E. coli ColE1 plasmid DNA strands as well as nuclear DNA of rat hepatocytes damaged by oxidative stress. In addition, a reaction mixture of PLE and RGE showed synergistic inhibitory effect against DNA damage. These results suggest that PLE and RGE have a cellular defensive effect against DNA damage induced by ROS.

  19. Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity

    Directory of Open Access Journals (Sweden)

    Nolte Ingo

    2011-10-01

    Full Text Available Abstract Background Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare. Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD, FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2, FHD and commercially available AuNPs (Plano-AuNP, and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI% positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence. Results The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%, whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50

  20. Photoresponsive Bridged Silsesquioxane Nanoparticles with Tunable Morphology for Light-Triggered Plasmid DNA Delivery

    KAUST Repository

    Fatieiev, Yevhen

    2015-09-25

    Bridged silsesquioxane nanocomposites with tunable morphologies incorporating o-nitrophenylene-ammonium bridges are described. The systematic screening of the sol-gel parameters allowed the material to reach the nanoscale –unlike most reported bridged silsesquioxane materials– with controlled dense and hollow structures of 100 to 200 nm. The hybrid composition of silsesquioxanes with 50% of organic content homogenously distributed in the nanomaterials endowed them with photoresponsive properties. Light irradiation was performed to reverse the surface charge of nanoparticles from +46 to -39 mV via the photoreaction of the organic fragments within the particles, as confirmed by spectroscopic monitorings. Furthermore, such NPs were ap-plied for the first time for the on-demand delivery of plasmid DNA in HeLa cancer cells via light actuation.

  1. Experimental Study of Plasmid TGF-β1 DNA Gene Transfer with Lipofectamine into Rabbit Corneal Epithelial Cells In Vitro

    Institute of Scientific and Technical Information of China (English)

    黄琼; 胡燕华; 姜发纲; 陈宏

    2002-01-01

    To investigate whether the TGF-β1 plasmid DNA carried by lipofectamine could be introduced into cultured rabbit corneal epithelial cells, specific expression of the plasmid pMAM TGF-β1in the cultured corneal epithelial cells was studied. Two days after 12 h of transfection of pMAMTGF-β1 mediated by lipofectamine into the cultured corneal epithelial cells, the TGF-β1 protein expression specific for pMAMTGF-β1 in the cells was detected by means of immunohistochemical staining and the positive rate was 23. 37 %. The results suggested that foreign plasmid DNA could be effectively delivered into cultured rabbit corneal epithelial cells by means of lipofectamine, and this will provide a promising method of studying TGF-β1 on the mechanism of physiology and pathology concerned with corneal epithelial cells.

  2. DNA sequence analysis of plasmids from multidrug resistant Salmonella enterica serotype Heidelberg isolates.

    Directory of Open Access Journals (Sweden)

    Jing Han

    Full Text Available Salmonella enterica serovar Heidelberg is among the most detected serovars in swine and poultry, ranks among the top five serotypes associated with human salmonellosis and is disproportionately associated with invasive infections and mortality in humans. Salmonella are known to carry plasmids associated with antimicrobial resistance and virulence. To identify plasmid-associated genes in multidrug resistant S. enterica serovar Heidelberg, antimicrobial resistance plasmids from five isolates were sequenced using the 454 LifeSciences pyrosequencing technology. Four of the isolates contained incompatibility group (Inc A/C multidrug resistance plasmids harboring at least eight antimicrobial resistance genes. Each of these strains also carried a second resistance plasmid including two IncFIB, an IncHI2 and a plasmid lacking an identified Inc group. The fifth isolate contained an IncI1 plasmid, encoding resistance to gentamicin, streptomycin and sulfonamides. Some of the IncA/C plasmids lacked the full concert of transfer genes and yet were able to be conjugally transferred, likely due to the transfer genes carried on the companion plasmids in the strains. Several non-IncA/C resistance plasmids also carried putative virulence genes. When the sequences were compared to previously sequenced plasmids, it was found that while all plasmids demonstrated some similarity to other plasmids, they were unique, often due to differences in mobile genetic elements in the plasmids. Our study suggests that Salmonella Heidelberg isolates harbor plasmids that co-select for antimicrobial resistance and virulence, along with genes that can mediate the transfer of plasmids within and among other bacterial isolates. Prevalence of such plasmids can complicate efforts to control the spread of S. enterica serovar Heidelberg in food animal and human populations.

  3. Dead-end hollow-fiber ultrafiltration for concentration and enumeration of Escherichia coli and broad-host-range plasmid DNA from wastewater

    Science.gov (United States)

    Asfahl, Kyle L.; Savin, Mary C.

    2012-01-01

    Broad-host-range plasmids can facilitate dissemination of antibiotic resistance determinants among diverse bacterial populations. We evaluated hollow-fiber ultrafiltration for increases in detection efficiency of broad-host-range plasmids and Escherichia coli DNA in wastewater. Ultrafiltration followed by PCR showed limited increases in DNA detection and quantification in effluent compared with membrane filtration alone. PMID:22251424

  4. Preparation and characterization of chitosan/β-cyclodextrin nanoparticles containing plasmid DNA encoding interleukin-12.

    Science.gov (United States)

    Nahaei, M; Valizadeh, H; Baradaran, B; Nahaei, M R; Asgari, D; Hallaj-Nezhadi, S; Dastmalchi, S; Lotfipour, F

    2013-01-01

    Interleukin-12 (IL-12) as a cytokine has been proved to possess antitumor effects via stimulating the immune system. Non-viral gene delivery systems offer several advantages, including easiness in production, low cost, safety; low immunogenicity and can carry higher amounts of genetic material without limitation on their sizes.pUMVC3-hIL12 loaded Low Molecular Weight chitosan/β-cyclodextrin (LMW CS/CD) nanoparticles were prepared using ionotropic gelation method and characterized in terms of size, zeta potential, polydispersity index, morphology, loading efficiency and cytotoxicity against the CT-26 colon carcinoma cell line.All prepared particles were spherical in shape and nano-sized (171.3±2.165 nm, PdI: 0.231±0.014) and exhibited a positive zeta potential (34.3±1.55). The nanoparticles demonstrated good DNA encapsulation efficiencies (83.315%±2.067). Prepared pUMVC3-hIL12 loaded LMW CS/CD nanoparticles showed no cell toxicity in murine CT-26 colon carcinoma cells. At the concentration of 0.1 µg/ml of nanoparticles, the transfection ability was obviously higher than that of the naked DNA.LMW CS/CD-plasmid DNA nanoparticles encoding IL-12 prepared using ionotropic gelation method with no toxic effect on the tested cells can be considered as a basis for further gene delivery studies both in vitro and in vivo to enhance the expression of IL-12.

  5. Plasmid DNA initiates replication of yellow fever vaccine in vitro and elicits virus-specific immune response in mice.

    Science.gov (United States)

    Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat; Jokinen, Jenny; Lukashevich, Igor S; Pushko, Peter

    2014-11-01

    Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20 μg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF.

  6. Gene therapy of experimental autoimmune thyroiditis mice by in vivo administration of plasmid DNA coding for human interleukin-10

    Institute of Scientific and Technical Information of China (English)

    ZHANGZhen-Lin; LINBo; YULu-Yang; SHENShui-Xian; ZHULi-Hua; WANGWui-Ping; GUOLi-He

    2003-01-01

    AIM: To investigate the effect of interleukin-10 (IL-10) gene on experimental autoimmune thyroiditis mice.METHODS: Mice were immunized to induce autoimmune thyroiditis with porcine thyroglobulin (pTg), and thyroids of mice were injected with IL-10 DNA. On d 28 after immunization with pTg, mRNA expression of IL-10 inthyroid glands was detected and thyroid specimens were histopathological studied. RESULTS: The mRNA expression of IL-10 was detected in thyroid glands on d 7 and 14 after injection of IL-10 plasmid DNA or on COS-7 cells48 h after IL-10 plasmid DNA transfection. In addition, hlL-10 levels in culture media significantly increased 48 hand 72 h after IL-10 plasmid DNA transfection. Infiltration index of lymphocytes (1.1±0.4) in thyroids ofIL-10-treated mice was significantly lower than that of pcDNA3-null-treated mice (2.2±0.5) (P<0.01). Comparedwith pcDNA3-null control mice, IL-10-treated mice had lower levels of serum IFN-γ(P<0.01). CONCLUSION:The direct injection of DNA expression vectors encoding IL-10 into thyroid significantly inhibited development oflymphocytic infiltration of thyroid of autoimmune th,yroiditis mice, and alleviated the progression of this disease.

  7. [Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells].

    Science.gov (United States)

    Misic, V; El-Mogy, M; Geng, S; Haj-Ahmad, Y

    2016-01-01

    Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.

  8. Advances in the research of adjuvants for plasmid DNA vaccines%DNA疫苗佐剂的研究进展

    Institute of Scientific and Technical Information of China (English)

    蒋丽明; 叶琳

    2009-01-01

    DNA疫苗是一种很有希望的免疫方法,经多途径接种质粒DNA能引起有效的免疫应答,重复给予不会产生抗载体免疫.然而,质粒DNA疫苗在小型实验动物中诱导的免疫应答远强于在人类和其他非人灵长类动物中.已设计多种佐剂通过直接刺激免疫系统或增强DNA表达来提高疫苗的免疫原性,这些佐剂包括免疫协同刺激分子、细胞因子、补体分子、脂质体、核酸、聚合物、纳米粒和微粒类佐剂.此文对DNA疫苗佐剂的研究进展作一综述.%Plasmid DNA vaccine is a promising modality for immunization. Immunization with plasmid DNA by various routes can trigger effective iimnune responses. The immunogens can be administered repeatedly without inducing anti-vector immunity. However, the immune responses induced by plasmid DNA vaccines are much stronger in small laboratory animal models than in non-human primates and humans. A number of adjuvants, including immune co-stimulatory molecules, cytokines, complement molecules, liposomes, nucleic acids, polymers, micro-and nano-particles, have been designed to improve the immunogenicity of DNA vaccines by directly stimulating the immune system or by enhancing plasmid DNA expression. This review introduces the progress in development of these adjuvants for plasmid DNA vaccines.

  9. Fetal Gene Therapy for Ornithine Transcarbamylase Deficiency by Intrahepatic Plasmid DNA-Micro-Bubble Injection Combined with Hepatic Ultrasound Insonation.

    Science.gov (United States)

    Oishi, Yoshie; Kakimoto, Takashi; Yuan, Wenji; Kuno, Shuichi; Yamashita, Hiromasa; Chiba, Toshio

    2016-06-01

    We evaluated the therapeutic efficacy of hepatic transfection of plasmid DNA using micro-bubbles and ultrasound insonation for fetal correction of ornithine transcarbamylase (OTC) deficiency in mice. Twenty-three sparse-fur heterozygous pregnant mice (day 16 of gestation) were divided into three groups: injection of plasmid-DNA micro-bubble mixture into fetal liver with ultrasound insonation (Tr, n = 11); control group 1 (C1), injection of plasmid-DNA micro-bubble mixture into fetal liver with no insonation (n = 5); and control group 2 (C2), injection of saline-micro-bubble mixture into fetal liver with ultrasound insonation (n = 7). Levels of blood ammonia and urinary orotic acid were significantly lower in the Tr group than in the C1 and C2 groups (p < 0.05, p < 0.01, respectively), whereas OTC activity was not different between groups. Therefore, ultrasound insonation with micro-bubbles enhanced plasmid DNA transfection into fetal mouse liver, leading to one of the therapeutic methods in ammonia metabolism. This might provide more time for OTC-deficient infants until liver transplantation. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  10. Recombinant goose-type lysozyme in channel catfish: Lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection

    Science.gov (United States)

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme g (CC-Lys-g) produced in E. coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme g plasmid DNA could be used as an immunostimulant to protect chann...

  11. Cu(II) complexes of glyco-imino-aromatic conjugates in DNA binding, plasmid cleavage and cell cytotoxicity

    Indian Academy of Sciences (India)

    Amit Kumar; Atanu Mitra; Amrendra Kumar Ajay; Manoj Kumar Bhat; Chebrolu P Rao

    2012-11-01

    Binding of metal complexes of C2-glucosyl conjugates with DNA has been established by absorption and fluorescence studies. Conformational changes occurred in DNA upon binding have been studied by circular dichroism. All these studies are suggestive that the metal complexes bind to DNA through intercalation. Binding of di-nuclear copper complex 5 was found to be stronger when compared to the other complexes studied. Copper complexes were found to cleave the plasmid DNA in the absence of oxidizing or reducing agent, whereas, zinc complexes do not cleave. Metal complexes have shown toxicity to the HeLa and MCF-7 cell lines.Morphological studies, western blot and FACS analysis are suggestive of apoptotic cell death induced by the metal complexes. Di-nuclear copper complexes were found to be better as compared to the mononuclear ones in binding, plasmid cleavage and also in causing more cell death.

  12. Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a

    Institute of Scientific and Technical Information of China (English)

    张继瑜; 刘红; 张笑兵; 杨剑; 杨帆; 杨国威; 沈岩; 侯云德; 金奇

    2003-01-01

    The complete nucleotide sequence and organization of the large virulence plasmid pCP301 (termed by us) of Shigella flexneri 2a strain 301 were determined and analyzed. The result showed that the entire DNA sequence of pCP301 is composed of 221618 bp which form a circular plasmid. Sequence analysis identified 272 open reading frames (ORFs), among which, 194 correspond to the proteins described previously, 61 have low identity (<60%) to known proteins and the rest 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include the genes associated with bacterial virulence, the genes associated with regulation and the genes relating to plasmid maintenance, stability and DNA metabolism. Insertion sequence (IS) elements are 68 kb in length and account for 30 percent of complete sequence of the plasmid which indicates that gene multiple rearrangements of the pCP301 have taken place in Shigella flexneri evolution history. The research result is helpful for interpreting the pathogenesis of Shigella, as well as the genetics and evolution of the plasmid.

  13. Microscope Titration and Extraction of DNA from Liver.

    Science.gov (United States)

    Mayo, Lois T.; And Others

    1993-01-01

    Describes a simple and inexpensive, one-period activity to extract DNA to make the study of DNA less abstract. A microscope titration is used to determine when cells are ready for DNA extraction. (PR)

  14. Microscope Titration and Extraction of DNA from Liver.

    Science.gov (United States)

    Mayo, Lois T.; And Others

    1993-01-01

    Describes a simple and inexpensive, one-period activity to extract DNA to make the study of DNA less abstract. A microscope titration is used to determine when cells are ready for DNA extraction. (PR)

  15. Screening of L-histidine-based ligands to modify monolithic supports and selectively purify the supercoiled plasmid DNA isoform.

    Science.gov (United States)

    Amorim, Lúcia F A; Sousa, Fani; Queiroz, João A; Cruz, Carla; Sousa, Ângela

    2015-06-01

    The growing demand of pharmaceutical-grade plasmid DNA (pDNA) suitable for biotherapeutic applications fostered the development of new purification strategies. The surface plasmon resonance technique was employed for a fast binding screening of l-histidine and its derivatives, 1-benzyl-L-histidine and 1-methyl-L-histidine, as potential ligands for the biorecognition of three plasmids with different sizes (6.05, 8.70, and 14 kbp). The binding analysis was performed with different isoforms of each plasmid (supercoiled, open circular, and linear) separately. The results revealed that the overall affinity of plasmids to l-histidine and its derivatives was high (KD  > 10(-8)  M), and the highest affinity was found for human papillomavirus 16 E6/E7 (K(D)  = 1.1 × 10(-10)  M and KD  = 3.34 × 10(-10)  M for open circular and linear plasmid isoforms, respectively). L-Histidine and 1-benzyl-L-histidine were immobilized on monolithic matrices. Chromatographic studies of L-histidine and 1-benzyl-L-histidine monoliths were also performed with the aforementioned samples. In general, the supercoiled isoform had strong interactions with both supports. The separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration in the eluent, in both supports, but a lower salt concentration was required in the 1-benzyl-L-histidine monolith because of stronger interactions promoted with pDNA. The efficiency of plasmid isoforms separation remained unchanged with flow rate variations. The binding capacity for pDNA achieved with the l-histidine monolith was 29-fold higher than that obtained with conventional L-histidine agarose. Overall, the combination of either L-histidine or its derivatives with monolithic supports can be a promising strategy to purify the supercoiled isoform from different plasmids with suitable purity degree for pharmaceutical applications.

  16. Effect of the caffeine on treated and non-treated plasmid DNA with stannic chloride; Efeito da cafeina em DNA plasmidial tratado e nao tratado com cloreto estanoso

    Energy Technology Data Exchange (ETDEWEB)

    Moreno, Silvana Ramos F. [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Inst. de Biologia. Dept. de Biofisica e Biometria]|[Universidade Federal Fluminense, Niteroi, RJ (Brazil). Faculdade de Ciencias Medicas. Curso de Pos-graduacao em Patologia Experimental; Mattos, Jose C.P. de; Dantas, Flavio; Araujo, Adriano Caldeira de; Bernardo-Filho, Mario [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Inst. de Biologia. Dept. de Biofisica e Biometria]. E-mail: bernardo@uerj.br

    2000-07-01

    Caffeine, a methilxantine drug is a component of coffee, tea, stimulants and other drinks. Caffeine inhibits phosphodiesterase leading to intracellular accumulation of cyclic AMP, blocks adenosine receptors, and increases the release of Ca{sup 2+}. We have studied the possible effect of caffeine in DNA plasmid treated or not with stannous chloride (SnCl{sub 2}). Previous evaluations of the effect of caffeine on the labeling of red blood cells and plasma proteins with technetium-99m have showed a decrease of % ATI in the insoluble fraction of plasma proteins. Samples of DNA were treated with SnCl{sub 2} (0 and 200{mu}g/ml) in 0.8% agarose. SnCl{sub 2} has induced break on DNA and caffeine has not showed effect on the DNA. This indicates that caffeine does not eliminate the oxidant action of SnCl{sub 2} and does not promote break in isolated DNA plasmid. (author)

  17. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.

    Science.gov (United States)

    Bierman, M; Logan, R; O'Brien, K; Seno, E T; Rao, R N; Schoner, B E

    1992-07-01

    We have constructed cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. All vectors contain the 760-bp oriT fragment from the IncP plasmid, RK2. Transfer functions need to be supplied in trans by the E. coli donor strain. We have incorporated into these vectors selectable antibiotic-resistance markers (AmR, ThR, SpR) that function in Streptomyces spp. and other features that should allow for: (i) integration via homologous recombination between cloned DNA and the Streptomyces spp. chromosome, (ii) autonomous replication, or (iii) site-specific integration at the bacteriophage phi C31 attachment site. Shuttle cosmids for constructing genomic libraries and bacteriophage P1 cloning vector capable of accepting approx. 100-kb fragments are also described. A simple mating procedure has been developed for the conjugal transfer of these vectors from E. coli to Streptomyces spp. that involves plating of the donor strain and either germinated spores or mycelial fragments of the recipient strain. We have shown that several of these vectors can be introduced into Streptomyces fradiae, a strain that is notoriously difficult to transform by PEG-mediated protoplast transformation.

  18. Structural features and oxidative stress towards plasmid DNA of apramycin copper complex.

    Science.gov (United States)

    Balenci, D; Bonechi, G; D'Amelio, N; Gaggelli, E; Gaggelli, N; Molteni, E; Valensin, G; Szczepanik, W; Dziuba, M; Swiecicki, G; Jezowska-Bojczuk, M

    2009-02-21

    The interaction of apramycin with copper at different pH values was investigated by potentiometric titrations and EPR, UV-vis and CD spectroscopic techniques. The Cu(II)-apramycin complex prevailing at pH 6.5 was further characterized by NMR spectroscopy. Metal-proton distances derived from paramagnetic relaxation enhancements were used as restraints in a conformational search procedure in order to define the structure of the complex. Longitudinal relaxation rates were measured with the IR-COSY pulse sequence, thus solving the problems due to signal overlap. At pH 6.5 apramycin binds copper(II) with a 2 : 1 stoichiometry, through the vicinal hydroxyl and deprotonated amino groups of ring III. Plasmid DNA electrophoresis showed that the Cu(II)-apramycin complex is more active than free Cu(II) in generating strand breakages. Interestingly, this complex in the presence of ascorbic acid damages DNA with a higher yield than in the presence of H(2)O(2).

  19. Non-DSB clustered DNA lesions induced by ionizing radiation are largely responsible for the loss of plasmid DNA functionality in the presence of cisplatin.

    Science.gov (United States)

    Kouass Sahbani, S; Rezaee, M; Cloutier, P; Sanche, L; Hunting, D J

    2014-06-25

    The combination of cisplatin and ionizing radiation (IR) increases cell toxicity by both enhancing DNA damage and inhibiting repair mechanisms. Although the formation of cluster DNA lesions, particularly double-strand breaks (DSB) at the site of cisplatin-DNA-adducts has been reported to induce cell death, the contribution of DSB and non-DSB cluster lesions to the cellular toxicity is still unknown. Although both lesions are toxic, it is not always possible to measure their frequency and cell survival in the same model system. To overcome this problem, here, we investigate the effect of cisplatin-adducts on the induction of DSB and non-DSB cluster DNA lesions by IR and determine the impact of such lesions on plasmid functionality. Cluster lesions are two or more lesions on opposite DNA strands with a short distance such that error free repair is difficult or impossible. At a ratio of two cisplatin per plasmid, irradiation of platinated DNA in solution with (137)Cs γ-rays shows enhancements in the formation of DNA DSB and non-DSB cluster lesions by factors of 2.6 and 2.1, respectively, compared to unmodified DNA. However, in absolute terms, the yield for non-DSB cluster lesions is far larger than that for DSB, by a factor of 26. Unmodified and cisplatin-modified DNA were irradiated and subsequently transformed into Escherichia coli to give survival curves representing the functionality of the plasmid DNA as a function of radiation dose. Our results demonstrate that non-DSB cluster lesions are the only toxic lesions present at a sufficient frequency to account for the loss of DNA functionality. Our data also show that Frank-DSB lesions are simply too infrequent to account for the loss of DNA functionality. In conclusion, non-DSB cluster DNA damage is known to be difficult to repair and is probably the lesion responsible for the loss of functionality of DNA modified by cisplatin.

  20. Recombinant goose-type lysozyme in channel catfish: lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection.

    Science.gov (United States)

    Pridgeon, Julia W; Klesius, Phillip H; Dominowski, Paul J; Yancey, Robert J; Kievit, Michele S

    2013-10-01

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme-g (CC-Lys-g) produced in Escherichia coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme-g plasmid DNA could be used as an immunostimulant to protect channel catfish against Aeromonas hydrophila infection. Recombinant CC-Lys-g produced in E. coli expression system exhibited significant (P recombinant channel catfish lysozyme-g (pcDNA-Lys-g) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-g offered significant (P DNA injection. Macrophages of fish injected with pcDNA-Lys-g produced significantly (P DNA injection. Taken together, our results suggest that pcDNA-Lys-g could be used as a novel immunostimulant to offer immediate protection to channel catfish against A. hydrophila infection.

  1. Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors

    Directory of Open Access Journals (Sweden)

    Lough John W

    2010-08-01

    Full Text Available Abstract Background The use of lentiviruses to reprogram human somatic cells into induced pluripotent stem (iPS cells could limit their therapeutic usefulness due to the integration of viral DNA sequences into the genome of the recipient cell. Recent work has demonstrated that human iPS cells can be generated using episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, or recombinant proteins. While these approaches offer an advance, the protocols have some drawbacks. Commonly the procedures require either subcloning to identify human iPS cells that are free of exogenous DNA, a knowledge of virology and safe handling procedures, or a detailed understanding of protein biochemistry. Results Here we report a simple approach that facilitates the reprogramming of human somatic cells using standard techniques to transfect expression plasmids that encode OCT4, NANOG, SOX2, and LIN28 without the need for episomal stability or selection. The resulting human iPS cells are free of DNA integration, express pluripotent markers, and form teratomas in immunodeficient animals. These iPS cells were also able to undergo directed differentiation into hepatocyte-like and cardiac myocyte-like cells in culture. Conclusions Simple transient transfection of plasmid DNA encoding reprogramming factors is sufficient to generate human iPS cells from primary fibroblasts that are free of exogenous DNA integrations. This approach is highly accessible and could expand the use of iPS cells in the study of human disease and development.

  2. Self-Assembled Functional Nanostructure of Plasmid DNA with Ionic Liquid [Bmim][PF₆]: Enhanced Efficiency in Bacterial Gene Transformation.

    Science.gov (United States)

    Soni, Sarvesh K; Sarkar, Sampa; Mirzadeh, Nedaossadat; Selvakannan, P R; Bhargava, Suresh K

    2015-04-28

    The electrostatic interaction between the negatively charged phosphate groups of plasmid DNA and the cationic part of hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF6]), initiates spontaneous self-assembly to form the functional nanostructures made up of DNA and ionic liquid (IL). These functional nanostructures were demonstrated as promising synthetic nonviral vectors for the efficient bacterial pGFP gene transformation in cells. In particular, the functional nanostructures that were made up of 1 μL of IL ([Bmim][PF6]) and 1 μg of plasmid DNA can increase the transformation efficiency by 300-400% in microbial systems, without showing any toxicity for E. coli DH5α cells. (31)P nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron (XPS) spectroscopic analysis revealed that the electrostatic interaction between negatively charged phosphate oxygen and cationic Bmim(+) tends to initiate the self-assembly process. Thermogravimetric analysis of the DNA-IL functional nanostructures showed that these nanostructures consist of ∼16 wt % ionic liquid, which is considered to provide the stability to the plasmid DNA that eventually enhanced the transformation efficiency.

  3. Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods.

    Science.gov (United States)

    Taverniers, Isabel; Van Bockstaele, Erik; De Loose, Marc

    2004-03-01

    Analytical real-time PCR technology is a powerful tool for implementation of the GMO labeling regulations enforced in the EU. The quality of analytical measurement data obtained by quantitative real-time PCR depends on the correct use of calibrator and reference materials (RMs). For GMO methods of analysis, the choice of appropriate RMs is currently under debate. So far, genomic DNA solutions from certified reference materials (CRMs) are most often used as calibrators for GMO quantification by means of real-time PCR. However, due to some intrinsic features of these CRMs, errors may be expected in the estimations of DNA sequence quantities. In this paper, two new real-time PCR methods are presented for Roundup Ready soybean, in which two types of plasmid DNA fragments are used as calibrators. Single-target plasmids (STPs) diluted in a background of genomic DNA were used in the first method. Multiple-target plasmids (MTPs) containing both sequences in one molecule were used as calibrators for the second method. Both methods simultaneously detect a promoter 35S sequence as GMO-specific target and a lectin gene sequence as endogenous reference target in a duplex PCR. For the estimation of relative GMO percentages both "delta C(T)" and "standard curve" approaches are tested. Delta C(T) methods are based on direct comparison of measured C(T) values of both the GMO-specific target and the endogenous target. Standard curve methods measure absolute amounts of target copies or haploid genome equivalents. A duplex delta C(T) method with STP calibrators performed at least as well as a similar method with genomic DNA calibrators from commercial CRMs. Besides this, high quality results were obtained with a standard curve method using MTP calibrators. This paper demonstrates that plasmid DNA molecules containing either one or multiple target sequences form perfect alternative calibrators for GMO quantification and are especially suitable for duplex PCR reactions.

  4. Correction of the lack of commutability between plasmid DNA and genomic DNA for quantification of genetically modified organisms using pBSTopas as a model.

    Science.gov (United States)

    Zhang, Li; Wu, Yuhua; Wu, Gang; Cao, Yinglong; Lu, Changming

    2014-10-01

    Plasmid calibrators are increasingly applied for polymerase chain reaction (PCR) analysis of genetically modified organisms (GMOs). To evaluate the commutability between plasmid DNA (pDNA) and genomic DNA (gDNA) as calibrators, a plasmid molecule, pBSTopas, was constructed, harboring a Topas 19/2 event-specific sequence and a partial sequence of the rapeseed reference gene CruA. Assays of the pDNA showed similar limits of detection (five copies for Topas 19/2 and CruA) and quantification (40 copies for Topas 19/2 and 20 for CruA) as those for the gDNA. Comparisons of plasmid and genomic standard curves indicated that the slopes, intercepts, and PCR efficiency for pBSTopas were significantly different from CRM Topas 19/2 gDNA for quantitative analysis of GMOs. Three correction methods were used to calibrate the quantitative analysis of control samples using pDNA as calibrators: model a, or coefficient value a (Cva); model b, or coefficient value b (Cvb); and the novel model c or coefficient formula (Cf). Cva and Cvb gave similar estimated values for the control samples, and the quantitative bias of the low concentration sample exceeded the acceptable range within ±25% in two of the four repeats. Using Cfs to normalize the Ct values of test samples, the estimated values were very close to the reference values (bias -13.27 to 13.05%). In the validation of control samples, model c was more appropriate than Cva or Cvb. The application of Cf allowed pBSTopas to substitute for Topas 19/2 gDNA as a calibrator to accurately quantify the GMO.

  5. Microbial DNA extraction from samples of varied origin

    National Research Council Canada - National Science Library

    S. Ray Chaudhuri; A. K. Pattanayak; A. R. Thakur

    2006-01-01

    The impact of four different soil DNA extraction methods on the quantity and quality of isolated community DNA was evaluated using agarose gel electrophoresis, DNA spectrum study and PCR-based 16S ribosomal DNA analysis...

  6. Coupling of importin beta binding peptide on plasmid DNA: transfection efficiency is increased by modification of lipoplex's physico-chemical properties

    Directory of Open Access Journals (Sweden)

    Escriou Virginie

    2003-09-01

    Full Text Available Abstract Background Non-viral vectors for gene transfer are less immunogenic than viral vectors but also less efficient. Significant effort has focused on enhancing non-viral gene transfer efficiency by increasing nuclear import of plasmid DNA, particularly by coupling nuclear localization peptidic sequences to plasmid DNA. Results We have coupled a 62-aminoacid peptide derived from hSRP1α importin beta binding domain, called the IBB peptide to plasmid DNA by using the heterobifunctional linker N-(4-azido-2,3,5,6 tetrafluorobenzyl-6-maleimidyl hexanamide (TFPAM-6. When covalently coupled to plasmid DNA, IBB peptide did not increase the efficiency of cationic lipid mediated transfection. The IBB peptide was still able to interact with its nuclear import receptor, importin β, but non-specifically. However, we observed a 20-fold increase in reporter gene expression with plasmid DNA / IBB peptide complexes under conditions of inefficient transfection. In which case, IBB was associated with plasmid DNA through self assembling ionic interaction. Conclusions The improvement of transfection activity was not due to an improved nuclear import of DNA, but rather by the modification of physicochemical properties of IBB peptide / plasmid complexes. IBB peptide increased lipoplex size and these larger complexes were more efficient for gene transfer.

  7. Toward the identification of a type I toxin-antitoxin system in the plasmid DNA of dairy Lactobacillus rhamnosus.

    Science.gov (United States)

    Folli, Claudia; Levante, Alessia; Percudani, Riccardo; Amidani, Davide; Bottazzi, Stefania; Ferrari, Alberto; Rivetti, Claudio; Neviani, Erasmo; Lazzi, Camilla

    2017-09-21

    Plasmids carry genes that give bacteria beneficial traits and allow them to survive in competitive environments. In many cases, they also harbor toxin-antitoxin (TA) systems necessary for plasmid maintenance. TA systems are generally characterized by a stable "toxin", a protein or peptide capable of killing the cell upon plasmid loss and by an unstable "antitoxin", a protein or a non-coding RNA that inhibits toxin activity. Here we report data toward the identification of a RNA-regulated TA system in the plasmid DNA of L. rhamnosus isolated from cheese. The proposed TA system comprises two convergently transcribed RNAs: a toxin RNA encoding a 29 amino acid peptide named Lpt and an antitoxin non-coding RNA. Both toxin and antitoxin RNAs resulted upregulated under conditions mimicking cheese ripening. The toxicity of the Lpt peptide was demonstrated in E. coli by cloning the Lpt ORF under the control of an inducible promoter. Bioinformatics screening of the bacterial nucleotide database, shows that regions homologous to the Lpt TA locus are widely distributed in the Lactobacillus genus, particularly within the L. casei group, suggesting a relevant role of TA systems in plasmid maintenance of cheese microbiota.

  8. Size effect on transfection and cytotoxicity of nanoscale plasmid DNA/polyethyleneimine complexes for aerosol gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Hoon Byeon, Jeong, E-mail: jbyeon@purdue.edu [Department of Chemistry, Purdue University, West Lafayette, Indiana 47907 (United States); Kim, Jang-Woo, E-mail: jwkim@hoseo.edu [Department of Digital Display Engineering, Hoseo University, Asan 336-795 (Korea, Republic of)

    2014-02-03

    Nanoscale plasmid DNA (pDNA)/polyethyleneimine (PEI) complexes were fabricated in the aerosol state using a nebulization system consisting of a collison atomizer and a cool-walled diffusion dryer. The aerosol fabricated nanoscale complexes were collected and employed to determine fundamental properties of the complexes, such as size, structure, surface charge, and in vitro gene transfection efficiency and cytotoxicity. The results showed that mass ratio between pDNA and PEI should be optimized to enhance gene transfection efficiency without a significant loss of cell viability. These findings may support practical advancements in the field of nonviral gene delivery.

  9. Integral parametrization of the Kinetics of Crosslink production in plasmid DNA as a function of 8-methoxypsoralen concentration

    Energy Technology Data Exchange (ETDEWEB)

    Vidania, R. de; Paramio, J. M.; Bauluz, C.

    1986-07-01

    In this paper we present results of crosslink production in pBR322 DNA along a wide range of 8-methoxypsoralen (8-MOP) concentration. Experimental data were obtained as DNA renaturation percentages, from the shift in hyperchromicity after a temperature-dependent denaturation-renaturation process. the experimental results showed a three-stage profile when represented as a function of the natural logarithms of 8-MOP concentration. an integral parametrization which allows a simultaneous fit of the three observed stages is presented here. the theoretical values of crosslink production determined from the fit are useful to asses the genotoxicity of psoralen-induced crosslinks in plasmid DNA. (Author) 24 refs.

  10. Optimization of supercoiled HPV-16 E6/E7 plasmid DNA purification with arginine monolith using design of experiments.

    Science.gov (United States)

    Almeida, A M; Queiroz, J A; Sousa, F; Sousa, A

    2015-01-26

    The progress of DNA vaccines is dependent on the development of suitable chromatographic procedures to successfully purify genetic vectors, such as plasmid DNA. Human Papillomavirus is associated with the development of tumours due to the oncogenic power of E6 and E7 proteins, produced by this virus. The supercoiled HPV-16 E6/E7 plasmid-based vaccine was recently purified with the arginine monolith, with 100% of purity, but only 39% of recovery was achieved. Therefore, the present study describes the application of experimental design tools, a newly explored methodology in preparative chromatography, in order to improve the supercoiled plasmid DNA recovery with the arginine monolith, maintaining the high purity degree. In addition, the importance and influence of pH in the pDNA retention to the arginine ligand was also demonstrated. The Composite Central Face design was validated and the recovery of the target molecule was successfully improved from 39% to 83.5%, with an outstanding increase of more than double, while maintaining 100% of purity.

  11. PROTECTION AGAINST LEPTOSPIROSIS BY IMMUNIZATION WITH PLASMID DNA ENCODING 33 kDa ENDOFLAGELLIN OF L.INTERROGANS SEROVAR LAI

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective. To evaluate how the efficacy of DNA inocutation affects the ability to raise protective immunity against Leptospira.Methods. A pair of oligonucleotide primers were designed to amplify the endoflagellar gene of L. interrogans sensu stricto serovar lai. An approximately 840bp fragment was generated with PCR and inserted into VR1012, a plasmid DNA expression vector, after the fragment and VR1012 were digested respectively with EcoRV and Sal I. A recombinant plasmid designated as VR1012+flaB2 was obtained. The vector, VR1012 consits of a pUC18 backbone with the cytomegalovirus(CMV) IE1 enhancer, promoter, and intron A, transcription regulatory elements and the BGH polyadenylation sequences driving the expressing of leptospiral endoflagellar gene of L. interrogans sensu stricto serovar lai. Plasmid encoding leptospiral endoflagellin gene was injected into quadriceps of NZW rabbits.Results.This resulted in the generation of specific leptospiral antibody with high ELISA titer (1:32768) in the rabbits. Immuno/protection was performed in guinea pigs without adjuvant. The group"VR1012+flaB2" showed higher survival rate(90%,9/10 animals),compared with the group "VR1012 lack flaB2" and the group "normal saline".Conclusion.The technique of DNA vaccine has potential advantages over certain other vaccine preparation technologies. However whether DNA vaccine will be useful for vaccine development remains to be tested.

  12. Extraction of Chromosomal DNA from Schizosaccharomyces pombe.

    Science.gov (United States)

    Murray, Johanne M; Watson, Adam T; Carr, Antony M

    2016-05-02

    Extraction of DNA from Schizosaccharomyces pombe cells is required for various uses, including templating polymerase chain reactions (PCRs), Southern blotting, library construction, and high-throughput sequencing. To purify high-quality DNA, the cell wall is removed by digestion with Zymolyase or Lyticase and the resulting spheroplasts lysed using sodium dodecyl sulfate (SDS). Cell debris, SDS, and SDS-protein complexes are subsequently precipitated by the addition of potassium acetate and removed by centrifugation. Finally, DNA is precipitated using isopropanol. At this stage, purity is usually sufficient for PCR. However, for more sensitive procedures, such as restriction enzyme digestion, additional purification steps, including proteinase K digestion and phenol-chloroform extraction, are recommended. All of these steps are described in detail here.

  13. Amplification of plasmid DNA bound on soil colloidal particles and clay minerals by the polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Polymerase chain reaction (PCR) was used to amplify a 600-base pair (bp) sequence of plasmid pGEX-2T DNA bound on soil colloidal particles from Brown soil (Alfisol) and Red soil (Ultisol), and three different minerals (goethite, kaolinite, montmorillonite). DNA bound on soil colloids, kaolinite, and montmorillonite was not amplified when the complexes were used directly but amplification occurred when the soil colloid or kaolinite-DNA complex was diluted, 10- and 20-fold. The montmorillonite-DNA complex required at least 100-fold dilution before amplification could be detected. DNA bound on goethite was amplified irrespective of whether the complex was used directly, or diluted 10- and 20-fold. The amplification of mineral-bound plasmid DNA by PCR is, therefore, markedly influenced by the type and concentration of minerals used. This information is of fundamental importance to soil molecular microbial ecology with particular reference to monitoring the fate of genetically engineered microorganisms and their recombinant DNA in soil environments.

  14. Breakthrough performance of plasmid DNA on ion-exchange membrane columns.

    Science.gov (United States)

    Montesinos-Cisneros, Rosa Ma; Olivas, Jonathan de la Vega; Ortega, Jaime; Guzmán, Roberto; Tejeda-Mansir, Armando

    2007-01-01

    Breakthrough performance of plasmid DNA adsorption on ion-exchange membrane columns was theoretically and experimentally investigated using batch and fixed-bed systems. System dispersion curves showed the absence of flow non-idealities in the experimental arrangement. Breakthrough curves (BTC) were significantly affected by inlet flow rate and solute concentration. In the theoretical analysis, a model was integrated by the serial coupling of the membrane transport model and the system dispersion model. A transport model that considers finite kinetic rate and column dispersed flow was used in the study. A simplex optimization routine, coupled to the solution of the partial differential model equations, was employed to estimate the maximum adsorption capacity constant, the equilibrium desorption constant, and the forward interaction rate constant, which are the parameters of the membrane transport model. The analysis shows that as inlet concentration or flow rate increases, the deviation of the model from the experimental behavior decreases. The BTCs displacement as inlet concentration increases was explained in terms of a greater degree of column saturation reached and more efficient operation accomplished. The degree of column saturation was not influenced by inlet flow rate. It was necessary to consider in the column model the slight variation in the BTC produced by the axial dispersion, in order to accomplish the experimental curve dispersion. Consequently, the design criteria that for Pe > 40 the column axial dispersion can be neglected should be taken with precaution.

  15. Evaluation of Maltose-Based Cationic Liposomes with Different Hydrophobic Tails for Plasmid DNA Delivery

    Directory of Open Access Journals (Sweden)

    Bo Li

    2017-03-01

    Full Text Available In this paper, three cationic glycolipids with different hydrophobic chains Malt-DiC12MA (IX a, Malt-DiC14MA (IX b and Malt-DiC16MA (IX c were constructed by using maltose as starting material via peracetylation, selective 1-O-deacetylation, trichloroacetimidation, glycosylation, azidation, deacetylation, Staudinger reaction, tertiary amination and quaternization. Target compounds and some intermediates were characterized by 1H-NMR, 13C-NMR, 1H-1H COSY and 1H-13C HSQC. The results of gel electrophoresis assay, atomic force microscopy images (AFM and dynamic light scattering (DLS demonstrate that all the liposomes could efficiently bind and compact DNA (N/P ratio less than 2 into nanoparticles with proper size (88 nm–146 nm, PDI < 0.4 and zeta potential (+15 mV–+26 mV. The transfection efficiency and cellular uptake of glycolipids in HEK293 cell were evaluated through the enhanced green fluorescent protein (EGFP expression and Cy3-labeled pEGFP-C1 (Enhanced Green Fluorescent Protein plasmid images, respectively. Importantly, it indicated that Malt-DiC14MA exhibited high gene transfer efficiency and better uptake capability at N/P ratios of 8:1. Additionally, the result of cell viability showed glycolipids exhibited low biotoxicity and good biocompatibility by thiazolyl blue tetrazolium bromide (MTT assay.

  16. A seven-year storage report of good manufacturing practice-grade naked plasmid DNA: stability, topology, and in vitro/in vivo functional analysis.

    Science.gov (United States)

    Walther, Wolfgang; Schmeer, Marco; Kobelt, Dennis; Baier, Ruth; Harder, Alexander; Walhorn, Volker; Anselmetti, Dario; Aumann, Jutta; Fichtner, Iduna; Schleef, Martin

    2013-12-01

    The great interest for naked plasmid DNA in gene therapy studies is reflected by the fact that it is currently used in 18% of all gene therapy trials. Therefore, validation of topology and functionality of DNA resulting from its long-term stability is an essential requirement for safe and effective gene transfer. To this aim, we analyzed the stability of good manufacturing practice-grade pCMVβ reporter plasmid DNA by capillary gel electrophoresis, agarose gel electrophoresis, and atomic force microscopy. The plasmid DNA was produced for a clinical gene transfer study started in 2005 and was stored for meanwhile 7 years under continuously monitored conditions at -20 °C. The stability of plasmid DNA was monitored by LacZ transgene expression functional assays performed in vitro and in vivo on the 7-year-old plasmid DNA samples compared with plasmid batches newly produced in similar experimental conditions and quality standards. The analyses revealed that during the overall storage time and conditions, the proportion of open circular and supercoiled or covalently closed circular forms is conserved without linearization or degradation of the plasmid. The in vitro transfection and the in vivo jet-injection of DNA showed unaltered functionality of the long-stored plasmid. In summary, the 7-year-old and the newly produced plasmid samples showed similar topology and expression performance. Therefore, our stable storage conditions are effective to preserve the integrity of the DNA to be used in clinical studies. This is an important prerequisite for the long-term performance of gene transfer materials used in trials of long duration as well as of the reference material used in standardization procedures and assays.

  17. OPTIMIZATION OF ELECTROPORATION PARAMETERS FOR TRANSFECTION OF PLASMID DNA INTO MURINE BONE MARROW-DERIVED DENDRITIC CELL

    Institute of Scientific and Technical Information of China (English)

    KE Shan; CHEN Xue-hua; LI Hao; LI Jian-fang; GU Qin-long; ZHU Zheng-gang; LIU Bing-ya

    2006-01-01

    Objective To explore the optimal electroporation parameters for transfection of plasmid DNA into murine bone marrow-derived dendritic cells. Methods Murine bone marrow-derived dendritic cells (DCs) were electroporated with plasmid DNA in varied conditions, such as electrical voltage, pulse time,pre-electroporation cell condition and serum concentration in electrical buffer. Inverted fluorescence microscope and flow cytometer were used to determine the transfection efficiency. Some of the DCs genetically modified under different conditions were stained with trypan-blue and its viability was observed microscopically 48h after electroporation. Results Highest transfection efficiency (22.10%) could be reached when electrical voltage was 250V and pulse time was 20ms. Refreshing the culture medium pre-electroporation may help the cells recover more easily from gene transfer.Besides, electrical buffer containing serum may benefit the viability of DC after electroporation and temperature may has little influence on transfection efficiency. Conclusion Our observations demonstrated plasmid DNA could be efficiently transferred into murine bone marrow-derived DCs by electroporation. These data may helpful for cancer research related to murine DC transfection.

  18. Thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR.

    Science.gov (United States)

    Le, Yilin; Chen, Huayou; Zagursky, Robert; Wu, J H David; Shao, Weilan

    2013-08-01

    Polymerase chain reaction (PCR) is a powerful method to produce linear DNA fragments. Here we describe the Tma thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR. In this thermostable DNA ligase-mediated whole-plasmid amplification method, the resultant DNA nick between the 5' end of the PCR primer and the extended newly synthesized DNA 3' end of each PCR cycle is ligated by Tma DNA ligase, resulting in circular plasmid DNA product that can be directly transformed. The template plasmid DNA is eliminated by 'selection marker swapping' upon transformation. When performed under an error-prone condition with Taq DNA polymerase, PPCP allows one-step construction of mutagenesis libraries based on in situ error-prone PCR so that random mutations are introduced into the target gene without altering the expression vector plasmid. A significant difference between PPCP and previously published methods is that PPCP allows exponential amplification of circular DNA. We used this method to create random mutagenesis libraries of a xylanase gene and two cellulase genes. Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PPCP for creating random mutagenesis libraries for directed evolution.

  19. Supercoiled plasmid DNA as a model target for assessing the generation of free radicals at the surface of fibres.

    Science.gov (United States)

    Donaldson, K; Gilmour, P S; Beswick, P H

    1995-09-01

    The ability of respirable amosite and crocidolite asbestos, refractory ceramic fibres (RCFs) and man made vitreous fibres (MMVFs) to cause free radical injury to plasmid, phiX174 RFI DNA was assessed. The amosite and crocidolite asbestos caused substantial damage to the DNA and, in the main, the free radicals responsible for the asbestos-mediated DNA damage were hydroxyl radicals as determined by inhibition with mannitol. Asbestos fibre-mediated damage to the DNA was completely ameliorated by the chelation of fibre-associated iron by pre-treatment of fibres with desferrioxamine-B, confirming the importance of iron in the production of free radicals. MMVFs and RCFs produced modest free radical damage to the DNA, which was prevented by mannitol but not by iron chelation.

  20. Antioxidant, DNA protective efficacy and HPLC analysis of Annona muricata (soursop) extracts.

    Science.gov (United States)

    George, V Cijo; Kumar, D R Naveen; Suresh, P K; Kumar, R Ashok

    2015-04-01

    Annona muricata is a naturally occurring edible plant with wide array of therapeutic potentials. In India, it has a long history of traditional use in treating various ailments. The present investigation was carried out to characterize the phytochemicals present in the methanolic and aqueous leaf extracts of A. muricata, followed by validation of its radical scavenging and DNA protection activities. The extracts were also analyzed for its total phenolic contents and subjected to HPLC analysis to determine its active metabolites. The radical scavenging activities were premeditated by various complementary assays (DRSA, FRAP and HRSA). Further, its DNA protection efficacy against H2O2 induced toxicity was evaluated using pBR322 plasmid DNA. The results revealed that the extracts were highly rich in various phytochemicals including luteolin, homoorientin, tangeretin, quercetin, daidzein, epicatechin gallate, emodin and coumaric acid. Both the extracts showed significant (p < 0.05) radical scavenging activities, while methanolic extract demonstrated improved protection against H2O2-induced DNA damage when compared to aqueous extract. A strong positive correlation was observed for the estimated total phenolic contents and radical scavenging potentials of the extracts. Further HPLC analysis of the phyto-constituents of the extracts provides a sound scientific basis for compound isolation.

  1. Chemotherapy of Bacterial Plasmids

    Science.gov (United States)

    1979-01-29

    render them non-susceptible to K: z plasmid-encoded enzymes. (3) Development of drugs which are selective inhibitor! 1 4, of plasmid DNA replication. (4... Development of drugs which inhibit phenotypic as expression of plasmid genes, and (5) Development of drugs which are inhibitors o, drug-inactivating...Barnes [2] them non-susceptible to plasmid-encoded enzymes, tabulated data on the incidence of Gram-negative 3) development of drugs which are

  2. Extraction of ultrashort DNA molecules from herbarium specimens.

    Science.gov (United States)

    Gutaker, Rafal M; Reiter, Ella; Furtwängler, Anja; Schuenemann, Verena J; Burbano, Hernán A

    2017-02-01

    DNA extracted from herbarium specimens is highly fragmented; therefore, it is crucial to use extraction protocols that retrieve short DNA molecules. Improvements in extraction and DNA library preparation protocols for animal remains have allowed efficient retrieval of molecules shorter than 50 bp. Here, we applied these improvements to DNA extraction protocols for herbarium specimens and evaluated extraction performance by shotgun sequencing, which allows an accurate estimation of the distribution of DNA fragment lengths. Extraction with N-phenacylthiazolium bromide (PTB) buffer decreased median fragment length by 35% when compared with cetyl-trimethyl ammonium bromide (CTAB); modifying the binding conditions of DNA to silica allowed for an additional decrease of 10%. We did not observe a further decrease in length for single-stranded DNA (ssDNA) versus double-stranded DNA (dsDNA) library preparation methods. Our protocol enables the retrieval of ultrashort molecules from herbarium specimens, which will help to unlock the genetic information stored in herbaria.

  3. Methods for microbial DNA extraction from soil for PCR amplification

    Directory of Open Access Journals (Sweden)

    Yeates C

    1998-01-01

    Full Text Available Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1. DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size.

  4. Methods for microbial DNA extraction from soil for PCR amplification

    OpenAIRE

    Yeates C; Gillings, MR; Davison AD; Altavilla N; Veal DA

    1998-01-01

    Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1). DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol pr...

  5. Use of sperm plasmid DNA lipofection combined with REMI (restriction enzyme-mediated insertion) for production of transgenic chickens expressing eGFP (enhanced green fluorescent protein) or human follicle-stimulating hormone.

    Science.gov (United States)

    Harel-Markowitz, Eliane; Gurevich, Michael; Shore, Laurence S; Katz, Adi; Stram, Yehuda; Shemesh, Mordechai

    2009-05-01

    Linearized p-eGFP (plasmid-enhanced green fluorescent protein) or p-hFSH (plasmid human FSH) sequences with the corresponding restriction enzyme were lipofected into sperm genomic DNA. Sperm transfected with p-eGFP were used for artificial insemination in hens, and in 17 out of 19 of the resultant chicks, the exogenous DNA was detected in their lymphocytes as determined by PCR and expressed in tissues as determined by (a) PCR, (b) specific emission of green fluorescence by the eGFP, and (c) Southern blot analysis. A complete homology was found between the Aequorea Victoria eGFP DNA and a 313-bp PCR product of extracted DNA from chick blood cells. Following insemination with sperm lipofected with p-hFSH, transgenic offspring were obtained for two generations as determined by detection of the transgene for human FSH (PCR) and expression of the gene (RT-PCR and quantitative real-time PCR) and the presence of the protein in blood (radioimmunoassay). Data demonstrate that lipofection of plasmid DNA with restriction enzyme is a highly efficient method for the production of transfected sperm to produce transgenic offspring by direct artificial insemination.

  6. Characterisation of LMD virus-like nanoparticles self-assembled from cationic liposomes, adenovirus core peptide mu and plasmid DNA.

    Science.gov (United States)

    Tagawa, T; Manvell, M; Brown, N; Keller, M; Perouzel, E; Murray, K D; Harbottle, R P; Tecle, M; Booy, F; Brahimi-Horn, M C; Coutelle, C; Lemoine, N R; Alton, E W F W; Miller, A D

    2002-05-01

    Liposome:mu:DNA (LMD) is a ternary nucleic acid delivery system built around the mu peptide associated with the condensed core complex of the adenovirus. LMD is prepared by precondensing plasmid DNA (D) with mu peptide (M) in a 1:0.6 (w/w) ratio and then combining these mu:DNA (MD) complexes with extruded cationic liposomes (L) resulting in a final lipid:mu:DNA ratio of 12:0.6:1 (w/w/w). Correct buffer conditions, reagent concentrations and rates of mixing are all crucial to success. However, once optimal conditions are established, homogeneous LMD particles (120 +/- 30 nm) will result that each appear to comprise an MD particle encapsulated within a cationic bilammellar liposome. LMD particles can be formulated reproducibly, they are amenable to long-term storage (>1 month) at -80 degrees C and are stable to aggregation at a plasmid DNA concentration up to 5 mg/ml (15 mM nucleotide concentration). Furthermore, LMD transfections are significantly more time and dose efficient in vitro than cationic liposome-plasmid DNA (LD) transfections. Transfection times as short as 10 min and plasmid DNA doses as low as 0.001 microg/well result in significant gene expression. LMD transfections will also take place in the presence of biological fluids (eg up to 100% serum) giving 15-25% the level of gene expression observed in the absence of serum. Results from confocal microscopy experiments using fluorescent-labelled LMD particles suggest that endocytosis is not a significant barrier to LMD transfection, although the nuclear membrane still is. We also confirm that topical lung transfection in vivo by LMD is at least equal in absolute terms with transfection mediated by GL-67:DOPE:DMPE-PEG(5000) (1:2:0.05 m/m/m), an accepted 'gold-standard' non-viral vector system for topical lung transfection, and is in fact at least six-fold more dose efficient. All these features make LMD an important new non-viral vector platform system from which to derive tailor-made non-viral delivery

  7. A Simplified Rice DNA Extraction Protocol for PCR Analysis

    Institute of Scientific and Technical Information of China (English)

    CHEN Wen-yue; CUI Hai-rui; BAO Jin-song; ZHOU Xiang-sheng; SHU Qing-yao

    2006-01-01

    A simple protocol was established for DNA extraction using etiolated rice seedlings, whereby rice DNA was directly extracted ir 0.5 mol/L NaOH solution in a single eppendorf tube. Results of comparative PCR analyses and electrophoresis showed that the DNA extracted using this method was as good and useful as that using standard CTAB method.

  8. Arginine-based cationic liposomes for efficient in vitro plasmid DNA delivery with low cytotoxicity

    Directory of Open Access Journals (Sweden)

    Sarker SR

    2013-04-01

    Full Text Available Satya Ranjan Sarker, Yumiko Aoshima, Ryosuke Hokama, Takafumi Inoue, Keitaro Sou, Shinji Takeoka Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University (TWIns, Tokyo, Japan Background: Currently available gene delivery vehicles have many limitations such as low gene delivery efficiency and high cytotoxicity. To overcome these drawbacks, we designed and synthesized two cationic lipids comprised of n-tetradecyl alcohol as the hydrophobic moiety, 3-hydrocarbon chain as the spacer, and different counterions (eg, hydrogen chloride [HCl] salt or trifluoroacetic acid [TFA] salt in the arginine head group. Methods: Cationic lipids were hydrated in 4-(2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES buffer to prepare cationic liposomes and characterized in terms of their size, zeta potential, phase transition temperature, and morphology. Lipoplexes were then prepared and characterized in terms of their size and zeta potential in the absence or presence of serum. The morphology of the lipoplexes was determined using transmission electron microscopy and atomic force microscopy. The gene delivery efficiency was evaluated in neuronal cells and HeLa cells and compared with that of lysine-based cationic assemblies and Lipofectamine™ 2000. The cytotoxicity level of the cationic lipids was investigated and compared with that of Lipofectamine™ 2000. Results: We synthesized arginine-based cationic lipids having different counterions (ie, HCl-salt or TFA-salt that formed cationic liposomes of around 100 nm in size. In the absence of serum, lipoplexes prepared from the arginine-based cationic liposomes and plasmid (p DNA formed large aggregates and attained a positive zeta potential. However, in the presence of serum, the lipoplexes were smaller in size and negative in zeta potential. The morphology of the lipoplexes was vesicular. Arginine-based cationic liposomes with HCl-salt showed the

  9. Molecular characterization of Syrian date palm cultivars using plasmid-like DNA markers.

    Science.gov (United States)

    Haider, N; Nabulsi, I

    2012-02-01

    Date palm (Phoenix dactylifera L.) is one of the most important domesticated fruit trees in the Near East and North African countries. This tree has been, for several decades, in serious threat of being completely destroyed by the "Bayoud" disease caused by Fusarium oxysporum f. sp. albedinis. In this study, 18 Syrian date palm cultivars and four male trees were analyzed according to the identity of mitochondrial plasmid-like DNAs. A PCR strategy that employs plasmid-like DNAs-specific primer pair was used. These primers amplify a product of either 373-bp or 265-bp that corresponds to the S-(Bayoud-susceptible) or the R-plasmid (Bayoud-resistant), respectively. Generated data revealed that only six cultivars ('Medjool', 'Ashrasi', 'Gish Rabi', 'Khineze', and yellow- and red-'Kabkab') have the S-plasmid, suggesting their susceptibility to the fusariosis, while the remaining 12 cultivars and the four male trees contain the R-plasmid, suggesting their resistance to the fusariosis. The PCR process applied here has been proved efficient for the rapid screening for the presence of the S and R DNAs in Syrian date palm. PCR markers developed in this study could be useful for the screening of date palm lines growing in the field. The availability of such diagnostic tool for plasmid characterization in date palm would also be of great importance in establishing propagation and breeding programs of date palm in Syria.

  10. Recombination frequency in plasmid DNA containing direct repeats--predictive correlation with repeat and intervening sequence length.

    Science.gov (United States)

    Oliveira, Pedro H; Lemos, Francisco; Monteiro, Gabriel A; Prazeres, Duarte M F

    2008-09-01

    In this study, a simple non-linear mathematical function is proposed to accurately predict recombination frequencies in bacterial plasmid DNA harbouring directly repeated sequences. The mathematical function, which was developed on the basis of published data on deletion-formation in multicopy plasmids containing direct-repeats (14-856 bp) and intervening sequences (0-3872 bp), also accounts for the strain genotype in terms of its recA function. A bootstrap resampling technique was used to estimate confidence intervals for the correlation parameters. More than 92% of the predicted values were found to be within a pre-established +/-5-fold interval of deviation from experimental data. The correlation does not only provide a way to predict, with good accuracy, the recombination frequency, but also opens the way to improve insight into these processes.

  11. Multiple Antibiotic Resistance Plasmids Allow Scalable, PCR-Mediated DNA Manipulation and Near-Zero Background Cloning

    Directory of Open Access Journals (Sweden)

    Remigiusz Arnak

    2016-01-01

    Full Text Available We have constructed two plasmids that can be used for cloning as templates for PCR-based gene disruption, mutagenesis and the construction of DNA chromosome translocation cassettes. To our knowledge, these plasmids are the first vectors that confer resistance to ampicillin, kanamycin and hygromycin B in bacteria, and to geneticin (G418 and hygromycin B in Saccharomyces cerevisiae simultaneously. The option of simultaneously using up to three resistance markers provides a highly stringent control of recombinant selection and the almost complete elimination of background resistance, while unique restriction sites allow easy cloning of chosen genetic material. Moreover, we successfully used these new vectors as PCR templates for the induction of chromosome translocation in budding yeast by the bridge-induced translocation system. Cells in which translocation was induced carried chromosomal rearrangements as expected and exhibited resistance to both, G418 and hygromycin B. These features make our constructs very handy tools for many molecular biology applications.

  12. Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping

    Science.gov (United States)

    Müller, Vilhelm; Rajer, Fredrika; Frykholm, Karolin; Nyberg, Lena K.; Quaderi, Saair; Fritzsche, Joachim; Kristiansson, Erik; Ambjörnsson, Tobias; Sandegren, Linus; Westerlund, Fredrik

    2016-12-01

    Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples.

  13. Investigation into the itegrons on plasmid DNA of Salmonella%沙门菌质粒DNA上整合子的分析

    Institute of Scientific and Technical Information of China (English)

    蔡俊源; 杨荣兴

    2009-01-01

    Objective To analyze the molecular feature of integrons on plasmid DNA in Salmonella. Methods The susceptibility of 32 Salmonella isolates to antibiotics were tested by Microscan WalkAway40 and the disk diffusion assay. Plasmid DNA was extracted. Class 1 integrons,intI2 and intI3 were detected by PCR. The purified amplicons of class 1 integrons were sequenced,and gene cassettes were identified. Results There 12 isolates carried class 1 integrons on plasmid DNA. The sizes of class 1 integrons were 1 000bp and 1 700bp respectively. 1 000bp integron carried gene cassette aadA1,1 700bp integron carried gene cassette dfr17-aadA5. There were no IntI2 and intI3. Conclusion Integrons are widely carried by Salmonella. Integrons are important molecular mechanism in mediation and dissemination of antibiotic resistance in Salmonella.%目的 分析沙门菌质粒DNA上整合子的分子特征.方法 用全自动细菌分析仪Microscan WalkAway40和纸片扩散法,对32株沙门菌进行抗生素敏感性测定,提取细菌质粒DNA,PCR扩增Ⅰ类整合子、Ⅱ类和Ⅲ类整合醇,对扩增产物进行序列分析.结果 12株细菌含有一个Ⅰ类整合子,整合子大小分别为1 000bp和1 700bp.1 000bp整合子含基因盒aadA1,1 700bp整合子含基因盒dfr17-aadA5,未检测到Ⅱ类和Ⅲ类整合酶.结论 整合子在沙门菌中广泛存在,整合子是介导和传播细菌耐药性的重要分子机制.

  14. Microbial diversity in fecal samples depends on DNA extraction method

    DEFF Research Database (Denmark)

    Mirsepasi, Hengameh; Persson, Søren; Struve, Carsten

    2014-01-01

    BACKGROUND: There are challenges, when extracting bacterial DNA from specimens for molecular diagnostics, since fecal samples also contain DNA from human cells and many different substances derived from food, cell residues and medication that can inhibit downstream PCR. The purpose of the study...... was to evaluate two different DNA extraction methods in order to choose the most efficient method for studying intestinal bacterial diversity using Denaturing Gradient Gel Electrophoresis (DGGE). FINDINGS: In this study, a semi-automatic DNA extraction system (easyMag®, BioMérieux, Marcy I'Etoile, France......) and a manual one (QIAamp DNA Stool Mini Kit, Qiagen, Hilden, Germany) were tested on stool samples collected from 3 patients with Inflammatory Bowel disease (IBD) and 5 healthy individuals. DNA extracts obtained by the QIAamp DNA Stool Mini Kit yield a higher amount of DNA compared to DNA extracts obtained...

  15. Nanospines incorporation into the structure of the hydrophobic cryogels via novel cryogelation method: an alternative sorbent for plasmid DNA purification.

    Science.gov (United States)

    Üzek, Recep; Uzun, Lokman; Şenel, Serap; Denizli, Adil

    2013-02-01

    In this study, it was aimed to prepare hydrophobic cryogels for plasmid DNA (pDNA) purification from Escherichia coli lysate. The hydrophobicity was achieved by incorporating a hydrophobic ligand, N-methacryloyl-(L)-phenylalanine (MAPA), into the cryogel backbone. In addition to the conventional cryogelation process, freeze-drying step was included to create nanospines. Three different cryogels {poly(2-hydoxyethyl methacrylate-N-methacryloyl-L-phenylalanine)-freeze dried, [P(HEMA-MAPA)-FD]; poly(2-hydoxyethyl methacrylate-N-methacryloyl-L-phenylalanine, [P(HEMA-MAPA)] and poly(2-hydoxyethyl methacrylate)-freeze dried, [P(HEMA)-FD]} were prepared, characterized, and used for DNA (salmon sperm DNA) adsorption studies from aqueous solution. The specific surface areas of cryogels were determined to be 21.4 m(2)/g for P(HEMA)-FD, 17.65 m(2)/g for P(HEMA-MAPA) and 36.0 m(2)/g for P(HEMA-MAPA)-FD. The parameters affecting adsorption such as temperature, initial DNA concentration, salt type and concentration were examined in continuous mode. The maximum adsorption capacities were observed as 45.31 mg DNA/g, 27.08 mg DNA/g and 1.81 mg DNA/g for P(HEMA-MAPA)-FD, P(HEMA-MAPA) and P(HEMA)-FD, respectively. Desorption process was performed using acetate buffer (pH 5.50) without salt. First, pDNA was isolated from E. coli lysate and the purity of pDNA was then determined by agarose gel electrophoresis. Finally, the chromatographic performance of P(HEMA-MAPA)-FD cryogel for pDNA purification was tested in FPLC. The resolution (R(s)) was 2.84, and the specific selectivity for pDNA was 237.5-folds greater than all impurities.

  16. A Histone-Like Protein Induces Plasmid DNA to Form Liquid Crystals in Vitro and Gene Compaction in Vivo

    Directory of Open Access Journals (Sweden)

    Shiyong Sun

    2013-12-01

    Full Text Available The liquid crystalline state is a universal phenomenon involving the formation of an ordered structure via a self-assembly process that has attracted attention from numerous scientists. In this study, the dinoflagellate histone-like protein HCcp3 is shown to induce super-coiled pUC18 plasmid DNA to enter a liquid crystalline state in vitro, and the role of HCcp3 in gene condensation in vivo is also presented. The plasmid DNA (pDNA-HCcp3 complex formed birefringent spherical particles with a semi-crystalline selected area electronic diffraction (SAED pattern. Circular dichroism (CD titrations of pDNA and HCcp3 were performed. Without HCcp3, pUC18 showed the characteristic B conformation. As the HCcp3 concentration increased, the 273 nm band sharply shifted to 282 nm. When the HCcp3 concentration became high, the base pair (bp/dimer ratio fell below 42/1, and the CD spectra of the pDNA-HCcp3 complexes became similar to that of dehydrated A-form DNA. Microscopy results showed that HCcp3 compacted the super-coiled gene into a condensed state and that inclusion bodies were formed. Our results indicated that HCcp3 has significant roles in gene condensation both in vitro and in histone-less eukaryotes in vivo. The present study indicates that HCcp3 has great potential for applications in non-viral gene delivery systems, where HCcp3 may compact genetic material to form liquid crystals.

  17. Photosensitization of plasmid-DNA loaded with platinum nano-particles and irradiated by low energy X-rays

    Science.gov (United States)

    Porcel, E.; Kobayashi, K.; Usami, N.; Remita, H.; Le Sech, C.; Lacombe, S.

    2011-01-01

    Damage in DNA plasmids (pBR322) loaded with platinum nanoparticles (NP-Pt) DNA-NP and irradiated with monochromatic X-rays tuned to the resonant photoabsorption energy of the LIII and MIII electronic inner-shell of platinum - respectively 11556 eV and 2649 eV - and off-resonant X-rays - 11536 eV and 2639 eV- is investigated. In all the experiments, an enhancement of the single and double strand break - SSB and DSB - yields is observed when NP-Pt are present. Amplification effects are almost similar for the irradiations performed at on and off the L or M shell resonance suggesting that a non resonant mechanism is responsible for the major part of the DNA breaks enhancement.The amount of DNA breaks measured in the present work is compared to the results in similar experiments made with complexes of plasmid-DNA containing platinum molecule : chloroterpyridine platinum (PtTC). The average number of PtTC molecules in the solution is the same as in the experiments made with NP-Pt in order to study a possible difference in the radiosensitization efficiency when the high-Z atoms are clustered (NP-Pt) or dispersed in the system (PtTC). A mechanism is suggested involving photoelectrons which can efficiently ionize the platinum atoms. These results are consistent with those observed when DNA-NP complexes are irradiated by fast atomic ions. These findings suggest that any nanoparticle made of high-Z atoms might behaves as radiation enhancer whatever the ionizing radiation is electromagnetic or charged particle source.

  18. Optimized microbial DNA extraction from diarrheic stools

    Directory of Open Access Journals (Sweden)

    Donatin Emilie

    2012-12-01

    Full Text Available Abstract Background The detection of enteropathogens in stool specimens increasingly relies on the detection of specific nucleic acid sequences. We observed that such detection was hampered in diarrheic stool specimens and we set-up an improved protocol combining lyophilization of stools prior to a semi-automated DNA extraction. Findings A total of 41 human diarrheic stool specimens comprising of 35 specimens negative for enteropathogens and six specimens positive for Salmonella enterica in culture, were prospectively studied. One 1-mL aliquot of each specimen was lyophilised and total DNA was extracted from lyophilised and non-lyophilised aliquots by combining automatic and phenol-chloroform DNA extraction. DNA was incorporated into real-time PCRs targeting the 16S rRNA gene of Bacteria and the archaea Methanobrevibacter smithii and the chorismate synthase gene of S. enterica. Whereas negative controls consisting in DNA-free water remained negative, M. smithii was detected in 26/41 (63.4% non-lyophilised (Ct value 28.78 ± 9.1 versus 39/41 (95.1% lyophilised aliquots (Ct value 22.04 ± 5.5; bacterial 16S rRNA was detected in 33/41 (80.5% non-lyophilised (Ct value 28.11 ± 5.9 versus 40/41 (97.6% lyophilised aliquots (Ct value 24.94 ± 6.6; and S. enterica was detected in 6/6 (100% non-lyophilized and lyophilized aliquots (Ct value 26.98 ± 4.55 and 26.16 ± 4.97, respectively. S. enterica was not detected in the 35 remaining diarrheal-stool specimens. The proportion of positive specimens was significantly higher after lyophilization for the detection of M. smithii (p = 0.00043 and Bacteria (p = 0.015. Conclusion Lyophilization of diarrheic stool specimens significantly increases the PCR-based detection of microorganisms. The semi-automated protocol described here could be routinely used for the molecular diagnosis of infectious diarrhea.

  19. Successive DNA extractions improve characterization of soil microbial communities

    Directory of Open Access Journals (Sweden)

    Mauricio R. Dimitrov

    2017-02-01

    Full Text Available Currently, characterization of soil microbial communities relies heavily on the use of molecular approaches. Independently of the approach used, soil DNA extraction is a crucial step, and success of downstream procedures will depend on how well DNA extraction was performed. Often, studies describing and comparing soil microbial communities are based on a single DNA extraction, which may not lead to a representative recovery of DNA from all organisms present in the soil. The use of successive DNA extractions might improve soil microbial characterization, but the benefit of this approach has only been limitedly studied. To determine whether successive DNA extractions of the same soil sample would lead to different observations in terms of microbial abundance and community composition, we performed three successive extractions, with two widely used commercial kits, on a range of clay and sandy soils. Successive extractions increased DNA yield considerably (1–374%, as well as total bacterial and fungal abundances in most of the soil samples. Analysis of the 16S and 18S ribosomal RNA genes using 454-pyrosequencing, revealed that microbial community composition (taxonomic groups observed in the successive DNA extractions were similar. However, successive DNA extractions did reveal several additional microbial groups. For some soil samples, shifts in microbial community composition were observed, mainly due to shifts in relative abundance of a number of microbial groups. Our results highlight that performing successive DNA extractions optimize DNA yield, and can lead to a better picture of overall community composition.

  20. The Inhibitory Effect of Extracts From Fructus Iycii and Rhizoma Polygonati on in vitro DNA B reakage by Alternariol

    Institute of Scientific and Technical Information of China (English)

    ZUDAO-SONG; KONGTIAN-QING; 等

    1996-01-01

    Alternariol caused DNA single-strand breakage.Conversion of the colsed circular double-stranded supercoiled DNA(pBR 322)to the nicked circular form and linear form was used to investigate the effect of extracts of some Chinese medical herbs on DNA nicking induced by alternariol.Some substances in the extracts of Rhizoma polygonati(RP) and Fructus lycii(FL)were shown to protect DNA from the attack by alternariol.Some substance in the RP may bind to plasmid NA,and this binding reduces the electophoretic mobility of DNA.These results indicate that substances from FL and RP may be used as DNA protectors.It is possible that they play an important role in preventing cancer.

  1. Purification of total DNA extracted from activated sludge

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis treatments (lysozyme, sodium dodecyl sulfate (SDS), sonication, mechanical mill and thermal shock) on yield and purity of the total DNA extracted from activated sludge were investigated. It was found that SDS and mechanical mill were the most effective ways for cell lysis, and both gave the highest DNA yields, while by SDS and thermal shock, the purest DNA extract could be obtained. The combination of SDS with other lysis treatment, such as sonication and thermal shock, could apparently increase the DNA yields but also result in severe shearing. For the purification of the crude DNA extract, polyvinyl polypyrrolidone was used for the removal of humic contaminants. Cetyltrimethyl ammonium bromide, potassium acetate and phenol/chloroform were used to remove proteins and polysaccharides from crude DNA. Crude DNA was further purified by isopropanol precipitation. Thus, a suitable protocol was proposed for DNA extraction, yielding about 49.9 mg (DNA)/g volatile suspended solids, and the DNA extracts were successfully used in PCR amplifications for 16S rDNA and 16S rDNA V3 region. The PCR products of 16S rDNA V3 region allowed the DGGE analysis (denatured gradient gel electrophoresis) to be possible.

  2. Effect of intramuscular injection of hepatocyte growth factor plasmid DNA with electroporation on bleomycin-induced lung fibrosis in rats

    Institute of Scientific and Technical Information of China (English)

    LONG Xiang; XIONG Sheng-dao; XIONG Wei-ning; XU Yong-jian

    2007-01-01

    Background So far, there is no efficient treatment for pulmonary fibrosis. The objective of this study was to determine whether intramuscular injection of the hepatocyte growth factor (HGF) plasmid DNA by in vivo electroporation could prevent bleomycin-induced pulmonary fibrosis in rats, and to investigate the possible mechanisms.Methods Twenty male Wistar rats were randomly divided into four groups: control group(group C), model group (group M), early intervention group (group Ⅰ ) and late intervention group (groupⅡ). Groups M, Ⅰ and Ⅱ were intratracheally infused with bleomycin, then injected the plasmid pcDNA3.1-hHGF to group Ⅰ on day 7, 14 and 21. Group Ⅱ received the same treatment like Group Ⅰ on day 14 and 21. All the rats were killed on day 28 after bleomycin injection. We detected Homo HGF expression in the rats with ELISA method and estimated the pathological fibrosis score of lung tissue using hematoxylin eosin (HE) and Massion staining. The mRNA expression of transforming growth factor-β1 (TGF-β1),cycloxygenase-2 (COX-2), and rat HGF in rat pulmonary parenchyma were evaluated by RT-PCR.Immunohistochemistry and Western blotting were performed to determine the protein expression of transforming TGF-β1 and COX-2 in lung parenchyma.Results The plasmid pcDNA3.1-hHGF could express hHGF in NIH3T3 cells and the hHGF protein is secreted into the culture medium. The expression of hHGF protein could be monitored in quadriceps muscle, plasma and lung in Groups Ⅰ and Ⅱ. Pulmonary fibrosis levels of Groups Ⅰ and Ⅱ were obviously lower than that of group M (P<0.05).Expression of TGF-β1 protein and mRNA in lung tissue was markedly decreased in Groups Ⅰ and Ⅱ compared with Group M (P<0.05). The level of expression of HGF and COX-2 mRNA was higher in Groups Ⅰ and Ⅱ than in Group M (P<0.05).Conclusions Injection of the plasmid pcDNA3.1-hHGF into skeletal muscle with electroporation has a potential role in the treatment of bleomycin

  3. Characterization of a targeted gene carrier, lactose-polyethylene glycol-grafted poly-L-lysine and its complex with plasmid DNA.

    Science.gov (United States)

    Choi, Y H; Liu, F; Choi, J S; Kim, S W; Park, J S

    1999-11-01

    The physicochemical properties and gene transfer ability of lactose-polyethylene glycol-grafted poly-L-lysine (Lac-PEG-PLL) were investigated. A dye displacement assay showed that plasmid DNA self-assembled with Lac-PEG-PLL, and condensation began at a <1:1 charge ratio of plasmid DNA to polymer. In atomic force microscopy, spontaneously assembled Lac-PEG-PLL/DNA complexes revealed a compact structure, with a size of about 100-200 nm. Circular dichroism spectra of Lac-PEG-PLL/DNA complexes revealed that the secondary structure of DNA was altered by complex formation and was similar to that of the poly-L-lysine/DNA complex. Lac-PEG-PLL was shown to protect DNA against nuclease action in a DNase I protection assay. The cytotoxicity test demonstrated that the complex composed of plasmid DNA and Lac-PEG-PLL had little influence on the viability of HepG2 cells, especially in comparison with that of poly-L-lysine/DNA complexes. In conclusion, our copolymer, Lac-PEG-PLI, formed complexes with plasmid DNA (on average, 150 nm), gave little cytotoxicity, and showed increased efficiency of gene transfer into hepatoma cells in vitro. Lactose-polyethylene glycol was grafted to poly-L-lysine to be used as a gene carrier for hepatoma cell targeting and to improve the solubility of the polyplexes. The average size of the carrier/DNA complexes was about 150 nm. The complexes also proved to have high resistance against nuclease attack and little cytotoxicity. The polymer also delivered plasmid DNA efficiently into a HepG2 cell line. Lac-PEG-PLL was more efficient than Lipofectin or galactose-PEG-PLL in transfection efficiency.

  4. STUDY REGARDING EFFICIENCY OF INDUCED GENETIC TRANSFORMATION IN BACILLUS LICHENIFORMIS WITH PLASMID DNA

    Directory of Open Access Journals (Sweden)

    VINTILĂ T.

    2007-01-01

    Full Text Available A strain of Bacillus licheniformis was subject to genetic transformation with plasmidvectors (pLC1 and pNC61, using electroporation technique, protoplasttransformation and bivalent cations (CaCl2 mediated transformation. In the case oftransformation by electroporation of Bacillus licheniformis B40, the highest numberof transformed colonies (3 were obtained only after a 1,79 KV electric shock, for 2,2milliseconds. Using this transformation technique we have obtained six kanamycinresistant transformants. The frequency of Bacillus licheniformis B40 protoplaststransformation using pLC1 and pNC61 plasmid vectors is approximately 10% (TF =10%. As a result of pLC1 plasmid integration in Bacillus licheniformis protoplasts,six kanamycin resistant transformants were obtained. The pNC61 plasmid, whichconfers trimethoprim resistance, does not integrate in receiver cells by protoplasttransformation. The direct genetic transformation in the presence of bivalent cations(CaCl2, mediated by pLC1 and pNC61 plasmid vectors, produce a lowtransformation frequency. Using this technique, we have obtained three trimethoprimresistant colonies and four kanamycin resistant colonies. The chemical way oftransformation is the only technique, which realizes the integration of pNC61 in B.licheniformis B40 cells.

  5. TOL Plasmid Carriage Enhances Biofilm Formation and Increases Extracellular DNA Content in Pseudomonas Putida KT2440

    DEFF Research Database (Denmark)

    Smets, Barth F.; D'Alvise, Paul; Yankelovich, T.

    Adherent growth of Pseudomonas putida KT2440 with and without the TOL plasmid (pWWO) at the solid-liquid and air-liquid interface was examined. We compared biofilm formation on glass in flow cells, and assayed pellicle (air-liquid interface biofilm) formation in stagnant liquid cultures by confoc...

  6. Food Fish Identification from DNA Extraction through Sequence Analysis

    Science.gov (United States)

    Hallen-Adams, Heather E.

    2015-01-01

    This experiment exposed 3rd and 4th y undergraduates and graduate students taking a course in advanced food analysis to DNA extraction, polymerase chain reaction (PCR), and DNA sequence analysis. Students provided their own fish sample, purchased from local grocery stores, and the class as a whole extracted DNA, which was then subjected to PCR,…

  7. Food Fish Identification from DNA Extraction through Sequence Analysis

    Science.gov (United States)

    Hallen-Adams, Heather E.

    2015-01-01

    This experiment exposed 3rd and 4th y undergraduates and graduate students taking a course in advanced food analysis to DNA extraction, polymerase chain reaction (PCR), and DNA sequence analysis. Students provided their own fish sample, purchased from local grocery stores, and the class as a whole extracted DNA, which was then subjected to PCR,…

  8. Genomic DNA extraction protocols from ovine hair

    Directory of Open Access Journals (Sweden)

    Jennifer Nonato da Silva Prate

    2013-12-01

    Full Text Available Genomic DNA extracted from animal cells can be used for several purposes, for example, to know genetic variability and genetic relationships between individuals, breeds and/or species, paternity tests, to describe the genetic profile for registration of the animal at association of breeders, detect genetic polymorphisms (SNP related to characteristics of commercial interest, disease diagnose, assess resistance or susceptibility to pathogens, etc. For such evaluations, in general, DNA is amplified by PCR (polymerase chain reaction, and then subjected to various techniques as RFLP (restriction fragments length polymorphism, SSCP (single strand conformation polymorphism, and sequencing. The DNA may be obtained from blood, buccal swabs, meat, cartilage or hair bulb. Among all, the last biological material has been preferred by farmers for its ease acquisition. Several methods for extracting DNA from hair bulb were reported without any consensus for its implementation. This study aimed to optimize a protocol for efficient DNA extraction for use in PCR-RFLP analysis of the Prion gene. For this study, were collected hair samples containing hair bulb from 131 Santa Inês sheep belonging to the Institute of Zootechny, Nova Odessa - SP. Two DNA extraction protocols were evaluated. The first, called phenol-chloroform-isoamyl alcohol (PCIA has long been used by Animal Genetic Laboratories, whose procedures are described below: in each microtube (1.5 mL containing 500 µL of TE-Tween solution (Tris-HCl 50 mM, EDTA 1 mM and 0.5% Tween 20 were added to approximately 30 hair bulb per animal which was incubated at 65°C with shaking at 170 rpm for 2 hours. Then was added 15 µL of proteinase K [10 mg mL-1] and incubated at 55°C at 170 rpm for 6-12 hours. At the end of digestion was added 1 volume of solution phenol-chloroform-isoamyl alcohol (25:24:1 followed by vigorous shaking for 10 seconds and centrifuged at 8000 rpm and 4°C for 10 minutes. The upper phase

  9. Purification of supercoiled plasmid DNA from clarified bacterial lysate by arginine-affinity chromatography: effects of spacer arms and ligand density.

    Science.gov (United States)

    Bai, Jin-Shan; Bai, Shu; Shi, Qing-Hong; Sun, Yan

    2014-06-01

    Efficient loading on a chromatographic column is the dilemma of the process development faced by engineers in plasmid DNA purification. In this research, novel arginine-affinity chromatographic beads were prepared to investigate the effect of spacer arm and ligand density to their chromatographic performance for the purification of plasmid. The result indicated that dynamic binding capacity for plasmid increased with an increasing ligand density and carbon number of spacer arm, and the highest binding capacity for plasmid of 6.32 mg/mL bead was observed in the column of arginine bead with a ligand density of 47 mmol/L and 10-atom carbon spacer. Furthermore, this arginine bead exhibited better selectivity to supercoiled (sc) plasmid. The evidence of a linear gradient elution suggested further that the binding of plasmid on arginine beads was driven by electrostatic interaction and hydrogen bonding. Hence, sc plasmid could successfully be purified from clarified lysate by two-stepwise elution of salt concentration. By the refinement of the elution scheme and loading volume of clarified lysate, the column of arginine bead with a ligand density of 47 mmol/L exhibited the highest recovery yield and a much higher productivity among arginine-affinity columns. Therefore, reshaped arginine beads provided more feasible and practical application in the preparation of sc plasmid from clarified lysate. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Leukocyte telomere length variation due to DNA extraction method.

    Science.gov (United States)

    Denham, Joshua; Marques, Francine Z; Charchar, Fadi J

    2014-12-04

    Telomere length is indicative of biological age. Shorter telomeres have been associated with several disease and health states. There are inconsistencies throughout the literature amongst relative telomere length measured by quantitative PCR (qPCR) and different extraction methods or kits used. We quantified whole-blood leukocyte telomere length using the telomere to single copy gene (T/S) ratio by qPCR in 20 young (18-25 yrs) men after extracting DNA using three common extraction methods: Lahiri and Nurnberger (high salt) method, PureLink Genomic DNA Mini kit (Life Technologies) and QiaAmp DNA Mini kit (Qiagen). Telomere length differences of DNA extracted from the three extraction methods was assessed by one-way analysis of variance (ANOVA). DNA purity differed between extraction methods used (P=0.01). Telomere length was impacted by the DNA extraction method used (P=0.01). Telomeres extracted using the Lahiri and Nurnberger method (mean T/S ratio: 2.43, range: 1.57-3.02) and PureLink Genomic DNA Mini Kit (mean T/S ratio: 2.57, range: 2.24-2.80) did not differ (P=0.13). Likewise, QiaAmp and Purelink-extracted telomeres were not statistically different (P=0.14). The Lahiri-extracted telomeres, however, were significantly shorter than those extracted using the QiaAmp DNA Mini Kit (mean T/S ratio: 2.71, range: 2.32-3.02; P=0.003). DNA purity was associated with telomere length. There are discrepancies between the length of leukocyte telomeres extracted from the same individuals according to the DNA extraction method used. DNA purity could be responsible for the discrepancy in telomere length but this will require validation studies. We recommend using the same DNA extraction kit when quantifying leukocyte telomere length by qPCR or when comparing different cohorts to avoid erroneous associations between telomere length and traits of interest.

  11. Well-defined star polymers for co-delivery of plasmid DNA and imiquimod to dendritic cells.

    Science.gov (United States)

    Lin, Wenjing; Hanson, Samuel; Han, Wenqing; Zhang, Xiaofang; Yao, Na; Li, Hongru; Zhang, Lijuan; Wang, Chun

    2017-01-15

    Co-delivery of antigen-encoding plasmid DNA (pDNA) and immune-modulatory molecules has importance in advancing gene-based immunotherapy and vaccines. Here novel star polymer nanocarriers were synthesized for co-delivery of pDNA and imiquimod (IMQ), a poorly soluble small-molecule adjuvant, to dendritic cells. Computational modeling and experimental results revealed that the polymers formed either multimolecular or unimolecular core-shell-type micelles in water, depending on the nature of the outer hydrophilic shell. Micelles loaded with both IMQ and pDNA were able to release IMQ in response to intracellular pH of the endo-lysosome and transfect mouse dendritic cells (DC2.4 line) in vitro. Importantly, IMQ-loaded micelle/pDNA complexes displayed much enhanced transfection efficiency than IMQ-free complexes. These results demonstrate the feasibility of co-delivery of pDNA and IMQ to antigen-presenting cells by multifunctional polymer nanocarriers with potential use in gene-based vaccine approaches.

  12. Efficient genomic DNA extraction from low target concentration bacterial cultures using SCODA DNA extraction technology.

    Science.gov (United States)

    So, Austin; Pel, Joel; Rajan, Sweta; Marziali, Andre

    2010-10-01

    Methods for the extraction of nucleic acids are straightforward in instances where there is ample nucleic acid mass in the sample and contamination is minimal. However, applications in areas such as metagenomics, life science research, clinical research, and forensics, that are limited by smaller amounts of starting materials or more dilute samples, require sample preparation methods that are more efficient at extracting nucleic acids. Synchronous coefficient of drag alteration (SCODA) is a novel electrophoretic nucleic acid purification technology that has been tested successfully with both highly contaminated and dilute samples and is a promising candidate for new sample preparation challenges. In this article, as an example of SCODA's performance with limited sample material, we outline a genomic DNA (gDNA) extraction protocol from low abundance cultures of Escherichia coli DH10B. This method is equally well suited to high biomass samples.

  13. Overcoming DNA extraction problems from carnivorous plants

    Directory of Open Access Journals (Sweden)

    Fleischmann, Andreas

    2009-12-01

    Full Text Available We tested previously published protocols for DNA isolation from plants with high contents of polyphenols and polysaccharides for several taxa of carnivorous plants. However, we did not get satisfying results with fresh or silica dried leaf tissue obtained from field collected or greenhouse grown plants, nor from herbarium specimens. Therefore, we have developed a simple modified protocol of the commercially available Macherey- Nagel NucleoSpin® Plant kit for rapid, effective and reproducible isolation of high quality genomic DNA suitable for PCR reactions. DNA extraction can be conducted from both fresh and dried leaf tissue of various carnivorous plant taxa, irrespective of high contents of polysaccharides, phenolic compounds and other secondary plant metabolites that interfere with DNA isolation and amplification.

    Probamos algunos protocolos publicados previamente para el aislamiento del ADN de plantas con alto contenido de polifenoles y polisacáridos para varios táxones de plantas carnívoras. Sin embargo, no conseguimos muy buenos resultados ni con tejidos de hojas frescas, ni con tejidos de hojas secadas en gel de sílice obtenidas de plantas colectadas en el campo o cultivadas en los invernaderos, ni de especímenes de herbario. Por lo tanto, hemos desarrollado un protocolo sencillo, modificado del Macherey- Nagel NucleoSpin® Plant kit disponible en el mercado para el aislamiento rápido, eficaz y reproducible de ADN genómico de alta calidad conveniente para la reacción en cadena de la polimerasa. La extracción del ADN se puede realizar en tejidos de hojas frescas o secas de varios táxones de plantas carnívoras, sin importar el grado de contenido de polisacáridos, compuestos fenólicos u otros metabolitos secundarios que interfieren con el aislamiento y la amplificación del ADN.

  14. Construction of adiponectin-encoding plasmid DNA and gene therapy of non-obese type 2 diabetes mellitus.

    Science.gov (United States)

    Nan, Mei Hua; Park, Jeong-Sook; Myung, Chang-Seon

    2010-01-01

    Adiponectin (ADN), an insulin-sensitizing adipokine, stimulates glucose uptake, inhibits gluconeogenesis, and plays an important role in improving insulin sensitivity. Since blood levels of ADN are low in type 2 diabetes mellitus (DM), this study was designed to investigate the therapeutic effectiveness of increasing the ADN level through injection of plasmid DNA encoding ADN in type 2 DM. A non-obese type 2 DM mouse model was established via combined administration of streptozotocin with nicotinamide and exhibited significantly higher plasma glucose concentration and insulin resistance compared with normal controls according to oral glucose tolerance and insulin challenge tests. Plasmid DNA encoding mouse ADN from differentiated NIH3T3 adipocytes was constructed in pVAX1 (pVAX/ADN). Transfection of pVAX/ADN into various cell lines including HeLa, HT22, HEK293, HepG2, and SK-Hep1 cells, increased ADN mRNA expression levels in a dose-dependent manner. The administration of pVAX/ADN into non-obese type 2 DM mice via tail vein significantly increased the blood level of ADN and decreased the plasma glucose concentration. Moreover, the parameters related to insulin resistance (HOMA-IR) and insulin sensitivity (QUICKI) were significantly improved. These results suggest that ADN gene therapy could be a clinically effective tool for the treatment of type 2 DM.

  15. DNA sequence conservation between the Bacillus anthracis pXO2 plasmid and genomic sequence from closely related bacteria

    Directory of Open Access Journals (Sweden)

    Sabin Robert

    2002-12-01

    Full Text Available Abstract Background Complete sequencing and annotation of the 96.2 kb Bacillus anthracis plasmid, pXO2, predicted 85 open reading frames (ORFs. Bacillus cereus and Bacillus thuringiensis isolates that ranged in genomic similarity to B. anthracis, as determined by amplified fragment length polymorphism (AFLP analysis, were examined by PCR for the presence of sequences similar to 47 pXO2 ORFs. Results The two most distantly related isolates examined, B. thuringiensis 33679 and B. thuringiensis AWO6, produced the greatest number of ORF sequences similar to pXO2; 10 detected in 33679 and 16 in AWO6. No more than two of the pXO2 ORFs were detected in any one of the remaining isolates. Dot-blot DNA hybridizations between pXO2 ORF fragments and total genomic DNA from AWO6 were consistent with the PCR assay results for this isolate and also revealed nine additional ORFs shared between these two bacteria. Sequences similar to the B. anthracis cap genes or their regulator, acpA, were not detected among any of the examined isolates. Conclusions The presence of pXO2 sequences in the other Bacillus isolates did not correlate with genomic relatedness established by AFLP analysis. The presence of pXO2 ORF sequences in other Bacillus species suggests the possibility that certain pXO2 plasmid gene functions may also be present in other closely related bacteria.

  16. High quality genomic DNA extraction from postmortem fetal tissue.

    Science.gov (United States)

    Addison, S; Sebire, N J; Taylor, A M; Abrams, D; Peebles, D; Mein, C; Munroe, P B; Thayyil, S

    2012-11-01

    We examined the yield and quality of genomic deoxyribonucleic acid (DNA) extracted from various postmortem fetal tissues. Fetal tissues were collected at the time of autopsy, and DNA was subsequently extracted. The yield and DNA quality was assessed using ultraviolet spectrometry and agarose gel electrophoresis. We used polymerase chain reaction (PCR) to assess the DNA extracted for genomic testing. The median (range) gestation of the fetuses was 22 (16-41) weeks and the postmortem interval was 5.5 (2-10) days. Non-degraded genomic DNA was successfully extracted from all fetal tissues. Liver tissue had the lowest quality and muscle the highest quality. DNA yield or purity was not influenced by the postmortem interval. High quality genomic DNA can be extracted from fetal muscle, despite postmortem intervals of several days.

  17. Purification of plasmid DNA from clarified and non-clarified Escherichia coli lysates by berenil pseudo-affinity chromatography.

    Science.gov (United States)

    Caramelo-Nunes, C; Gabriel, M F; Almeida, P; Marcos, J C; Tomaz, C T

    2012-09-01

    In this study, berenil was tested as a ligand, specifically to purify plasmids of different sizes pVAX1-LacZ (6.05 Kbp) and pCAMBIA-1303 (12.361 Kbp) from clarified Escherichia coli alkaline lysates. For this purpose, chromatographic experiments were performed using Sepharose derivatized with berenil. The results showed that both pDNA molecules are completely purified using lower amounts of salt in the eluent than those previously reported for other pseudo-affinity and hydrophobic interaction chromatography based processes. Total retention of all lysate components was achieved with 1.3M ammonium sulphate in the eluent buffer and pDNA elution was obtained by decreasing the salt concentration to 0.55 M. All impurities were eluted after decreasing the concentration to 0M. The recovery yield for pCAMBIA-1303 (45%) was lower than that obtained for pVAX1-LacZ (85%), however the larger pDNA showed a higher purity level. Purification of pVAX1-LacZ was also performed using non-clarified E. coli process streams, replacing the clarification step with a second chromatographic run on the berenil-Sepharose. Using the same binding and elution conditions as before, a pure plasmid sample was obtained with a 33% yield and with all host impurity levels in accordance with the requirements established by the regulatory agencies. These results suggest that this chromatographic method is a promising alternative to purify pDNA for therapeutic use. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Optimization of the Phenol -Chloroform Silica DNA Extraction Method in Ancient Bones DNA Extraction

    Directory of Open Access Journals (Sweden)

    Morteza Sadeghi

    2014-04-01

    Full Text Available Introduction: DNA extraction from the ancient bones tissues is currently very difficult. Phenol chloroform silica method is one of the methods currently used for this aim. The purpose of this study was to optimize the assessment method. Methods: DNA of 62 bone tissues (average 3-11 years was first extracted with phenol chloroform silica methods and then with changing of some parameters of the methods the extracted DNA was amplified in eight polymorphisms area including FES, F13, D13S317, D16, D5S818, vWA and CD4. Results from samples gained by two methods were compared in acrylamide gel. Results: The average of PCR yield for new method and common method in eight polymorphism regions was 75%, 78%, 81%, 76%, 85%, 71%, 89%, 86% and 64%, 39%, 70%, 49%, 68%, 76%, 71% and 28% respectively. The average of DNA in optimized (in 35l silica density and common method were 267.5 µg/ml with 1.12 purity and 192.76 g/ml with 0.84 purity respectively. Conclusions: According to the findings of this study, it is estimated that longer EDTA attendance is an efficient agent in removing calcium and also adequate density of silica particles can be efficient in removal of PCR inhibitors.

  19. Targeted Multifunctional Lipid ECO Plasmid DNA Nanoparticles as Efficient Non-viral Gene Therapy for Leber's Congenital Amaurosis.

    Science.gov (United States)

    Sun, Da; Sahu, Bhubanananda; Gao, Songqi; Schur, Rebecca M; Vaidya, Amita M; Maeda, Akiko; Palczewski, Krzysztof; Lu, Zheng-Rong

    2017-06-16

    Development of a gene delivery system with high efficiency and a good safety profile is essential for successful gene therapy. Here we developed a targeted non-viral delivery system using a multifunctional lipid ECO for treating Leber's congenital amaurosis type 2 (LCA2) and tested this in a mouse model. ECO formed stable nanoparticles with plasmid DNA (pDNA) at a low amine to phosphate (N/P) ratio and mediated high gene transfection efficiency in ARPE-19 cells because of their intrinsic properties of pH-sensitive amphiphilic endosomal escape and reductive cytosolic release (PERC). All-trans-retinylamine, which binds to interphotoreceptor retinoid-binding protein (IRBP), was incorporated into the nanoparticles via a polyethylene glycol (PEG) spacer for targeted delivery of pDNA into the retinal pigmented epithelium. The targeted ECO/pDNA nanoparticles provided high GFP expression in the RPE of 1-month-old Rpe65(-/-) mice after subretinal injection. Such mice also exhibited a significant increase in electroretinographic activity, and this therapeutic effect continued for at least 120 days. A safety study in wild-type BALB/c mice indicated no irreversible retinal damage following subretinal injection of these targeted nanoparticles. All-trans-retinylamine-modified ECO/pDNA nanoparticles provide a promising non-viral platform for safe and effective treatment of RPE-specific monogenic eye diseases such as LCA2. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  20. Comparative analysis of protocols for DNA extraction from soybean caterpillars.

    Science.gov (United States)

    Palma, J; Valmorbida, I; da Costa, I F D; Guedes, J V C

    2016-04-07

    Genomic DNA extraction is crucial for molecular research, including diagnostic and genome characterization of different organisms. The aim of this study was to comparatively analyze protocols of DNA extraction based on cell lysis by sarcosyl, cetyltrimethylammonium bromide, and sodium dodecyl sulfate, and to determine the most efficient method applicable to soybean caterpillars. DNA was extracted from specimens of Chrysodeixis includens and Spodoptera eridania using the aforementioned three methods. DNA quantification was performed using spectrophotometry and high molecular weight DNA ladders. The purity of the extracted DNA was determined by calculating the A260/A280 ratio. Cost and time for each DNA extraction method were estimated and analyzed statistically. The amount of DNA extracted by these three methods was sufficient for PCR amplification. The sarcosyl method yielded DNA of higher purity, because it generated a clearer pellet without viscosity, and yielded high quality amplification products of the COI gene I. The sarcosyl method showed lower cost per extraction and did not differ from the other methods with respect to preparation times. Cell lysis by sarcosyl represents the best method for DNA extraction in terms of yield, quality, and cost effectiveness.

  1. The angiogenic response to PLL-g-PEG-mediated HIF-1α plasmid DNA delivery in healthy and diabetic rats.

    Science.gov (United States)

    Thiersch, Markus; Rimann, Markus; Panagiotopoulou, Vasiliki; Öztürk, Ece; Biedermann, Thomas; Textor, Marcus; Lühmann, Tessa C; Hall, Heike

    2013-05-01

    Impaired angiogenesis is a major clinical problem and affects wound healing especially in diabetic patients. Improving angiogenesis is a reasonable strategy to increase diabetes-impaired wound healing. Recently, our lab described a system of transient gene expression due to pegylated poly-l-lysine (PLL-g-PEG) polymer-mediated plasmid DNA delivery in vitro. Here we synthesized peptide-modified PLL-g-PEG polymers with two functionalities, characterized them in vitro and utilized them in vivo via a fibrin-based delivery matrix to induce dermal wound angiogenesis in diabetic rats. The two peptides were 1) a TG-peptide to covalently bind these nanocondensates to the fibrin matrix (TG-peptide) for a sustained release and 2) a polyR peptide to improve cellular uptake of these nanocondensates. In order to induce angiogenesis in vivo we condensed modified and non-modified polymers with plasmid DNA encoding a truncated form of the therapeutic candidate gene hypoxia-inducible transcription factor 1α (HIF-1α). HIF-1α is the primarily oxygen-dependent regulated subunit of the heterodimeric transcription factor HIF-1, which controls angiogenesis among other physiological pathways. The truncated form of HIF-1α lacks the oxygen-dependent degradation domain (ODD) and therefore escapes degradation under normoxic conditions. PLL-g-PEG polymer-mediated HIF-1α-ΔODD plasmid DNA delivery was found to lead to a transiently induced gene expression of angiogenesis-related genes Acta2 and Pecam1 as well as the HIF-1α target gene Vegf in vivo. Furthermore, HIF-1α gene delivery was shown to enhance the number endothelial cells and smooth muscle cells - precursors for mature blood vessels - during wound healing. We show that - depending on the selection of the therapeutic target gene - PLL-g-PEG nanocondensates are a promising alternative to viral DNA delivery approaches, which might pose a risk to health. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Auto-assembly of nanometer thick, water soluble layers of plasmid DNA complexed with diamines and basic amino acids on graphite: Greatest DNA protection is obtained with arginine.

    Science.gov (United States)

    Khalil, T T; Boulanouar, O; Heintz, O; Fromm, M

    2017-02-01

    We have investigated the ability of diamines as well as basic amino acids to condense DNA onto highly ordered pyrolytic graphite with minimum damage after re-dissolution in water. Based on a bibliographic survey we briefly summarize DNA binding properties with diamines as compared to basic amino acids. Thus, solutions of DNA complexed with these linkers were drop-cast in order to deposit ultra-thin layers on the surface of HOPG in the absence or presence of Tris buffer. Atomic Force Microscopy analyses showed that, at a fixed ligand-DNA mixing ratio of 16, the mean thickness of the layers can be statistically predicted to lie in the range 0-50nm with a maximum standard deviation ±6nm, using a simple linear law depending on the DNA concentration. The morphology of the layers appears to be ligand-dependent. While the layers containing diamines present holes, those formed in the presence of basic amino acids, except for lysine, are much more compact and dense. X-ray Photoelectron Spectroscopy measurements provide compositional information indicating that, compared to the maximum number of DNA sites to which the ligands may bind, the basic amino acids Arg and His are present in large excess. Conservation of the supercoiled topology of the DNA plasmids was studied after recovery of the complex layers in water. Remarkably, arginine has the best protection capabilities whether Tris was present or not in the initial solution. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Plasmid DNA Supercoiling and Gyrase Activity in Escherichia coli Wild-Type and rpoS Stationary-Phase Cells

    Science.gov (United States)

    Reyes-Domínguez, Yazmid; Contreras-Ferrat, Gabriel; Ramírez-Santos, Jesús; Membrillo-Hernández, Jorge; Gómez-Eichelmann, M. Carmen

    2003-01-01

    Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase rpoS cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that rpoS plays a role in the regulation of plasmid topology during the stationary phase. PMID:12533486

  4. DNA rearrangement has occurred in the carbazole-degradative plasmid pCAR1 and the chromosome of its unsuitable host, Pseudomonas fluorescens Pf0-1.

    Science.gov (United States)

    Shintani, Masaki; Matsumoto, Takashi; Yoshikawa, Hirofumi; Yamane, Hisakazu; Ohkuma, Moriya; Nojiri, Hideaki

    2011-12-01

    The carbazole-degradative plasmid pCAR1 carries the class II transposon Tn4676, which contains the car and ant genes, essential for conversion of carbazole into anthranilate, and anthranilate into catechol, respectively. In our previous study, DNA rearrangements in pCAR1 were frequently detected in the host Pseudomonas fluorescens Pf0-1 in the presence of carbazole, resulting in the improvement of host survivability. Several Pf0-1 mutants harbouring pCAR1 were isolated, and deletion of DNA in the plasmid ant gene was found. Here, we compared genome sequences of the parent strain Pf0-1L(pCAR1::rfp) and one of its mutants, 5EP83, to assess whether other DNA rearrangements occurred in either the plasmid or the host chromosome. We found transposition of Tn4676 into the 5EP83 chromosome. In addition, ISPre1 had transposed into the car gene intergenic region on the pCAR1-derivative plasmid of 5EP83, which inhibited car transcription. As a result of these transpositions, 5EP83 was able to metabolize carbazole due to the Tn4676 on its chromosome, although the car genes on its plasmid were non-functional. We also found that one copy of duplicate carAa genes had been deleted, and that ISPre4 had transposed into both the host chromosome and the plasmid. Our findings suggest that Pf0-1 harbouring pCAR1 is subjected to DNA rearrangements not only on the plasmid but also on its chromosome in the presence of carbazole.

  5. An experimental study on use of 7T MRI for evaluation of myocardial infarction in SD rats transfected with pcDNA 3.1(+)/VEGF121 plasmid

    Science.gov (United States)

    Zhang, Yan; Tian, Ruiqing; Shen, Xiangchun; Chen, Yushu; Chen, Wei; Gan, Lu; Shen, Guiquan; Ju, Haiyue; Yang, Li; Gao, Fabao

    2016-01-01

    This study aims to build the myocardial infarction model in SD rats transfected with pcDNA 3.1(+)/VEGF121 plasmid and study the effect of the transfection using 7T MRI. Twenty-four male SD rats were randomly divided into 2 groups, pcDNA 3.1(+)/VEGF121 plasmid transfection group (with improved coronary perfusion delivery) and myocardial infarction model group. Cardiac cine magnetic resonance imaging (Cine-MRI), T2-mapping and late gadolinium enhancement (LGE) cardiac imaging were performed at 24 h, 48 h, 72 h and 7 d after myocardial infarction, respectively. The signal intensity, area at risk (AAR), myocardium infarction core (MIC) and salvageable myocardial zone (SMZ) were compared. The hearts were harvested for anatomic characterization, which was related to pathological examination (TTC staining, HE staining, Masson staining and immunohistochemical staining). The Cine-MRI results showed that pcDNA 3.1(+)/VEGF121 plasmid transfection group had higher end-diastolic volume (EDV) with a reduction in MIC and SMZ, as compared with the myocardial infarction model group. MIC, SMZ and AAR of the plasmid transfection declined over time. At 7 d, the two groups did not differ significantly in AAR and T2 value. According to Western Blotting, VEGF was up-regulated, while CaSR and caspase-3 were downregulated in the plasmid transfection group, as compared with the model group. In conclusion, a good treatment effect was achieved by coronary perfusion of pcDNA 3.1(+)/VEGF121 plasmid. 7T CMR sequences provide a non-invasive quantification of the treatment efficacy. However, the assessment of myocardial injury using T2 value and AAR in the presence of edema is less accurate. The myocardial protection of the plasmid transfection group may be related to the inhibition of myocardial apoptosis, vascular endothelial cell (VEC) proliferation and collagen proliferation. The CaSR signaling pathway may contribute to reversing the apoptosis. PMID:27648128

  6. In vitro antioxidant and DNA damage inhibition activity of aqueous extract of Lantana camara L. (Verbenaceae) leaves

    Institute of Scientific and Technical Information of China (English)

    Kokati Venkata Bhaskara Rao

    2012-01-01

    Objective: To investigate the in vitro antioxidant and DNA damage inhibition potential of aqueous extract of Lantana camara leaves. Methods: Antioxidant activity of the aqueous extract of L. camara was estimated by 2, 2-diphenyl-1-picrylhydrazyl radical scavenging assay, metal chelating activity and reducing power assay. DNA damage inhibition was performed by photolysing H2O2 by UV radiation in the presence of pBR322 and extract. Estimation of phenolic content was carried out by Folin-Ciocalteau assay. Results: Extract exhibited high antioxidant activity in DPPH radical scavenng assay (IC50= 42.66 μg/ml), metal chelating activity (IC50= 1036.4μg/ml) and reducing power assay. Extract also exhibited the complete protection of pBR322 plasmid DNA during DNA damage inhibition assay. Extract showed high phenolic content which justified the antioxidant and DNA damage inhibition properties of the plant. Conclusions:These observations emphasize that aqueous extract of L. camara possess high antioxidant and DNA damage inhibition potential, thus, the plant can be used to develop natural antioxidant compounds for therapeutic use.

  7. Comparison of plasmid DNA versus PCR amplified gene of insert DNA for nucleofection in Kasumi-1 cells.

    Science.gov (United States)

    Wu, Kang; Zhao, Xu-Jie; Wong, Ka-Wing; Fan, Xiao-Yong

    2015-03-01

    Plasmid electroporation, or its optimized version nucleofection, is an important technique for gene transfection of cells in suspension. However, substantial cell death and/or low transfection efficiency are still common for some cell lines. By using enhanced green fluorescent protein (EGFP) as a reporter, we compared the use of PCR amplified EGFP (PaEGFP) and its parental plasmid (pEGFP-N2) for nucleofection in Kasumi-1 cells. We found that PaEGFP induced significantly lower cell death but had similar transfection efficiency compared to its parent plasmid (pEGFP-N2). Most importantly, contrary to the pEGFP-N2-nucleofected cells, the PaEGFP-nucleofected cells subsequently grew properly. Tests in other cell lines also implied that PaEGFP indeed induced consistently less cell death, but transfection efficiencies varied, being good in suspension cell lines but lower in adhesive cell lines. We suggest that direct transfection with PCR amplified genes can be a simple and useful approach for optimization of electropulse-based transfection not only of Kasumi-1 cells, but also may be useful for other cell lines that are difficult to transfect in suspension.

  8. Comparison of three DNA extraction methods for recovery of soil protist DNA.

    Science.gov (United States)

    Santos, Susana S; Nielsen, Tue Kjærgaard; Hansen, Lars H; Winding, Anne

    2015-08-01

    The use of molecular methods to investigate protist communities in soil is in rapid development this decade. Molecular analysis of soil protist communities is usually dependant on direct genomic DNA extraction from soil and inefficient or differential DNA extraction of protist DNA can lead to bias in downstream community analysis. Three commonly used soil DNA extraction methods have been tested on soil samples from three European Long-Term Observatories (LTOs) with different land-use and three protist cultures belonging to different phylogenetic groups in different growth stages. The methods tested were: ISOm-11063 (a version of the ISO-11063 method modified to include a FastPrep ®-24 mechanical lysis step), GnS-GII (developed by the GenoSol platform to extract soil DNA in large-scale soil surveys) and a commercial DNA extraction kit - Power Lyzer™ PowerSoil® DNA Isolation Kit (MoBio). DNA yield and quality were evaluated along with DNA suitability for amplification of 18S rDNA fragments by PCR. On soil samples, ISOm-11063 yields significantly higher DNA for two of the three soil samples, however, MoBio extraction favors DNA quality. This method was also more effective to recover copies of 18S rDNA numbers from all soil types. In addition and despite the lower yields, higher DNA quality was observed with DNA extracted from protist cultures with the MoBio method. Likewise, a bead-beating step shows to be a good solution for DNA extraction of soil protists, since the recovery of DNA from protist cultures and from the different soil samples with the ISOm method proved to be efficient in recovering PCR-amplifiable DNA. This study showed that soil DNA extraction methods provide biased results towards the cyst stages of protist organism. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Preliminary assessment for DNA extraction on microfluidic channel

    Science.gov (United States)

    Gopinath, Subash C. B.; Hashim, Uda; Uda, M. N. A.

    2017-03-01

    The aim of this research is to extract, purify and yield DNA in mushroom from solid state mushroom sample by using fabricated continuous high-capacity sample delivery microfluidic through integrated solid state extraction based amino-coated silica bead. This device is made to specifically extract DNA in mushroom sample in continuous inflow process with energy and cost consumption. In this project, we present two methods of DNA extraction and purification which are by using centrifuge (complex and conventional method) and by using microfluidic biosensor (new and fast method). DNA extracted can be determined by using ultraviolet-visible spectroscopy (UV-VIS). The peak obtained at wavelength 260nm after measuring the absorbance of sample proves that DNA is successfully extracted from the mushroom.

  10. Plasmid DNA Vaccine Co-Immunisation Modulates Cellular and Humoral Immune Responses Induced by Intranasal Inoculation in Mice.

    Directory of Open Access Journals (Sweden)

    Deborah F L King

    Full Text Available An effective HIV vaccine will likely require induction of both mucosal and systemic cellular and humoral immune responses. We investigated whether intramuscular (IM delivery of electroporated plasmid DNA vaccine and simultaneous protein vaccinations by intranasal (IN and IM routes could be combined to induce mucosal and systemic cellular and humoral immune responses to a model HIV-1 CN54 gp140 antigen in mice.Co-immunisation of DNA with intranasal protein successfully elicited both serum and vaginal IgG and IgA responses, whereas DNA and IM protein co-delivery did not induce systemic or mucosal IgA responses. Cellular IFNγ responses were preserved in co-immunisation protocols compared to protein-only vaccination groups. The addition of DNA to IN protein vaccination reduced the strong Th2 bias observed with IN protein vaccination alone. Luminex analysis also revealed that co-immunisation with DNA and IN protein induced expression of cytokines that promote B-cell function, generation of TFH cells and CCR5 ligands that can reduce HIV infectivity.These data suggest that while IN inoculation alone elicits both cellular and humoral responses, co-administration with homologous DNA vaccination can tailor these towards a more balanced Th1/Th2 phenotype modulating the cellular cytokine profile while eliciting high-levels of antigen-specific antibody. This work provides insights on how to generate differential immune responses within the same vaccination visit, and supports co-immunisation with DNA and protein by a mucosal route as a potential delivery strategy for HIV vaccines.

  11. DermaVir: a plasmid DNA-based nanomedicine therapeutic vaccine for the treatment of HIV/AIDS.

    Science.gov (United States)

    Lori, Franco

    2011-10-01

    The HIV global pandemic continues to rage with over 33 million people living with the disease. Although multidrug therapy has improved the prognosis for those infected by the virus, it has not eradicated the infection. Immunological therapies, including therapeutic vaccines, are needed to supplement drug therapy in the search for a 'functional cure' for HIV. DermaVir (Genetic Immunity Kft, Budapest, Hungary and McLean, Virginia, USA), an experimental HIV/AIDS therapeutic vaccine, combines three key elements of rational therapeutic vaccine design: a single plasmid DNA (pDNA) immunogen expressing 15 HIV antigens, a synthetic pDNA nanomedicine formulation and a dendritic cell-targeting topical-vaccine administration. DermaVir's novel mechanism of action, natural transport by epidermal Langerhans cells to the lymph nodes to express the pDNA-encoded HIV antigens and induce precursor/memory T cells with high proliferation capacity, has been consistently demonstrated in mouse, rabbit, primate and human subjects. Safety, immunogenicity and preliminary efficacy of DermaVir have been clinically demonstrated in HIV-infected human subjects. The DermaVir technology platform for dendritic cell-based therapeutic vaccination might offer a new treatment paradigm for cancer and infectious diseases.

  12. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo.

    Science.gov (United States)

    Wegman, F; Bijenhof, A; Schuijff, L; Oner, F C; Dhert, W J A; Alblas, J

    2011-03-15

    Bone regeneration is one of the major focus points in the field of regenerative medicine. A well-known stimulus of bone formation is bone morphogenetic protein-2 (BMP-2), which has already been extensively used in clinical applications. We investigated the possibility of achieving osteogenic differentiation both in vitro and in vivo as a result of prolonged presence of BMP-2 using plasmid DNA-based gene therapy. By delivering BMP-2 cDNA in an alginate hydrogel, a versatile formulation is developed. High transfection efficiencies of up to 95% were obtained in both human multipotent stromal cells (MSCs) and MG-63 cells using naked DNA in vitro. Over a period of 5 weeks, an increasing amount of biologically active BMP-2 was released from the cells and remained present in the gel. In vivo, transfected cells were found after both two and six weeks implantation in naked mice, even in groups without seeded cells, thus indicating in vivo transfection of endogenous cells. The protein levels were effective in inducing osteogenic differentiation in vitro, as seen by elevated alkaline phosphatase (ALP) production and in vivo, as demonstrated by the production of collagen I and osteocalcin in a mineralised alginate matrix. We conclude that BMP-2 cDNA incorporated in alginate hydrogel appears to be a promising new strategy for minimal-invasive delivery of growth factors in bone regeneration.

  13. Novel PEI/Poly-γ-Gutamic Acid Nanoparticles for High Efficient siRNA and Plasmid DNA Co-Delivery

    Directory of Open Access Journals (Sweden)

    Shu-Fen Peng

    2017-01-01

    Full Text Available The efficient delivery of sufficient amounts of nucleic acids into target cells is critical for successful gene therapy and gene knockdown. The DNA/siRNA co-delivery system has been considered a promising approach for cancer therapy to simultaneously express and inhibit tumor suppressor genes and overexpressed oncogenes, respectively, triggering synergistic anti-cancer effects. Polyethylenimine (PEI has been identified as an efficient non-viral vector for transgene expression. In this study, we created a very high efficient DNA/siRNA co-delivery system by incorporating a negatively-charged poly-γ-glutamic acid (γ-PGA into PEI/nucleic acid complexes. Spherical nanoparticles with about 200 nm diameter were formed by mixing PEI/plasmid DNA/siRNA/γ-PGA (dual delivery nanoparticles; DDNPs with specific ratio (N/P/C ratio and the particles present positive surface charge under all manufacturing conditions. The gel retardation assay shows both nucleic acids were effectively condensed by PEI, even at low N/P ratios. The PEI-based DDNPs reveal excellent DNA/siRNA transfection efficiency in the human hepatoma cell line (Hep 3B by simultaneously providing high transgene expression efficiency and high siRNA silencing effect. The results indicated that DDNP can be an effective tool for gene therapy against hepatoma.

  14. Novel PEI/Poly-γ-Gutamic Acid Nanoparticles for High Efficient siRNA and Plasmid DNA Co-Delivery.

    Science.gov (United States)

    Peng, Shu-Fen; Hsu, Hung-Kun; Lin, Chun-Cheng; Cheng, Ya-Ming; Hsu, Kuang-Hsing

    2017-01-04

    The efficient delivery of sufficient amounts of nucleic acids into target cells is critical for successful gene therapy and gene knockdown. The DNA/siRNA co-delivery system has been considered a promising approach for cancer therapy to simultaneously express and inhibit tumor suppressor genes and overexpressed oncogenes, respectively, triggering synergistic anti-cancer effects. Polyethylenimine (PEI) has been identified as an efficient non-viral vector for transgene expression. In this study, we created a very high efficient DNA/siRNA co-delivery system by incorporating a negatively-charged poly-γ-glutamic acid (γ-PGA) into PEI/nucleic acid complexes. Spherical nanoparticles with about 200 nm diameter were formed by mixing PEI/plasmid DNA/siRNA/γ-PGA (dual delivery nanoparticles; DDNPs) with specific ratio (N/P/C ratio) and the particles present positive surface charge under all manufacturing conditions. The gel retardation assay shows both nucleic acids were effectively condensed by PEI, even at low N/P ratios. The PEI-based DDNPs reveal excellent DNA/siRNA transfection efficiency in the human hepatoma cell line (Hep 3B) by simultaneously providing high transgene expression efficiency and high siRNA silencing effect. The results indicated that DDNP can be an effective tool for gene therapy against hepatoma.

  15. A comparison of DNA extraction methods using Petunia hybrida tissues.

    Science.gov (United States)

    Tamari, Farshad; Hinkley, Craig S; Ramprashad, Naderia

    2013-09-01

    Extraction of DNA from plant tissue is often problematic, as many plants contain high levels of secondary metabolites that can interfere with downstream applications, such as the PCR. Removal of these secondary metabolites usually requires further purification of the DNA using organic solvents or other toxic substances. In this study, we have compared two methods of DNA purification: the cetyltrimethylammonium bromide (CTAB) method that uses the ionic detergent hexadecyltrimethylammonium bromide and chloroform-isoamyl alcohol and the Edwards method that uses the anionic detergent SDS and isopropyl alcohol. Our results show that the Edwards method works better than the CTAB method for extracting DNA from tissues of Petunia hybrida. For six of the eight tissues, the Edwards method yielded more DNA than the CTAB method. In four of the tissues, this difference was statistically significant, and the Edwards method yielded 27-80% more DNA than the CTAB method. Among the different tissues tested, we found that buds, 4 days before anthesis, had the highest DNA concentrations and that buds and reproductive tissue, in general, yielded higher DNA concentrations than other tissues. In addition, DNA extracted using the Edwards method was more consistently PCR-amplified than that of CTAB-extracted DNA. Based on these results, we recommend using the Edwards method to extract DNA from plant tissues and to use buds and reproductive structures for highest DNA yields.

  16. Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein

    Science.gov (United States)

    Lipps, Georg; Stegert, Mario; Krauss, Gerhard

    2001-01-01

    There is still a lack of information on the specific characteristics of DNA-binding proteins from hyperthermophiles. Here we report on the product of the gene orf56 from plasmid pRN1 of the acidophilic and thermophilic archaeon Sulfolobus islandicus. orf56 has not been characterised yet but low sequence similarily to several eubacterial plasmid-encoded genes suggests that this 6.5 kDa protein is a sequence-specific DNA-binding protein. The DNA-binding properties of ORF56, expressed in Escherichia coli, have been investigated by EMSA experiments and by fluorescence anisotropy measurements. Recombinant ORF56 binds to double-stranded DNA, specifically to an inverted repeat located within the promoter of orf56. Binding to this site could down-regulate transcription of the orf56 gene and also of the overlapping orf904 gene, encoding the putative initiator protein of plasmid replication. By gel filtration and chemical crosslinking we have shown that ORF56 is a dimeric protein. Stoichiometric fluorescence anisotropy titrations further indicate that ORF56 binds as a tetramer to the inverted repeat of its target binding site. CD spectroscopy points to a significant increase in ordered secondary structure of ORF56 upon binding DNA. ORF56 binds without apparent cooperativity to its target DNA with a dissociation constant in the nanomolar range. Quantitative analysis of binding isotherms performed at various salt concentrations and at different temperatures indicates that approximately seven ions are released upon complex formation and that complex formation is accompanied by a change in heat capacity of –6.2 kJ/mol. Furthermore, recombinant ORF56 proved to be highly thermostable and is able to bind DNA up to 85°C. PMID:11160922

  17. Effect of intense, ultrashort laser pulses on DNA plasmids in their native state: strand breakages induced by {\\it in-situ} electrons and radicals

    CERN Document Server

    D'Souza, J S; Dharmadhikari, A K; Rao, B J; Mathur, D

    2011-01-01

    Single strand breaks are induced in DNA plasmids, pBR322 and pUC19, in aqueous media exposed to strong fields generated using ultrashort laser pulses (820 nm wavelength, 45 fs pulse duration, 1 kHz repetition rate) at intensities of 1-12 TW cm$^{-2}$. The strong fields generate, {\\it in situ}, electrons and radicals that induce transformation of supercoiled DNA into relaxed DNA, the extent of which is quantified. Introduction of electron and radical scavengers inhibits DNA damage; results indicate that OH radicals are the primary (but not sole) cause of DNA damage.

  18. [DNA extraction methods of compost for molecular ecology analysis].

    Science.gov (United States)

    Yang, Zhao-Hui; Xiao, Yong; Zeng, Guang-Ming; Liu, Yun-Guo; Deng, Jiu-Hua

    2006-08-01

    Molecular ecology provides new techniques for studying compost microbes, and the DNA extraction is the basis of molecular techniques. Because of the contamination of humic acids, it turns to be more difficult for compost microbial DNA extraction. Three different approaches, named as lysozyme lysis, ultrasonic lysis and proteinase K lysis with CTAB, were used to extract the total DNA from compost. The detection performed on a nucleic acids and protein analyzer showed that all the three approaches produced high DNA yields. The agarose gel electrophoresis showed that the DNA fragments extracted from compost had a length of about 23 kb. A eubacterial 16S rRNA gene targeted primer pair (27F and 1 495R) was used for PCR amplification, and all the samples got almost the full length 16S rDNA sequence (about 1.5 kb). After digested by restriction endonucleases (Hae Ill and Alu I), the restriction map showed relatively identical microbial diversity in the DNA, which was extracted by the three different approaches. All the compost microbial DNA extracted by the three different approaches could be used for molecular ecological study, and researchers should choose the right approach for extracting microbial DNA from compost based on the facts.

  19. Successive DNA extractions improve characterization of soil microbial communities

    NARCIS (Netherlands)

    Dimitrov, M.R.; Veraart, A.J.; De Hollander, M.; Smidt, H.; van Veen, J.A.; Kuramae, E.E.

    2017-01-01

    Currently, characterization of soil microbial communities relies heavily on the use of molecular approaches. Independently of the approach used, soil DNA extraction is a crucial step, and success of downstream procedures will depend on how well DNA extraction was performed. Often, studies describing

  20. Successive DNA extractions improve characterization of soil microbial communities

    NARCIS (Netherlands)

    Rocha Dimitrov, Mauricio; Veraart, Annelies J.; Hollander, de Mattias; Smidt, Hauke; Veen, van Johannes A.; Kuramae, Eiko E.

    2017-01-01

    Currently, characterization of soil microbial communities relies heavily on the use of molecular approaches. Independently of the approach used, soil DNA extraction is a crucial step, and success of downstream procedures will depend on how well DNA extraction was performed. Often, studies

  1. An efficient method for DNA extraction from Cladosporioid fungi

    NARCIS (Netherlands)

    Moslem, M.A.; Bahkali, A.H.; Abd-Elsalam, K.A.; Wit, de P.J.G.M.

    2010-01-01

    We developed an efficient method for DNA extraction from Cladosporioid fungi, which are important fungal plant pathogens. The cell wall of Cladosporioid fungi is often melanized, which makes it difficult to extract DNA from their cells. In order to overcome this we grew these fungi for three days on

  2. An efficient method for DNA extraction from Cladosporioid fungi

    NARCIS (Netherlands)

    Moslem, M.A.; Bahkali, A.H.; Abd-Elsalam, K.A.; Wit, de P.J.G.M.

    2010-01-01

    We developed an efficient method for DNA extraction from Cladosporioid fungi, which are important fungal plant pathogens. The cell wall of Cladosporioid fungi is often melanized, which makes it difficult to extract DNA from their cells. In order to overcome this we grew these fungi for three days on

  3. Protocol for extraction of genomic DNA from swine solid tissues

    Directory of Open Access Journals (Sweden)

    Fernando Henrique Biase

    2002-01-01

    Full Text Available Molecular diagnostics are performed by using DNA from different body tissues. However, it is necessary to obtain genomic DNA of good quality. Due to the impossibility of collecting blood from slaughtered animals, DNA extraction from solid tissues is necessary. The objective of this study was to describe a protocol of DNA extraction from swine skin, adipose, brain, liver, kidney and muscle tissues. We obtained high molecular weight DNA of good quality, shown by agarose gel and amplification of two DNA fragments, 605bp and 891pb, by PCR. Spectrophotometric analysis of DNA concentration showed variation among the DNA from different tissues, with the liver and adipose tissues presenting the greatest and the smallest concentration, respectively. The described protocol has proven to be advantageous due to its simplicity, quickness, affordable reagents and absence of phenol, resulting in a high molecular weight DNA of good quality from several tissues.

  4. Construction and expression of recombinant plasmid TAZ-pcDNA3 .1 and TAZ-pEGFP-C2%T AZ 基因重组质粒的构建与表达

    Institute of Scientific and Technical Information of China (English)

    仲念念; 朱伶俐; 王旋; 房娜

    2015-01-01

    Objective Two recombinant plasmids , TAZ‐pcDNA3 .1 and TAZ‐pEGFP‐C2 , were established . The protein expression of TAZ in HEK293 cells was detected by Western Blot and the roles of TAZ in promoting cell proliferation and migration were further explored . Methods AZ gene was amplified by PCR , fragments were recovered followed by connection with glue T carrier , blue‐white screening , transformation and extraction of plasmid DNA . Then the plasmid DNA was digested , connected by T 4 DNA Ligase , and then sub‐cloned into pEGFP‐C2 and pcDNA3 .1 to construct new recombinant plasmids . These plasmids were transfected into HEK293 cells to observe the distribution of TAZ using a fluorescence detector . The protein expression was detected by Western Blot .Results By restriction enzyme identification and sequence analysis , the recombinant plasmids were successfully constructed . Fluorescent photos show that the distribution of TAZ molecule was in the nucleus and cytoplasm . Western Blot test results showed that TAZ molecule could induce over‐expression of specific proteins . Conclusion Two recombinant plasmids were successfully constructed . The effects of TAZ over‐expression were validated , which will lay a foundation for revealing the mechanism of TAZ in promoting cell proliferation and migration .%目的:构建重组质粒TAZ‐pcDNA31.及 TAZ‐pEGFP‐C2,并应用Western Blot检测TAZ蛋白在细胞内的表达情况,初步探索TAZ分子促进细胞增殖和迁移的作用机制。方法通过PCR扩增获得 TAZ基因片段,胶回收后连接T载体,蓝白斑筛选,转化,提质粒,酶切,用T4 DNA Ligase连接,亚克隆进入pEGFP‐C2和pcDNA31.获得新的重组质粒,分别转染 HEK293细胞,智能型荧光细胞监测仪观察TAZ分子在细胞内的分布情况,Western Blot检测其在细胞内的表达情况。结果重组质粒经双酶切鉴定和测序证明构建成功,荧光照片显示 TAZ分子分布在

  5. The First Attested Extraction of Ancient DNA in Legumes (Fabaceae).

    Science.gov (United States)

    Mikić, Aleksandar M

    2015-01-01

    Ancient DNA (aDNA) is any DNA extracted from ancient specimens, important for diverse evolutionary researches. The major obstacles in aDNA studies are mutations, contamination and fragmentation. Its studies may be crucial for crop history if integrated with human aDNA research and historical linguistics, both general and relating to agriculture. Legumes (Fabaceae) are one of the richest end economically most important plant families, not only from Neolithic onwards, since they were used as food by Neanderthals and Paleolithic modern man. The idea of extracting and analyzing legume aDNA was considered beneficial for both basic science and applied research, with an emphasis on genetic resources and plant breeding. The first reported successful and attested extraction of the legume aDNA was done from the sample of charred seeds of pea (Pisum sativum) and bitter vetch (Vicia ervilia) from Hissar, southeast Serbia, dated to 1,350-1,000 Before Christ. A modified version of cetyltrimethylammonium bromide (CTAB) method and the commercial kit for DNA extraction QIAGEN DNAesy yielded several ng μl(-1) of aDNA of both species and, after the whole genome amplification and with a fragment of nuclear ribosomal DNA gene 26S rDNA, resulted in the detection of the aDNA among the PCR products. A comparative analysis of four informative chloroplast DNA regions (trnSG, trnK, matK, and rbcL) among the modern wild and cultivated pea taxa demonstrated not only that the extracted aDNA was genuine, on the basis of mutation rate, but also that the ancient Hissar pea was most likely an early domesticated crop, related to the modern wild pea of a neighboring region. It is anticipated that this premier extraction of legume aDNA may provide taxonomists with the answers to diverse questions, such as leaf development in legumes, as well as with novel data on the single steps in domesticating legume crops worldwide.

  6. Microcalorimetric Studies on Gene Promoter Function of Cloned DNA Fragements from Halobacterium halobium J7 Plasmid pHH205 in Escherichia coli TG1

    Institute of Scientific and Technical Information of China (English)

    LEI,Ke-Lin; HOU,Han-Na; LIU,Yi; YE,Xue-Cheng; SHEN,Ping

    2007-01-01

    Halobacterium halobium is a typical kind of extremely halophilic bacterium. Combined with the antibiotic resistance assay, the microcalorimetric method was used to study the promoter function of the cloned DNA fragments from Halobacterium halobium J7 plasmid pHH205 in Escherichia coli TG1. The promoter probe vector, plasmid pKK232-8, was used to form the recombinants. The DNA fragment, which is the promoter for the chloramphenicol acetyl transferase (CAT) gene in plasmid pKK232-8, is about 800 bp, and the chloramphenicol resistance level presented by IC50 is about 200 μg·mL-1, which suggests a high promoter activity. The conclusions show that there probably exist double-function or trinary-function gene promoters in Halobacterium halobium, and Archaea may contain rich genetic resources.

  7. Comparison of three methods of DNA extraction in endocervical specimens for Chlamydia trachomatis infection by spectrophotometry, agarose gel, and PCR.

    Science.gov (United States)

    Jenab, Anahita; Roghanian, Rasoul; Golbang, Naser; Golbang, Pouran; Chamani-Tabriz, Leili

    2010-06-01

    Chlamydia trachomatis is the major cause of sexually transmitted disease in the world. The aim of this study was to determine the best method of DNA extraction for detecting C. trachomatis by polymerase chain reaction (PCR) in sexually active women (n = 80) attending Shahid Beheshti Hospital in Isfahan, Iran. Endocervical swabs were collected from 80 women, 22 of whom were asymptomatic and 58 symptomatic. Three different DNA extraction methods were used in this study (phenol-chlorophorm, proteinase K, and boiling). DNA yield was evaluated by spectrophotometry, agarose gel, and PCR. The internal control was assayed by beta-globin primers (PCO4, GH20). The DNA cryptic plasmid was selected as the target for C. trachomatis and samples were examined by PCR using specific KL1 and KL2 primers. It was shown that DNA extraction by boiling was the most sensitive with the highest yield of DNA. Of the 80 samples, 17 (21.25%) showed positivity for C. trachomatis by PCR. The highest rate of C. trachomatis infection was found in the group aged between 35 and 45 years old and those who used withdrawal or an intrauterine device as methods of contraception. It was demonstrated that DNA extraction by boiling was the least expensive and a very rapid method that gave the highest DNA yield. The infection rate in the sexually active women, including symptomatic and asymptomatic, was 21.25%, with a presumably high prevalence compared with other studies done in this field.

  8. Enhancing yields of low and single copy number plasmid DNAs from Escherichia coli cells.

    Science.gov (United States)

    Wood, Whitney N; Smith, Kyle D; Ream, Jennifer A; Kevin Lewis, L

    2017-02-01

    Many plasmids used for gene cloning and heterologous protein expression in Escherichia coli cells are low copy number or single copy number plasmids. The extraction of these types of plasmids from small bacterial cell cultures produces low DNA yields. In this study, we have quantitated yields of low copy and single copy number plasmid DNAs after growth of cells in four widely used broths (SB, SOC, TB, and 2xYT) and compared results to those obtained with LB, the most common E. coli cell growth medium. TB (terrific broth) consistently generated the greatest amount of plasmid DNA, in agreement with its ability to produce higher cell titers. The superiority of TB was primarily due to its high levels of yeast extract (24g/L) and was independent of glycerol, a unique component of this broth. Interestingly, simply preparing LB with similarly high levels of yeast extract (LB24 broth) resulted in plasmid yields that were equivalent to those of TB. By contrast, increasing ampicillin concentration to enhance plasmid retention did not improve plasmid DNA recovery. These experiments demonstrate that yields of low and single copy number plasmid DNAs from minipreps can be strongly enhanced using simple and inexpensive media. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. A RAPID PCR-QUALITY DNA EXTRACTION METHOD IN FISH

    Institute of Scientific and Technical Information of China (English)

    LI Zhong; LIANG Hong-Wei; ZOU Gui-Wei

    2012-01-01

    PCR has been a general preferred method for biological research in fish, and previous research have enabled us to extract and purify PCR-quality DNA templates in laboratories[1-4]. The same problem among these procedures is waiting for tissue digesting for a long time. The overabundance time spent on PCR-quality DNA extraction restricts the efficiency of PCR assay, especially in large-scale PCR amplification, such as SSR-based genetic-mapping construction [5,6], identification of germ plasm resource[7,8] and evolution research [9,10], etc. In this study, a stable and rapid PCR-quality DNA extraction method was explored, using a modified alkaline lysis protocol. Extracting DNA for PCR only takes approximately 25 minutes. This stable and rapid DNA extraction method could save much laboratory time and promotes.%PCR has been a general preferred method for biological research in fish,and previous research have enabled us to extract and purify PCR-quality DNA templates in laboratories [1-4].The same problem among these procedures is waiting for tissue digesting for a long time.The overabundance time spent on PCR-quality DNA extraction restricts the efficiency of PCR assay,especially in large-scale PCR amplification,such as SSR-based genetic-mapping construction [5,6],identification of germ plasm resource[7,8] and evolution research [9,10],etc.In this study,a stable and rapid PCR-quality DNA extraction method was explored,using a modified alkaline lysis protocol.Extracting DNA for PCR only takes approximately 25 minutes.This stable and rapid DNA extraction method could save much laboratory time and promotes.

  10. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    F Wegman

    2011-03-01

    Full Text Available Bone regeneration is one of the major focus points in the field of regenerative medicine. A well-known stimulus of bone formation is bone morphogenetic protein-2 (BMP-2, which has already been extensively used in clinical applications. We investigated the possibility of achieving osteogenic differentiation both in vitro and in vivo as a result of prolonged presence of BMP-2 using plasmid DNA-based gene therapy. By delivering BMP-2 cDNA in an alginate hydrogel, a versatile formulation is developed. High transfection efficiencies of up to 95% were obtained in both human multipotent stromal cells (MSCs and MG-63 cells using naked DNA in vitro. Over a period of 5 weeks, an increasing amount of biologically active BMP-2 was released from the cells and remained present in the gel. In vivo, transfected cells were found after both two and six weeks implantation in naked mice, even in groups without seeded cells, thus indicating in vivo transfection of endogenous cells. The protein levels were effective in inducing osteogenic differentiation in vitro, as seen by elevated alkaline phosphatase (ALP production and in vivo, as demonstrated by the production of collagen I and osteocalcin in a mineralised alginate matrix.

  11. Lichen metabolites prevent UV light and nitric oxide-mediated plasmid DNA damage and induce apoptosis in human melanoma cells.

    Science.gov (United States)

    Russo, A; Piovano, M; Lombardo, L; Garbarino, J; Cardile, V

    2008-09-26

    In humans both UV-A and UV-B can cause gene mutations and suppress immunity, which leads to skin cancer, including melanoma. Inhibition of reactive oxygen species (ROS) and reactive nitrogen species (RNS) appears particularly promising as ROS and RNS production by both UV-A and UV-B contributes to inflammation, immunosuppression, gene mutation and carcinogenesis. We evaluated the effect of two lichen compounds, sphaerophorin (depside) and pannarin (depsidone) on pBR322 DNA cleavage induced by hydroxyl radicals (()OH), and by nitric oxide (NO), and their superoxide anion (O(2)(-)) scavenging capacity. In addition, we investigated the growth inhibitory activity of these compounds against human melanoma cells (M14 cell line). Sphaerophorin and pannarin showed a protective effect on plasmid DNA and exhibited a superoxide dismutase like effect. The data obtained in cell culture show that these lichen metabolites inhibit the growth of melanoma cells, inducing an apoptotic cell death, demonstrated by the fragmentation of genomic DNA (COMET and TUNEL Assays) and by a significant increase of caspase-3 activity, and correlated, at least in part, to the increase of ROS generation, These results confirm the promising biological properties of sphaerophorin and pannarin and encourage further investigations on their molecular mechanisms.

  12. Single primer-mediated circular polymerase chain reaction for hairpin DNA cloning and plasmid editing.

    Science.gov (United States)

    Huang, Jiansheng; Khan, Inamullah; Liu, Rui; Yang, Yan; Zhu, Naishuo

    2016-05-01

    We developed and validated a universal polymerase chain reaction (PCR) method, single primer circular (SPC)-PCR, using single primer to simultaneously insert and amplify a short hairpin sequence into a vector with a high success rate. In this method, the hairpin structure is divided into two parts and fused into a vector by PCR. Then, a single primer is used to cyclize the chimera into a mature short hairpin RNA (shRNA) expression vector. It is not biased by loop length or palindromic structures. Six hairpin DNAs with short 4-nucleotide loops were successfully cloned. Moreover, SPC-PCR was also applied to plasmid editing within 3 h with a success rate higher than 95%.

  13. 亲和色谱纯化超螺旋质粒DNA的研究进展%RESEARCH PROGRESS OF AFFINITY CHROMATOGRAPHY IN PURIFICATION OF SUPERCOILED PLASMID DNA

    Institute of Scientific and Technical Information of China (English)

    白金山; 白姝

    2013-01-01

    非病毒载体质粒DNA已被广泛应用于基因治疗和DNA疫苗,目前迫切需要开发其大规模制备和分离纯化方法.亲和色谱是一种高分辨率、高选择性的分离技术,在蛋白质、抗体、核酸等生物大分子的分离纯化方面显示了良好的应用前景.本文综述了亲和色谱技术在超螺旋质粒DNA分离纯化中的研究进展,总结了各种亲和色谱方法分离超螺旋质粒DNA的机理和优缺点,并展望了亲和纯化技术在质粒DNA生产和制备中的应用前景.%Non-viral vector,plasmid DNA has been widely used in gene therapy and DNA vaccines.It is imperative to develop large-scale preparation and purification methods of plasmid DNA at present.As a separation technology of high resolution and high selectivity,affinity chromatography shows great application potential in terms of separation and purification of biological macromolecules such as proteins,antibodies,nucleic acids and so on.The domestic and foreign research progress of High Performance Liquid Chromatography (HPLC) technology,used in separation and purification of supercoiled plasmid DNA was reviewed in this paper.The advantages and disadvantages of various affinity chromatographic methods for separating supercoiled plasmid DNA were also summarized.At last,the affinity chromatography technology for preparation and purification of plasmid DNA was prospected.

  14. Detection of surface free radical activity of respirable industrial fibres using supercoiled phi X174 RF1 plasmid DNA.

    Science.gov (United States)

    Gilmour, P S; Beswick, P H; Brown, D M; Donaldson, K

    1995-12-01

    The ability of a number of respirable industrial fibres, amosite and crocidolite asbestos, refractory ceramic fibres (RCFs) and man-made vitreous fibres (MMVFs) to cause free radical injury to plasmid phi X174 RFI DNA was assessed. The oxidative DNA damage was observed as depletion of supercoiled DNA after fibre treatment was quantified by scanning laser densitometry. The mechanism of fibre-mediated damage was determined by the use of the specific hydroxyl radical scavenger mannitol and the iron chelator desferrioxamine-B. The amosite and crocidolite asbestos caused substantial damage to DNA that was dose-related. The free radicals responsible for the asbestos-mediated DNA damage were hydroxyl radicals, as determined by inhibition with mannitol. Asbestos fibre-mediated damage to DNA was completely ameliorated by the chelation of fibre-associated iron with desferrioxamine-B. The amount of Fe(II) and Fe(III) released by equal numbers of the different fibre types at equal fibre number was determined. The fibres released very small amounts of Fe(II) and there were no significant differences between the fibre types. The fibres released substantial amounts of Fe(III); MMVF 21 released significantly more Fe(III) than any of the other fibres and short fibre amosite also released more Fe(III) than three of the MMVFs and two of the RCFs. When ability to release Fe(II) and Fe(III) was compared with ability to cause DNA damage there was not a good correlation, because only the long amosite and crocidolite caused substantial free radical injury to DNA; this contrasts with MMVF 21 and short amosite being the two fibres that released the greatest amounts of iron. The loss of ability to damage DNA in DSF-B-treated asbestos fibres shows that iron at the surface of asbestos fibres definitely has a role in generating hydroxyl radicals. However, it is clear that some fibres, such as short amosite and MMVF 21, release large quantities of iron without causing free radical damage, whilst

  15. Construction of targeted plasmid vector pcDNA3.1-Egr.1p-p16 and its expression in pancreatic cancer JF305 cells induced by radiation in vitro

    Institute of Scientific and Technical Information of China (English)

    Hong-Bing Ma; Ming-Hua Bai; Xi-Jing Wang; Zheng-Li Di; Hui Xia; Zheng Li; Jie Liu; Jie Ma; Hua-Fen Kang; Cong-Mei Wu

    2007-01-01

    AIM: To construct pcDNA3.1-Egr.1p-p16 recombinant plasmid and investigate the expression of p16 in pancreatic cancer JF305 cells induced by radiation and the feasibility of gene radiotherapy for pancreatic carcinoma.METHODS: Human p16 cDNA was ligated to th edownstream of Egr-1 promotor to construct pcDNA3.1-Egr.1p-p16 plasmid by restriction enzyme digested. The recombined plasmids were transfected into pancreatic cancer JF305 cells with lipofectamine. p16 mRNA level was detected by RT-PCR. The expression of p16 after different doses of X-ray radiation was detected by Western blot technique. Cell survival was assessed by clonogenic assays and cell viability was analysed by trypen blue exclusion. Flow cytometry was performed to study the apoptosis of JF305 cells.RESULTS: Restriction enzyme digestion showed the correctly constructed pcDNA3.1-Egr.1p-p16. The p16expression in cells transfected with pcDNA3.1-Egr.1p-p16induced by different doses of radiation was higher than that in the control group (P < 0.05). Eight hours after 2 Gy X-ray radiation, the expression reached its peak(87.00 ng/L), and was significantly higher than that in the control group (P < 0.0.5). Clonogenic analysis and trypan blue extraction test showed that the pcDNA3.1-Egr.1p-p16 transfer enhanced radiation-induced cell killing in p16-null JF305 cell lines. The induction of apoptosis was lower in combined transfection and irradiation group than that in irradiation alone.CONCLUSION: X-ray can induce the recombinant plasmid pcDNA3.1-Egr.1p-p16 expression in JF305 cells.The detection of dose and time provides an experimental basis for in vivo study in future.

  16. Evaluation of six commercial DNA extraction kits for recovery of Burkholderia pseudomallei DNA.

    Science.gov (United States)

    Marques, Maria Angela de Mello; Zimmermann, Pia; Messelhäußer, Ute; Sing, Andreas

    2012-12-01

    Six commercially available DNA extraction kits, as well as thermal lysis and proteinase K DNA extraction were evaluated regarding bacterial inactivation, DNA yield and purity, and their use in a Burkholderia pseudomallei real-time PCR. While all methods successfully inactivated the bacteria, by measuring DNA purity and the level of detection by real-time PCR, the proteinase K method was the most sensitive.

  17. Chemoaffinity material for plasmid DNA analysis by high-performance liquid chromatography with condition-dependent switching between isoform and topoisomer selectivity.

    Science.gov (United States)

    Mahut, Marek; Gargano, Andrea; Schuchnigg, Hermann; Lindner, Wolfgang; Lämmerhofer, Michael

    2013-03-05

    Plasmid DNA may exist in three isoforms, the linear, open-circular (oc, "nicked"), and covalently closed circular (ccc, "supercoiled") form. We have recently reported on the chromatographic separation of supercoiled plasmid topoisomers on cinchona-alkaloid modified silica-based stationary phases. Herein, we present a selectivity switching mechanism to achieve separation of isoforms and/or supercoiled topoisomers using the very same chromatographic column and system. While salt gradient elution facilitates topoisomer separation, the supercoiled species are eluting as a single peak upon elution by a mixed pH and organic modifier gradient, still well separated from the other isoforms. We have found that a mobile phase pH value near the pI of the zwitterionic adsorbent surface leads to full recovery of all plasmid DNA isoforms, which is a major issue when using anion exchange-based resins. Furthermore, the observed elution pattern, oc < linear < ccc, is constant upon changes of mobile phase composition, gradient slope, and plasmid size. The remarkable isoform selectivity found on quinine-based selectors is explained by van't Hoff plots, revealing a different binding mechanism between the supercoiled plasmid on one hand and the oc and linear isoforms on the other hand.

  18. Development of microfluidic modules for DNA purification via phenol extraction and analyte concentration using transverse electrokinetics

    Science.gov (United States)

    Morales, Mercedes C.

    In this work, microfluidic platforms have been designed and evaluated to demonstrate microscale DNA purification via organic (phenol) extraction as well as analyte trapping and concentration using a transverse electrokinetic force balance. First, in order to evaluate DNA purification via phenol extraction in a microdevice, an aqueous phase containing protein and DNA and an immiscible receiving organic phase were utilized to evaluate microfluidic DNA extraction under both stratified and droplet-based flow conditions using a serpentine microfluidic device. The droplet based flow resulted in a significant improvement of protein partitioning from the aqueous phase due to the flow recirculation inside each droplet improving material convective transport into the organic phase. The plasmid recovery from bacterial lysates using droplet-based flow was high (>92%) and comparable to the recovery achieved using commercial DNA purification kits and standard macroscale phenol extraction. Second, a converging Y-inlet microfluidic channel with integrated coplanar electrodes was used to investigate transverse DNA and protein migration under uniform direct current (DC) electric fields. Negatively charged samples diluted in low and high ionic strength buffers were co-infused with a receiving buffer of the same ionic strength into a main channel where transverse electric fields were applied. Experimental results demonstrated that charged analytes could traverse the channel width and accumulate at the positive bias electrode in a low electroosmotic mobility and high electrophoretic mobility condition (high ionic strength buffer) or migrated towards an equilibrium position within the channel when both electroosmotic mobility and electrophoretic mobility are high (low ionic strength buffer). The different behaviors are the result of a balance between the electrophoretic force and a drag force induced by a recirculating electroosmotic flow generated across the channel width due to the

  19. An easy and versatile 2-step protocol for targeted modification and subcloning of DNA from bacterial artificial chromosomes using non-commercial plasmids

    Directory of Open Access Journals (Sweden)

    Hartwich Heiner

    2012-03-01

    Full Text Available Abstract Background Promoter-specific expression of foreign DNA in transgenic organisms often relies on bacterial artificial chromosomes (BACs. This approach requires modification and subcloning of BAC-DNA by recombineering technologies in Escherichia coli. Most current protocols rely on commercial kits or isolation of BACs, their transfer between different host strains, and their restriction. Findings In this report we present a 2-step protocol for efficient modification and subcloning of DNA from bacterial artificial chromosomes using the non-commercial plasmids pKM208 and pTP223, distributed from addgene.com. A targeting cassette was successfully integrated into a BAC and 42 kb of this construct were subcloned. Both a plasmid-derived substrate with longer homology arms and a PCR-generated substrate with short homology arms (50 bp were used for recombination. pKM208 and pTP223 contain all required genes for recombineering, but differ in their antibiotic resistance genes. This makes the system independent of the selection markers on the DNA molecules targeted for recombination. Conclusions The time and cost saving protocol presented here compares favorably to currently used systems. Using non-commercial plasmids, it allows targeted modification and cloning of large DNA (> 40 kb fragments in vivo without restriction and ligation. Furthermore, both steps are performed in the same host eliminating the need to isolate BAC DNA and to use different bacterial strains.

  20. The influence of substrate on DNA transfer and extraction efficiency.

    Science.gov (United States)

    Verdon, Timothy J; Mitchell, R John; van Oorschot, Roland A H

    2013-01-01

    The circumstances surrounding deposition of DNA profiles are increasingly becoming an issue in court proceedings, especially whether or not the deposit was made by primary transfer. In order to improve the currently problematic evaluation of transfer scenarios in court proceedings, we examined the influence a variety of nine substrate types (six varieties of fabric, plywood, tarpaulin, and plastic sheets) has on DNA transfer involving blood. DNA transfer percentages were significantly higher (p=0.03) when the primary substrate was of non-porous material (such as tarpaulin, plastic or, to a lesser degree, wood) and the secondary substrate porous (such as fabrics). These findings on transfer percentages confirm the results of previous studies. Fabric composition was also shown to have a significant (p=0.03) effect on DNA transfer; when experiments were performed with friction from a variety of fabrics to a specific weave of cotton, transfer percentages ranged from 4% (flannelette) to 94% (acetate). The propensity for the same nine substrates to impact upon the efficiency of DNA extraction procedures was also examined. Significant (p=0.03) differences were found among the extraction efficiencies from different materials. When 15μL of blood was deposited on each of the substrates, the lowest quantity of DNA was extracted from plastic (20ng) and the highest quantities extracted from calico and flannelette (650ng). Significant (pDNA extraction yield from different initial blood volumes from all substrates. Also, significantly greater (pDNA was seen during concentration of extracts with higher compared to lower initial quantities of DNA. These findings suggest that the efficiency of extraction and concentration impacts upon the final amount of DNA available for analysis and that consideration of these effects should not be ignored. The application of correction factors to adjust for any variation among extraction and concentration efficiencies among substrates is

  1. Preparation of chitosan-plasmid DNA nanoparticles encoding interleukin-12 and their expression in CT-26 colon carcinoma cells.

    Science.gov (United States)

    Hallaj-Nezhadi, Somayeh; Valizadeh, Hadi; Dastmalchi, Siavoush; Baradaran, Behzad; Jalali, Mohammad Barzegar; Dobakhti, Faramarz; Lotfipour, Farzaneh

    2011-01-01

    Interleukin-12 (Il-12) as a cytokine has been proved to possess antitumor effects via stimulating the immune system. Non-viral gene delivery systems exhibit low toxicity and are easier to prepare compared to their viral counterparts. In this study, we aimed to prepare plasmid DNA loaded chitosan nanoparticles for expression of Il-12 and to evaluate their physicochemical characteristics, cytotoxicity and transfection efficiency in Murine CT-26 colon carcinoma cells. Nanoparticles were prepared using a complex coacervation process at different N/P ratios and characterized in terms of size, zeta potential, polydispersity index, morphology, encapsulation efficiency and polyplex formation. The cytotoxicities and transfection efficiencies of the prepared polyplexes were evaluated by MTT assay and ELISA (for hIL-12, p70), respectively. Size and zeta potential varied from 76.73 to 867.03 nm and between 5.68 and 16.77 mV, respectively. Strong attachment of the DNA to chitosan was observed after polyplex preparation. Encapsulation efficiencies were high (72.97-94.87%). The transfection efficiencies of the prepared complexes were obviously higher than those of naked pDNA when N/P ratios were between 16 and 60. Maximum level of phIL-12 expression was obtained at (N/P = 16) with mean particle size of 381.83±82.77 nm (polydispersity index=0.44) indicating the improved transfection of pUMVC3-hIL12 about 2.80 times compared to that of the naked pUMVC3-hIL12. Prepared polyplexes were nontoxic to CT-26 cells. Chitosan-DNA nanoparticles at N/P = 16 with minimal cytotoxicity, can be used as suitable candidate for Il-12 delivery. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

  2. Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA.

    Science.gov (United States)

    Atanesyan, Lilit; Steenkamer, Maryvonne J; Horstman, Anja; Moelans, Cathy B; Schouten, Jan P; Savola, Suvi P

    2017-01-01

    Molecular genetic analysis of formalin-fixed, paraffin-embedded (FFPE) tissues is of great importance both for research and diagnostics. Multiplex ligation-dependent probe amplification (MLPA) is a widely used technique for gene copy number determination, and it has been successfully used for FFPE tissue-extracted DNA analysis. However, there have been no studies addressing the effect of tissue fixation procedures and DNA extraction methods on MLPA. This study therefore focuses on selecting optimal preanalytic conditions such as FFPE tissue preparation conditions and DNA extraction methods. Healthy tissues were fixed in buffered or nonbuffered formalin for 1 hour, 12 to 24 hours, or 48 to 60 hours at 4 °C or at room temperature. DNA extracted from differently fixed and subsequently paraffin-embedded tissues was used for MLPA. Four commercial DNA extraction kits and one in-house method were compared. Tissues fixed for 12 to 24 hours in buffered formalin at room temperature produced DNA with the most optimal quality for MLPA. The in-house FFPE DNA extraction method was shown to perform as efficient as or even superior to other methods in terms of suitability for MLPA, time and cost-efficiency, and ease of performance. FFPE-extracted DNA is well suitable for MLPA analysis, given that optimal tissue fixation and DNA extraction methods are chosen.

  3. Extracting DNA of nematodes communities from Argentine Pampas agricultural soils

    Directory of Open Access Journals (Sweden)

    Eduardo A. Mondino

    2015-06-01

    Full Text Available We examined four strategies (Tris/EDTA, sodium dodecyl sulfate, Chelex 100 resin and cetyltrimethylammonium bromide -CTAB- for extracting nucleic acid (DNA from communities of nematodes. Nematodes were isolated from an agricultural area under different management of long-term crop rotation experiment from Argentina during three seasons. After DNA extraction, Polymerase Chain Reaction-amplifications were performed and considered as indicators of successful DNA extraction. The CTAB combined with proteinase K and phenol-chloroform-isoamyl alcohol was the unique successful method because positive amplifications were obtained by using both eukaryotic and nematode specific primers. This work could contribute to biodiversity studies of nematodes on agroecosystems.

  4. Extracting DNA of nematodes communities from Argentine Pampas agricultural soils.

    Science.gov (United States)

    Mondino, Eduardo A; Covacevich, Fernanda; Studdert, Guillermo A; Pimentel, João P; Berbara, Ricardo L L

    2015-01-01

    We examined four strategies (Tris/EDTA, sodium dodecyl sulfate, Chelex 100 resin and cetyltrimethylammonium bromide -CTAB-) for extracting nucleic acid (DNA) from communities of nematodes. Nematodes were isolated from an agricultural area under different management of long-term crop rotation experiment from Argentina during three seasons. After DNA extraction, Polymerase Chain Reaction-amplifications were performed and considered as indicators of successful DNA extraction. The CTAB combined with proteinase K and phenol-chloroform-isoamyl alcohol was the unique successful method because positive amplifications were obtained by using both eukaryotic and nematode specific primers. This work could contribute to biodiversity studies of nematodes on agroecosystems.

  5. Effect of serotonin on the expression of antigens and DNA levels in Yersinia pestis cells with different plasmid content

    Science.gov (United States)

    Klueva, Svetlana N.; Korsukov, Vladimir N.; Schukovskaya, Tatyana N.; Kravtsov, Alexander L.

    2004-08-01

    Using flow cytometry (FCM) the influence of exogenous serotonin on culture growth, DNA content and fluorescence intensity of cells binding FITC-labelled plague polyclonal immunoglobulins was studied in Yersinia pestis EV (pFra+, pCad+, pPst+), Yersinia pestis KM218 (pFra-, pCad-, pPst-), Yersinia pestis KM 216 (pFra-, pCad-, pPst+). The results have been obtained by FCM showed serotonin accelerated Yersinia pestis EV (pFra+, pCad+, pPst+), Yersinia pestis KM218 (pFra-, pCad-, pPst-) culture growth during cultivation in Hottinger broth pH 7.2 at 28°C at concentration of 10-5 M. The presence of 10-5 M serotonin in nutrient broth could modulate DNA content in 37°C growing population of plague microbe independently of their plasmid content. Serotonin have been an impact on the distribution pattern of the cells according to their phenotypical characteristics, which was reflected in the levels of population heterogeneity in the intensity of specific immunofluorescence determined by FMC.

  6. Extraction of high quality DNA from bloodstains using diatoms.

    Science.gov (United States)

    Günther, S; Herold, J; Patzelt, D

    1995-01-01

    A simple method is described for the extraction of high quality DNA for PCR amplification. The DNA was extracted by using Chelex-100 ion exchange resin or a special cell lysis buffer containing proteinase K. For further purification the DNA was bound to silica in the presence of a chaotrophic agent. Hence it is possible to unlimitedly wash the bound DNA and inhibitory substances are removed. By using diatoms as a source of silicates, this method is very economical and can therefore be used as a routine method.

  7. A rapid and efficient assay for extracting DNA from fungi

    Science.gov (United States)

    Griffin, Dale W.; Kellogg, C.A.; Peak, K.K.; Shinn, E.A.

    2002-01-01

    Aims: A method for the rapid extraction of fungal DNA from small quantities of tissue in a batch-processing format was investigated. Methods and Results: Tissue (DNA for PCR/ sequencing applications. Conclusions: The method allowed batch DNA extraction from multiple fungal isolates using a simple yet rapid and reliable assay. Significance and Impact of the Study: Use of this assay will allow researchers to obtain DNA from fungi quickly for use in molecular assays that previously required specialized instrumentation, was time-consuming or was not conducive to batch processing.

  8. Exploring Antibiotic Resistance Genes and Metal Resistance Genes in Plasmid Metagenomes from Wastewater Treatment Plants

    Directory of Open Access Journals (Sweden)

    An-Dong eLi

    2015-09-01

    Full Text Available Plasmids operate as independent genetic elements in microorganism communities. Through horizontal gene transfer, they can provide their host microorganisms with important functions such as antibiotic resistance and heavy metal resistance. In this study, six metagenomic libraries were constructed with plasmid DNA extracted from influent, activated sludge and digested sludge of two wastewater treatment plants. Compared with the metagenomes of the total DNA extracted from the same sectors of the wastewater treatment plant, the plasmid metagenomes had significantly higher annotation rates, indicating that the functional genes on plasmids are commonly shared by those studied microorganisms. Meanwhile, the plasmid metagenomes also encoded many more genes related to defense mechanisms, including ARGs. Searching against an antibiotic resistance genes (ARGs database and a metal resistance genes (MRGs database revealed a broad-spectrum of antibiotic (323 out of a total 618 subtypes and metal resistance genes (23 out of a total 23 types on these plasmid metagenomes. The influent plasmid metagenomes contained many more resistance genes (both ARGs and MRGs than the activated sludge and the digested sludge metagenomes. Sixteen novel plasmids with a complete circular structure that carried these resistance genes were assembled from the plasmid metagenomes. The results of this study demonstrated that the plasmids in wastewater treatment plants could be important reservoirs for resistance genes, and may play a significant role in the horizontal transfer of these genes.

  9. Exploring antibiotic resistance genes and metal resistance genes in plasmid metagenomes from wastewater treatment plants.

    Science.gov (United States)

    Li, An-Dong; Li, Li-Guan; Zhang, Tong

    2015-01-01

    Plasmids operate as independent genetic elements in microorganism communities. Through horizontal gene transfer (HGT), they can provide their host microorganisms with important functions such as antibiotic resistance and heavy metal resistance. In this study, six metagenomic libraries were constructed with plasmid DNA extracted from influent, activated sludge (AS) and digested sludge (DS) of two wastewater treatment plants (WWTPs). Compared with the metagenomes of the total DNA extracted from the same sectors of the wastewater treatment plant, the plasmid metagenomes had significantly higher annotation rates, indicating that the functional genes on plasmids are commonly shared by those studied microorganisms. Meanwhile, the plasmid metagenomes also encoded many more genes related to defense mechanisms, including ARGs. Searching against an antibiotic resistance genes (ARGs) database and a metal resistance genes (MRGs) database revealed a broad-spectrum of antibiotic (323 out of a total 618 subtypes) and MRGs (23 out of a total 23 types) on these plasmid metagenomes. The influent plasmid metagenomes contained many more resistance genes (both ARGs and MRGs) than the AS and the DS metagenomes. Sixteen novel plasmids with a complete circular structure that carried these resistance genes were assembled from the plasmid metagenomes. The results of this study demonstrated that the plasmids in WWTPs could be important reservoirs for resistance genes, and may play a significant role in the horizontal transfer of these genes.

  10. Necessity of Purification during Bacterial DNA Extraction with Environmental Soils.

    Science.gov (United States)

    Lim, Hyun Jeong; Choi, Jung-Hyun; Son, Ahjeong

    2017-08-08

    Complexity and heterogeneity of soil samples have often implied the inclusion of purification steps in conventional DNA extraction for PCR assays. Unfortunately the purification steps are also time and labor intensive. Therefore the necessity of DNA purification was re-visited and investigated for a variety of environmental soil samples that contained various amounts of PCR inhibitors. Bead beating and centrifugation was used as the baseline (without purification) method for DNA extraction. Its performance was compared with that of conventional DNA extraction kit (with purification). The necessity criteria for DNA purification were established with environmental soil samples. Using lysis conditions at 3000 rpm for 3 minutes with 0.1 mm glass beads, centrifugation time of 10 minutes and 1:10 dilution ratio, the baseline method outperformed conventional DNA extraction on cell seeded sand samples. Further investigation with PCR inhibitors (i.e., humic acids, clay, and magnesium) showed that sand samples containing less than 10 ug/g humic acids and 70% clay may not require purifications. Interestingly, the inhibition pattern of magnesium ion was different from other inhibitors due to the complexation interaction of magnesium ion with DNA fragments. It was concluded that DNA extraction method without purification is suitable for soil samples that have less than 10 ug/g of humic acids, less than 70% clay content and less than 0.01% magnesium ion content.

  11. Extraction of DNA from plant and fungus tissues in situ

    Directory of Open Access Journals (Sweden)

    Abu Almakarem Amal S

    2012-06-01

    Full Text Available Abstract Background When samples are collected in the field and transported to the lab, degradation of the nucleic acids contained in the samples is frequently observed. Immediate extraction and precipitation of the nucleic acids reduces degradation to a minimum, thus preserving accurate sequence information. An extraction method to obtain high quality DNA in field studies is described. Findings DNA extracted immediately after sampling was compared to DNA extracted after allowing the sampled tissues to air dry at 21°C for 48 or 72 hours. While DNA extracted from fresh tissues exhibited little degradation, DNA extracted from all tissues exposed to 21°C air for 48 or 72 hours exhibited varying degrees of degradation. Yield was higher for extractions from fresh tissues in most cases. Four microcentrifuges were compared for DNA yield: one standard electric laboratory microcentrifuge (max rcf = 16,000×g, two battery-operated microcentrifuges (max rcf = 5,000 and 3,000 ×g, and one manually-operated microcentrifuge (max rcf = 120×g. Yields for all centrifuges were similar. DNA extracted under simulated field conditions was similar in yield and quality to DNA extracted in the laboratory using the same equipment. Conclusions This CTAB (cetyltrimethylammonium bromide DNA extraction method employs battery-operated and manually-operated equipment to isolate high quality DNA in the field. The method was tested on plant and fungus tissues, and may be adapted for other types of organisms. The method produced high quality DNA in laboratory tests and under simulated field conditions. The field extraction method should prove useful for working in remote sites, where ice, dry ice, and liquid nitrogen are unavailable; where degradation is likely to occur due to the long distances between the sample site and the laboratory; and in instances where other DNA preservation and transportation methods have been unsuccessful. It may be possible to adapt

  12. An efficient genomic DNA extraction from whole blood using Nextractor.

    Science.gov (United States)

    Jeong, Tae-Dong; Cho, Young-Uk; Lee, Woochang; Chun, Sail; Min, Won-Ki

    2014-08-05

    We evaluated the performance of the Nextractor NX-48 nucleic acid extractor system for the extraction of genomic DNA from whole blood samples. We compared the performance of the Nextractor to that of the QIAamp DNA Blood Mini Kit and the Maxwell system, using five whole blood samples. Extraction efficiencies were compared based on the total amount of extracted DNA adjusted by input blood volume, and the purity was compared. Polymerase chain reaction analyses were performed using ACTB as a target. The real-time PCR assay was carried out for housekeeping gene GAPDH. Total elapsed time for DNA extraction was compared. Extraction efficiencies for the QIAamp, Maxwell, and Nextractor were 25.4±3.8ng/μL, 9.2±0.6ng/μL, and 31.0±5.6ng/μL, respectively. No significant differences in purity were observed among three methods. DNA extracted using the ACTB was successfully amplified in all three methods. There were no obvious differences in Ct values for GAPDH real-time PCR. Total elapsed time for DNA extraction was about 50min for the QIAamp, 40min for the Maxwell, and 20min for the Nextractor. As the Nextractor is faster and requires less hands-on time than manual procedures, it may be useful for molecular diagnostic testing in clinical laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Optimizing a Method for the Quantification by Quantitative Real-Time Polymerase Chain Reaction of Host Cell DNA in Plasmid Vector Batches Used in Human Gene Therapy.

    Science.gov (United States)

    Ferro, Serge; Fabre, Isabelle; Chenivesse, Xavier

    2016-08-01

    Gene therapy products are very complex advanced therapy medicinal products produced using different processes that require many chemical and biological reagents and production intermediates, such as producing cells. The quantification of residual impurities in gene therapy vectors is a major quality control step when these vectors are used for therapeutic purposes, whether or not they are derived from viruses. Indeed, in nonviral gene therapy products, particularly plasmid vectors used to transfer genetic material, the presence of host-cell DNA (HCDNA) from the bacterial cells used for the vector production is an important concern because of the risk of immunogenicity and insertional mutagenesis. Several methods have been developed to quantify residual HCDNA, but real-time quantitative polymerase chain reaction (qPCR) seems to be most suitable because it allows detecting traces of "contaminating" DNA. The French National Agency for Medicines and Health Products Safety (ANSM) ensures the quality and safety of gene transfer medicinal products and must be able to quantify, in its own laboratories, the amount of HCDNA present in plasmid vector batches. Therefore, we developed and validated a qPCR method to quantify at the femtogram level the presence of Escherichia coli residual DNA in plasmid vectors. This approach uses the capillary-based LightCycler 1.5 System (Roche) with SYBR Green I, a primer pair against the E. coli 23S ribosomal RNA gene and different concentrations of a linearized plasmid that contains the 23S target sequence, as standard. This qPCR method is linear on an 8-decade logarithmic scale, accurate, reproducible, and sensitive (quantification of up to 10 copies of 23S target sequence per reaction, or 1.4 E. coli genome, or 7 fg of bacterial DNA). This technique allows ensuring that batches of plasmid vectors to be used in clinical trials comply with the specifications on HCDNA content.

  14. Characterization of a linear DNA plasmid from the filamentous fungal plant pathogen Glomerella musae [Anamorph: Colletotrichum musae (Berk. and Curt.) arx.

    Science.gov (United States)

    Freeman, S.; Redman, R.S.; Grantham, G.; Rodriguez, R.J.

    1997-01-01

    A 7.4-kilobase (kb) DNA plasmid was isolated from Glomerella musae isolate 927 and designated pGML1. Exonuclease treatments indicated that pGML1 was a linear plasmid with blocked 5' termini. Cell-fractionation experiments combined with sequence-specific PCR amplification revealed that pGML1 resided in mitochondria. The pGML1 plasmid hybridized to cesium chloride-fractionated nuclear DNA but not to A + T-rich mitochondrial DNA. An internal 7.0-kb section of pGML1 was cloned and did not hybridize with either nuclear or mitochondrial DNA from G. musae. Sequence analysis revealed identical terminal inverted repeats (TIR) of 520 bp at the ends of the cloned 7.0-kb section of pGML1. The occurrence of pGML1 did not correspond with the pathogenicity of G. musae on banana fruit. Four additional isolates of G. musae possessed extrachromosomal DNA fragments similar in size and sequence to pGML1.

  15. Rapid extraction and preservation of genomic DNA from human samples.

    Science.gov (United States)

    Kalyanasundaram, D; Kim, J-H; Yeo, W-H; Oh, K; Lee, K-H; Kim, M-H; Ryew, S-M; Ahn, S-G; Gao, D; Cangelosi, G A; Chung, J-H

    2013-02-01

    Simple and rapid extraction of human genomic DNA remains a bottleneck for genome analysis and disease diagnosis. Current methods using microfilters require cumbersome, multiple handling steps in part because salt conditions must be controlled for attraction and elution of DNA in porous silica. We report a novel extraction method of human genomic DNA from buccal swab and saliva samples. DNA is attracted onto a gold-coated microchip by an electric field and capillary action while the captured DNA is eluted by thermal heating at 70 °C. A prototype device was designed to handle four microchips, and a compatible protocol was developed. The extracted DNA using microchips was characterized by qPCR for different sample volumes, using different lengths of PCR amplicon, and nuclear and mitochondrial genes. In comparison with a commercial kit, an equivalent yield of DNA extraction was achieved with fewer steps. Room-temperature preservation for 1 month was demonstrated for captured DNA, facilitating straightforward collection, delivery, and handling of genomic DNA in an environment-friendly protocol.

  16. A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples.

    Science.gov (United States)

    Claassen, Shantelle; du Toit, Elloise; Kaba, Mamadou; Moodley, Clinton; Zar, Heather J; Nicol, Mark P

    2013-08-01

    Differences in the composition of the gut microbiota have been associated with a range of diseases using culture-independent methods. Reliable extraction of nucleic acid is a key step in identifying the composition of the faecal microbiota. Five widely used commercial deoxyribonucleic acid (DNA) extraction kits (QIAsymphony® Virus/Bacteria Midi Kit (kit QS), ZR Fecal DNA MiniPrep™ (kit Z), QIAamp® DNA Stool Mini Kit (kit QA), Ultraclean® Fecal DNA Isolation Kit (kit U) and PowerSoil® DNA Isolation Kit (kit P)) were evaluated, using human faecal samples. Yield, purity and integrity of total genomic DNA were compared spectrophotometrically and using gel electrophoresis. Three bacteria, commonly found in human faeces were quantified using real time polymerase chain reaction (qPCR) and total bacterial diversity was studied using denaturing gradient gel electrophoresis (DGGE) as well as terminal restriction fragment length polymorphism (T-RFLP). The measurements of DNA yield and purity exhibited variations between the five kits tested in this study. Automated kit QS exhibited the best quality and highest quantity of DNA. All kits were shown to be reproducible with CV values≤0.46 for DNA extraction. qPCR results showed that all kits were uniformly efficient for extracting DNA from the selected target bacteria. DGGE and T-RFLP produced the highest diversity scores for DNA extracted using kit Z (H'=2.30 and 1.27) and kit QS (H'=2.16 and 0.94), which also extracted the highest DNA yields compared to the other kits assessed. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. A Novel Method of Genomic DNA Extraction for Cactaceae

    Directory of Open Access Journals (Sweden)

    Shannon D. Fehlberg

    2013-03-01

    Full Text Available Premise of the study: Genetic studies of Cactaceae can at times be impeded by difficult sampling logistics and/or high mucilage content in tissues. Simplifying sampling and DNA isolation through the use of cactus spines has not previously been investigated. Methods and Results: Several protocols for extracting DNA from spines were tested and modified to maximize yield, amplification, and sequencing. Sampling of and extraction from spines resulted in a simplified protocol overall and complete avoidance of mucilage as compared to typical tissue extractions. Sequences from one nuclear and three plastid regions were obtained across eight genera and 20 species of cacti using DNA extracted from spines. Conclusions: Genomic DNA useful for amplification and sequencing can be obtained from cactus spines. The protocols described here are valuable for any cactus species, but are particularly useful for investigators interested in sampling living collections, extensive field sampling, and/or conservation genetic studies.

  18. Comparison of four methods of DNA extraction from rice

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    @@ Polyphenols, teroens, and resins make it difficult to obtain high quality genomic DNA from rice. Four extraction methods were compared in our study, and CTAB precipitation was the most practical one.

  19. A novel method of genomic DNA extraction for Cactaceae1

    Science.gov (United States)

    Fehlberg, Shannon D.; Allen, Jessica M.; Church, Kathleen

    2013-01-01

    • Premise of the study: Genetic studies of Cactaceae can at times be impeded by difficult sampling logistics and/or high mucilage content in tissues. Simplifying sampling and DNA isolation through the use of cactus spines has not previously been investigated. • Methods and Results: Several protocols for extracting DNA from spines were tested and modified to maximize yield, amplification, and sequencing. Sampling of and extraction from spines resulted in a simplified protocol overall and complete avoidance of mucilage as compared to typical tissue extractions. Sequences from one nuclear and three plastid regions were obtained across eight genera and 20 species of cacti using DNA extracted from spines. • Conclusions: Genomic DNA useful for amplification and sequencing can be obtained from cactus spines. The protocols described here are valuable for any cactus species, but are particularly useful for investigators interested in sampling living collections, extensive field sampling, and/or conservation genetic studies. PMID:25202521

  20. Influence of EDTA and magnesium on DNA extraction from blood ...

    African Journals Online (AJOL)

    Influence of EDTA and magnesium on DNA extraction from blood samples and specificity of polymerase chain reaction. ... African Journal of Biotechnology ... of initial EDTA level added to blood samples on quantity and quality of genomic DNA ...

  1. DNA extraction for streamlined metagenomics of diverse environmental samples.

    Science.gov (United States)

    Marotz, Clarisse; Amir, Amnon; Humphrey, Greg; Gaffney, James; Gogul, Grant; Knight, Rob

    2017-06-01

    A major bottleneck for metagenomic sequencing is rapid and efficient DNA extraction. Here, we compare the extraction efficiencies of three magnetic bead-based platforms (KingFisher, epMotion, and Tecan) to a standardized column-based extraction platform across a variety of sample types, including feces, oral, skin, soil, and water. Replicate sample plates were extracted and prepared for 16S rRNA gene amplicon sequencing in parallel to assess extraction bias and DNA quality. The data demonstrate that any effect of extraction method on sequencing results was small compared with the variability across samples; however, the KingFisher platform produced the largest number of high-quality reads in the shortest amount of time. Based on these results, we have identified an extraction pipeline that dramatically reduces sample processing time without sacrificing bacterial taxonomic or abundance information.

  2. Radioprotection against DNA damage by an extract of Indian green mussel, Perna viridis (L.)

    Digital Repository Service at National Institute of Oceanography (India)

    Kumaran, S.P.; Kutty, B.C.; Chatterji, A.; Parameswaran, P.S.; Mishra, K.P.

    This study describes the radioprotective ability of a hydrolysate prepared using an enzymeacid hydrolysis method from the green mussel Perna viridis in terms of its ability to prevent radiation-induced damage in plasmid DNA, cell death, reactive...

  3. Preparation and Characterization of Cationic PLA-PEG Nanoparticles for Delivery of Plasmid DNA

    Science.gov (United States)

    Zou, Weiwei; Liu, Chunxi; Chen, Zhijin; Zhang, Na

    2009-09-01

    The purpose of the present work was to formulate and evaluate cationic poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) nanoparticles as novel non-viral gene delivery nano-device. Cationic PLA-PEG nanoparticles were prepared by nanoprecipitation method. The gene loaded nanoparticles were obtained by incubating the report gene pEGFP with cationic PLA-PEG nanoparticles. The physicochemical properties (e.g., morphology, particle size, surface charge, DNA binding efficiency) and biological properties (e.g., integrity of the released DNA, protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in Hela cells) of the gene loaded PLA-PEG nanoparticles were evaluated, respectively. The obtained cationic PLA-PEG nanoparticles and gene loaded nanoparticles were both spherical in shape with average particle size of 89.7 and 128.9 nm, polydispersity index of 0.185 and 0.161, zeta potentials of +28.9 and +16.8 mV, respectively. The obtained cationic PLA-PEG nanoparticles with high binding efficiency (>95%) could protect the loaded DNA from the degradation by nuclease and plasma. The nanoparticles displayed sustained-release properties in vitro and the released DNA maintained its structural and functional integrity. It also showed lower cytotoxicity than Lipofectamine 2000 and could successfully transfect gene into Hela cells even in presence of serum. It could be concluded that the established gene loaded cationic PLA-PEG nanoparticles with excellent properties were promising non-viral nano-device, which had potential to make cancer gene therapy achievable.

  4. Preparation and Characterization of Cationic PLA-PEG Nanoparticles for Delivery of Plasmid DNA

    Directory of Open Access Journals (Sweden)

    Zou Weiwei

    2009-01-01

    Full Text Available Abstract The purpose of the present work was to formulate and evaluate cationic poly(lactic acid-poly(ethylene glycol (PLA-PEG nanoparticles as novel non-viral gene delivery nano-device. Cationic PLA-PEG nanoparticles were prepared by nanoprecipitation method. The gene loaded nanoparticles were obtained by incubating the report gene pEGFP with cationic PLA-PEG nanoparticles. The physicochemical properties (e.g., morphology, particle size, surface charge, DNA binding efficiency and biological properties (e.g., integrity of the released DNA, protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in Hela cells of the gene loaded PLA-PEG nanoparticles were evaluated, respectively. The obtained cationic PLA-PEG nanoparticles and gene loaded nanoparticles were both spherical in shape with average particle size of 89.7 and 128.9 nm, polydispersity index of 0.185 and 0.161, zeta potentials of +28.9 and +16.8 mV, respectively. The obtained cationic PLA-PEG nanoparticles with high binding efficiency (>95% could protect the loaded DNA from the degradation by nuclease and plasma. The nanoparticles displayed sustained-release properties in vitro and the released DNA maintained its structural and functional integrity. It also showed lower cytotoxicity than Lipofectamine 2000 and could successfully transfect gene into Hela cells even in presence of serum. It could be concluded that the established gene loaded cationic PLA-PEG nanoparticles with excellent properties were promising non-viral nano-device, which had potential to make cancer gene therapy achievable.

  5. A Novel Method of Genomic DNA Extraction for Cactaceae

    OpenAIRE

    Fehlberg, Shannon D.; Jessica M. Allen; Kathleen Church

    2013-01-01

    • Premise of the study: Genetic studies of Cactaceae can at times be impeded by difficult sampling logistics and/or high mucilage content in tissues. Simplifying sampling and DNA isolation through the use of cactus spines has not previously been investigated. • Methods and Results: Several protocols for extracting DNA from spines were tested and modified to maximize yield, amplification, and sequencing. Sampling of and extraction from spines resulted in a simplified protocol overall and compl...

  6. Escherichia coli DNA helicase I catalyzes a sequence-specific cleavage/ligation reaction at the F plasmid origin of transfer.

    Science.gov (United States)

    Sherman, J A; Matson, S W

    1994-10-21

    Recent studies have shown that the Escherichia coli F plasmid-encoded traI gene product (TraIp), also known as DNA helicase I, catalyzes the formation of the site- and strand-specific nick that initiates F plasmid DNA transfer. Scission of the phosphodiester bond at the nic site within the origin of transfer (oriT) is accompanied by the covalent attachment of TraIp to the 5'-phosphate of the nicked DNA strand. This mechanism suggests that TraIp may also be capable of catalyzing a DNA ligation reaction using the energy stored in the protein-DNA intermediate. To test this possibility, an in vitro assay was designed that utilized short single-stranded DNA oligonucleotides of different lengths derived from the region within oriT that spanned the nic site. Purified TraIp was capable of efficiently cleaving single-stranded DNA that contained a nic site, and upon cleavage, the protein became covalently linked to the 5'-end of the nic site. When TraIp was incubated with two oligonucleotides of different length that contained the nic site, there was formation of novel recombinant products resulting from a TraIp-catalyzed cleavage/ligation reaction. Furthermore, the cleavage and ligation reactions were both sequence-specific. These data suggest that TraIp plays an important role in the initiation and termination of conjugative DNA transfer.

  7. Differential Salt-Induced Dissociation of the p53 Protein Complexes with Circular and Linear Plasmid DNA Substrates Suggest Involvement of a Sliding Mechanism

    Directory of Open Access Journals (Sweden)

    Peter Šebest

    2015-01-01

    Full Text Available A study of the effects of salt conditions on the association and dissociation of wild type p53 with different ~3 kbp long plasmid DNA substrates (supercoiled, relaxed circular and linear, containing or lacking a specific p53 binding site, p53CON using immunoprecipitation at magnetic beads is presented. Salt concentrations above 200 mM strongly affected association of the p53 protein to any plasmid DNA substrate. Strikingly different behavior was observed when dissociation of pre-formed p53-DNA complexes in increased salt concentrations was studied. While contribution from the p53CON to the stability of the p53-DNA complexes was detected between 100 and 170 mM KCl, p53 complexes with circular DNAs (but not linear exhibited considerable resistance towards salt treatment for KCl concentrations as high as 2 M provided that the p53 basic C-terminal DNA binding site (CTDBS was available for DNA binding. On the contrary, when the CTDBS was blocked by antibody used for immunoprecipitation, all p53-DNA complexes were completely dissociated from the p53 protein in KCl concentrations ≥200 mM under the same conditions. These observations suggest: (a different ways for association and dissociation of the p53-DNA complexes in the presence of the CTDBS; and (b a critical role for a sliding mechanism, mediated by the C-terminal domain, in the dissociation process.

  8. Effect of the Plasmid-DNA Vaccination on Macroscopic and Microscopic Damage Caused by the Experimental Chronic Trypanosoma cruzi Infection in the Canine Model

    Science.gov (United States)

    Rodríguez-Morales, Olivia; Carrillo-Sánchez, Silvia C.; García-Mendoza, Humberto; Aranda-Fraustro, Alberto; Ballinas-Verdugo, Martha A.; Alejandre-Aguilar, Ricardo; Rosales-Encina, José Luis; Arce-Fonseca, Minerva

    2013-01-01

    The dog is considered the main domestic reservoir for Trypanosoma cruzi infection and a suitable experimental animal model to study the pathological changes during the course of Chagas disease (CD). Vaccine development is one of CD prevention methods to protect people at risk. Two plasmids containing genes encoding a trans-sialidase protein (TcSP) and an amastigote-specific glycoprotein (TcSSP4) were used as DNA vaccines in a canine model. Splenomegaly was not found in either of the recombinant plasmid-immunized groups; however, cardiomegaly was absent in animals immunized only with the plasmid containing the TcSSP4 gene. The inflammation of subendocardial and myocardial tissues was prevented only with the immunization with TcSSP4 gene. In conclusion, the vaccination with these genes has a partial protective effect on the enlargement of splenic and cardiac tissues during the chronic CD and on microscopic hearth damage, since both plasmids prevented splenomegaly but only one avoided cardiomegaly, and the lesions in heart tissue of dog immunized with plasmid containing the TcSSP4 gene covered only subepicardial tissue. PMID:24163822

  9. Effect of the Plasmid-DNA Vaccination on Macroscopic and Microscopic Damage Caused by the Experimental Chronic Trypanosoma cruzi Infection in the Canine Model

    Directory of Open Access Journals (Sweden)

    Olivia Rodríguez-Morales

    2013-01-01

    Full Text Available The dog is considered the main domestic reservoir for Trypanosoma cruzi infection and a suitable experimental animal model to study the pathological changes during the course of Chagas disease (CD. Vaccine development is one of CD prevention methods to protect people at risk. Two plasmids containing genes encoding a trans-sialidase protein (TcSP and an amastigote-specific glycoprotein (TcSSP4 were used as DNA vaccines in a canine model. Splenomegaly was not found in either of the recombinant plasmid-immunized groups; however, cardiomegaly was absent in animals immunized only with the plasmid containing the TcSSP4 gene. The inflammation of subendocardial and myocardial tissues was prevented only with the immunization with TcSSP4 gene. In conclusion, the vaccination with these genes has a partial protective effect on the enlargement of splenic and cardiac tissues during the chronic CD and on microscopic hearth damage, since both plasmids prevented splenomegaly but only one avoided cardiomegaly, and the lesions in heart tissue of dog immunized with plasmid containing the TcSSP4 gene covered only subepicardial tissue.

  10. A simplified universal genomic DNA extraction protocol suitable for PCR.

    Science.gov (United States)

    Wang, T Y; Wang, L; Zhang, J H; Dong, W H

    2011-03-29

    Conventional genomic DNA extraction protocols need expensive and hazardous reagents for decontamination of phenolic compounds from the extracts and are only suited for certain types of tissue. We developed a simple, time-saving and cost-efficient method for genomic DNA extraction from various types of organisms, using relatively innocuous reagents. The protocol employs a single purification step to remove contaminating compounds, using a silica column and a non-hazardous buffer, and a chaotropic-detergent lysing solution that hydrolyzes RNA and allows the selective precipitation of DNA from cell lysates. We used this system to extract genomic DNA from different tissues of various organisms, including algae (Dunaliella salina), human peripheral blood, mouse liver, Escherichia coli, and Chinese hamster ovary cells. Mean DNA yields were 20-30 μg/cm(3) from fresh tissues (comparable to yields given by commercial extraction kits), and the 260/280 nm absorbance ratio was 1.8-2.0, demonstrating a good degree of purity. The extracted DNA was successfully used in PCR, restriction enzyme digestion and for recombinant selection studies.

  11. Anchoring of self-assembled plasmid DNA/ anti-DNA antibody/cationic lipid micelles on bisphosphonate-modified stent for cardiovascular gene delivery

    Directory of Open Access Journals (Sweden)

    Ma G

    2013-03-01

    Full Text Available Guilei Ma,1,# Yong Wang,1,# Ilia Fishbein,2 Mei Yu,1 Linhua Zhang,1 Ivan S Alferiev,2 Jing Yang,1 Cunxian Song,1 Robert J Levy2 1Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China; 2Children's Hospital of Philadelphia, Abramson Research Building, Philadelphia, PA, USA #These authors contributed equally to this work Purpose: To investigate the anchoring of plasmid DNA/anti-DNA antibody/cationic lipid tri-complex (DAC micelles onto bisphosphonate-modified 316 L coronary stents for cardiovascular site-specific gene delivery. Methods: Stents were first modified with polyallylamine bisphosphonate (PAA-BP, thereby enabling the retention of a PAA-BP molecular monolayer that permits the anchoring (via vector-binding molecules of DAC micelles. DAC micelles were then chemically linked onto the PAA-BP-modified stents by using N-succinimidyl-3-(2-pyridyldithiol-propionate (SPDP as a crosslinker. Rhodamine-labeled DNA was used to assess the anchoring of DAC micelles, and radioactive-labeled antibody was used to evaluate binding capacity and stability. DAC micelles (encoding green fluorescent protein were tethered onto the PAA-BP-modified stents, which were assessed in cell culture. The presence of a PAA-BP molecular monolayer on the steel surface was confirmed by X-ray photoelectron spectroscopy and atomic force microscope analysis. Results: The anchoring of DAC micelles was generally uniform and devoid of large-scale patches of defects. Isotopic quantification confirmed that the amount of antibody chemically linked on the stents was 17-fold higher than that of the physical adsorbed control stents and its retention time was also significantly longer. In cell culture, numerous green fluorescent protein-positive cells were found on the PAA-BP modified stents, which demonstrated high localization and efficiency of gene delivery. Conclusion: The DAC micelle

  12. Optimization of DNA extraction for advancing coral microbiota investigations.

    Science.gov (United States)

    Weber, Laura; DeForce, Emelia; Apprill, Amy

    2017-02-08

    DNA-based sequencing approaches are commonly used to identify microorganisms and their genes and document trends in microbial community diversity in environmental samples. However, extraction of microbial DNA from complex environmental samples like corals can be technically challenging, and extraction methods may impart biases on microbial community structure. We designed a two-phase study in order to propose a comprehensive and efficient method for DNA extraction from microbial cells present in corals and investigate if extraction method influences microbial community composition. During phase I, total DNA was extracted from seven coral species in a replicated experimental design using four different MO BIO Laboratories, Inc., DNA Isolation kits: PowerSoil®, PowerPlant® Pro, PowerBiofilm®, and UltraClean® Tissue & Cells (with three homogenization permutations). Technical performance of the treatments was evaluated using DNA yield and amplification efficiency of small subunit ribosomal RNA (SSU ribosomal RNA (rRNA)) genes. During phase II, potential extraction biases were examined via microbial community analysis of SSU rRNA gene sequences amplified from the most successful DNA extraction treatments. In phase I of the study, the PowerSoil® and PowerPlant® Pro extracts contained low DNA concentrations, amplified poorly, and were not investigated further. Extracts from PowerBiofilm® and UltraClean® Tissue and Cells permutations were further investigated in phase II, and analysis of sequences demonstrated that overall microbial community composition was dictated by coral species and not extraction treatment. Finer pairwise comparisons of sequences obtained from Orbicella faveolata, Orbicella annularis, and Acropora humilis corals revealed subtle differences in community composition between the treatments; PowerBiofilm®-associated sequences generally had higher microbial richness and the highest coverage of dominant microbial groups in comparison to the Ultra

  13. Rapid Extraction of Human DNA Containing Humic Acid

    OpenAIRE

    Sutlović, Davorka; Definis Gojanović, Marija; Anđelinović, Šimun

    2007-01-01

    The identification process of dead bodies or human remains is nowadays conducted in numerous fields of forensic science, archeology and other judicial cases. A particular problem is the isolation and DNA typing of human remains found in mass graves, due to the degradation process, as well as post mortal DNA contamination with bacteria, fungi, humic acids, metals, etc. In this study, the influence of humic acid (HA) on the DNA extraction and typing is investigated. If present in...

  14. A Comparison of DNA Extraction Methods using Petunia hybrida Tissues

    OpenAIRE

    Tamari, Farshad; Hinkley, Craig S.; Ramprashad, Naderia

    2013-01-01

    Extraction of DNA from plant tissue is often problematic, as many plants contain high levels of secondary metabolites that can interfere with downstream applications, such as the PCR. Removal of these secondary metabolites usually requires further purification of the DNA using organic solvents or other toxic substances. In this study, we have compared two methods of DNA purification: the cetyltrimethylammonium bromide (CTAB) method that uses the ionic detergent hexadecyltrimethylammonium brom...

  15. Plasmid DNA studies in Lactobacillus plantarum strains isolated from olive fermentations: production of and immunity to plantaricin OL15 is associated to a 9.6 Kb plasmid (pOL15

    Directory of Open Access Journals (Sweden)

    Mourad, Kacem

    2007-06-01

    Full Text Available Previously 12 Lactobacillus plantarum strains were isolated from fermented olives. Among these, only L. plantarum OL15 produced bacteriocin (plantaricin OL15. In this study, the 12 strains were examined for plasmid DNA content. Of these, 9 strains have shown one to three plasmid bands ranging in size from 5.4 to 12.2 kb. L. plantarum OL15 exhibited one plasmid (9.6 kb which was named pOL15. After curing with novobiocin and ethidium bromide, the plasmid profile analysis of non producing derivatives, showed that the 9.6 kb plasmid pOL15 harbored by the parental strain had been lost in all cases and none of them regained the ability to produce plantaricin OL15 suggesting that the production of plantaricin OL15 is plasmid linked. Plantaricin OL15 was not inactived by amylase and lipase suggesting that plantaricin OL15 activity was not dependent on the presence of either a carbohydrate or lipid moiety. Plantaricin OL15 showed activity against lactic acid bacteria of different species and also against olive spoilage and phytopathogenic bacteria, including Pseudomonas and Erwinia.En un estudio previo, se aislaron 12 cepas de Lactobacillus plantarum a partir de aceitunas fermentadas. Entre ellas, solo L. plantarum OL15 produjo bacteriocinas (plantaricin OL15. En este estudio, se examinó el contenido de AND plásmido en las 12 cepas citadas. Entre ellas, 9 cepas han mostrado de una a tres bandas de plásmido con tamaños en el rango de 5.4 a 12.2 kb. L. plantarum OL15 exhibió un plásmido (9.6 kb que se denominó pOL15. Después del curado con novobiocina y bromuro de etidio, la pérdida del plásmido pOL15 asociada a la pérdida de su facultad para producir plantaricin OL15, sugiere que la producción de plantaricina OL15 está ligada al plásmido. La plantaricin OL15 no se inactivó por amilasa ni por lipasa sugiriendo que su actividad no es dependiente de la presencia de carbohidratos o lípidos. La plantaricina OL15 mostró actividad frente a

  16. Extracting biological knowledge from DNA sequences

    Energy Technology Data Exchange (ETDEWEB)

    De La Vega, F.M. [CINVESTAV-IPN (Mexico); Thieffry, D. [Universite Libre de Bruxelles, Rhode-Saint-Genese (Belgium)]|[Universidad Nacional Autonoma de Mexico, Morelos (Mexico); Collado-Vides, J. [Universidad Nacional Autonoma de Mexico, Morelos (Mexico)

    1996-12-31

    This session describes the elucidation of information from dna sequences and what challenges computational biologists face in their task of summarizing and deciphering the human genome. Techniques discussed include methods from statistics, information theory, artificial intelligence and linguistics. 1 ref.

  17. Coimmunization with IL-15 plasmid enhances the longevity of CD8 T cells induced by DNA encoding hepatitis B virus core antigen

    Institute of Scientific and Technical Information of China (English)

    Wei Zhang; Sheng-Fu Dong; Shu-Hui Sun; Yuan Wang; Guang-Di Li; Di Qu

    2006-01-01

    AIM: To test the feasibility of delivering a plasmid encoding IL-15 as a DNA vaccine adjuvant for improving the immune responses induced by hepatitis B virus core gene DNA vaccine.METHODS: We used RT-PCR based strategies to develop IL-15 expression constructs. We first confirmed that the gene could be expressed in Escherichia coli due to the poor expression of IL-15. Then the bioactivity of IL-15 plasmid expression product was identified by CTLL-2 proliferation assay. One hundred micrograms of DNA from each of the IL-15 eukaryotic expressed plasmid and the recombinant plasmid harboring DNA encoding the 144 amino acids of the N-terminus of HBV core gene (abbreviated pHBc144) was used to co-immunize C57 BL/6 mice. The titer of anti-HBcIgG was detected by ELISA and the antigen-specific CD8+T cells (CD8+IFN-γ+ T cells) were detected by intracellular cytokine staining at different time points.RESULTS: After co-immunization by pIL-15 and pHBc144 DNA vaccine the antigen-specific CD8+ cells of mice increased gradually, the first peak of immune response appeared 14 d later, then the number of antigen-specific CD8+ Ts cells decreased gradually and maintained at a steady level in 3 mo. After boosting, the number of antigen-specific CD8+ T cells reached the second peak 10 d later with a double of the 1st peak, then the number of antigen-specific CD8+T cells decreased slowly. IL-15 as a gene adjuvant had no significant effect on humoral immune responses induced by hepatitis B virus core gene DNA vaccine, but increased the memory antigen-specific CD8+ T cells induced by hepatitis B virus core gene DNA vaccine.CONCLUSION: DNA vaccine constructed by HBc Ag 1-144 amino acid induces effective cell immunity, and cytokine plasmid-delivered IL-15 enhances the longevity of CD8+ T cells.

  18. A recombinant plasmid of composite cysteine proteinase inhibitor/glyceraldehyde-3-phosphate dehydrogenase gene of periodic Brugia malayi functions on DNA immunity in the host

    Directory of Open Access Journals (Sweden)

    Z Fang

    2016-01-01

    Full Text Available Objectives: Both cysteine proteinase inhibitors (CPIs and glyceraldehyde-3-phosphate dehydrogenase (GAPDH play important roles in the pathogenesis of parasites and their relationship with the hosts. We constructed a new eukaryotic recombinant expression plasmid pcDNA3.1(+-BmCPI/BmGAPDH of periodic Brugia malayi for investigation of the DNA vaccine-elicited immune responses. Materials and Methods: We cloned a gene encoding the CPIs and GAPDH from periodic B. malayi into vector pcDNA3.1. The composited plasmid or the control was injected into the tibialis anterior muscle of the hind leg in BALB/c mice, respectively. The target genes were detected by reverse transcription-polymerase chain reaction in muscle tissues. The stimulation index (SI of T-lymphocyte proliferation and the levels of interferon-gamma (INF-g and interleukin-4 ( IL-4 in serum were detected by thiazolyl blue tetrazolium blue and enzyme-linked immunosorbent assays. Results: The pcDNA3.1(+-BmCPI/BmGAPDH was amplified from muscle tissues of the mice after immunisation. The SI of the immunised group was significantly higher than that of the two control groups (P < 0.05. The levels of INF-g and IL-4 of pcDNA3.1(+-BmCPI/BmGAPDH group were both higher than those of the two control groups (P < 0.05. The level of INF-g of pcDNA3.1(+-BmCPI/BmGAPDH group was significantly higher than that of pcDNA3.1(+-BmCPI/CpG group (P < 0.05. Conclusions: We conclude that the recombinant plasmid pcDNA3.1(+-BmCPI/BmGAPDH could elicit specific humoural and cellular immune responses in mice.

  19. The first attested extraction of ancient DNA in legumes (Fabaceae

    Directory of Open Access Journals (Sweden)

    Aleksandar M. Mikić

    2015-11-01

    Full Text Available Ancient DNA (aDNA is any DNA extracted from ancient specimens, important for diverse evolutionary researches. The major obstacles in aDNA studies are mutations, contamination and fragmentation. Its studies may be crucial for crop history if integrated with human aDNA research and historical linguistics, both general and relating to agriculture. Legumes (Fabaceae are one of the richest end economically most important plant families, not only from Neolithic onwards, since they were used as food by Neanderthals and Paleolithic modern man. The idea of extracting and analysing legume aDNA was considered beneficial for both basic science and applied research, with an emphasis on genetic resources and plant breeding. The first reported successful and attested extraction of the legume aDNA was done from the sample of charred seeds of pea (Pisum sativum and bitter vetch (Vicia ervilia from Hissar, southeast Serbia, dated to 1,350 - 1,000 Before Christ. A modified version of cetyltrimethylammonium bromide (CTAB method and the commercial kit for DNA extraction QIAGEN DNAesy yielded several ng μl-1 of aDNA of both species and, after the whole genome amplification and with a fragment of nuclear ribosomal DNA gene 26S rDNA, resulted in the detection of the aDNA among the PCR products. A comparative analysis of four informative chloroplast DNA regions (trnSG, trnK, matK and rbcL among the modern wild and cultivated pea taxa demonstrated not only that the extracted aDNA was genuine, on the basis of mutation rate, but also that the ancient Hissar pea was most likely an early domesticated crop, related to the modern wild pea of a neighbouring region. It is anticipated that this premier extraction of legume aDNA may provide taxonomists with the answers to diverse questions, such as leaf development in legumes, as well as with novel data on the single steps in domesticating legume crops worldwide.

  20. Rapid DNA extraction of pig ear tissues.

    Science.gov (United States)

    Kunhareang, S; Zhou, H; Hickford, J G H

    2010-07-01

    A single-step DNA isolation procedure from pig tissues was developed and the product used directly for polymerase chain reaction (PCR) amplification, single-strand conformational polymorphism (SSCP) analysis and sequencing. The procedure consists of proteinase K digestion of 2-10mg of fresh tissue, at 55 degrees C for 1h, followed by application of the products of digestion to filter paper. A 1.2mm-diameter punch of that paper has sufficient DNA to act as a template for PCR amplification. The quality of the genomic DNA appeared to be high as the PCR amplicons produced sharp banding patterns on both agarose gel electrophoresis and on SSCP analysis, and they could be used for DNA sequencing following cloning. The dried genomic DNA on filter paper can be kept at room temperature. The procedure is considered effective as it is simple, fast and inexpensive. It would be useful for large-scale genotyping and could be used to obtain genomic DNA from various tissues.

  1. Novel actin filaments from Bacillus thuringiensis form nanotubules for plasmid DNA segregation.

    Science.gov (United States)

    Jiang, Shimin; Narita, Akihiro; Popp, David; Ghoshdastider, Umesh; Lee, Lin Jie; Srinivasan, Ramanujam; Balasubramanian, Mohan K; Oda, Toshiro; Koh, Fujiet; Larsson, Mårten; Robinson, Robert C

    2016-03-01

    Here we report the discovery of a bacterial DNA-segregating actin-like protein (BtParM) from Bacillus thuringiensis, which forms novel antiparallel, two-stranded, supercoiled, nonpolar helical filaments, as determined by electron microscopy. The BtParM filament features of supercoiling and forming antiparallel double-strands are unique within the actin fold superfamily, and entirely different to the straight, double-stranded, polar helical filaments of all other known ParMs and of eukaryotic F-actin. The BtParM polymers show dynamic assembly and subsequent disassembly in the presence of ATP. BtParR, the DNA-BtParM linking protein, stimulated ATP hydrolysis/phosphate release by BtParM and paired two supercoiled BtParM filaments to form a cylinder, comprised of four strands with inner and outer diameters of 57 Å and 145 Å, respectively. Thus, in this prokaryote, the actin fold has evolved to produce a filament system with comparable features to the eukaryotic chromosome-segregating microtubule.

  2. Photogeneration of reactive oxygen species and photoinduced plasmid DNA cleavage by novel synthetic chalcones.

    Science.gov (United States)

    Yesuthangam, Y; Pandian, S; Venkatesan, K; Gandhidasan, R; Murugesan, R

    2011-03-02

    This paper describes the synthesis and photodynamic properties of six different chalcone derivatives. Using N,N-dimethyl-4-nitrosoaniline (RNO) bleaching assay, the singlet oxygen generating efficiencies of these chalcones are determined relative to rose bengal (RB). Superoxide dismutase (SOD) inhibitable cytochrome c reduction assay and electron magnetic resonance (EMR) spin trapping techniques are used to determine the superoxide anion radical (O₂·⁻) yield upon photoirradiation. Photoinduced DNA scission studies show that O₂·⁻ is involved in the DNA strand break. In addition, antimicrobial activity of these chalcones is also investigated. Structure activity relationship accounts for the difference in the photogeneration of reactive oxygen species (ROS) by these sensitizers. Presence of electron releasing -OCH₃ groups enhances the photogeneration of ROS. Cyclic voltammetry studies indicate a correlation between enzymatic O₂·⁻ generation efficiency and redox potential of chalcones. Both O₂·⁻ (Type I) and ¹O₂ (Type II) paths are involved in the photosensitization of chalcones. The LUMO energies obtained by molecular modeling correlate with the one-electron reduction potentials.

  3. Induction of Th1-Type Immune Response by Chitosan Nanoparticles Containing Plasmid DNA Encoding House Dust Mite Allergen Der p 2 for Oral Vaccination in Mice

    Institute of Scientific and Technical Information of China (English)

    Guoping Li; Zhigang Liu; Bin Liao; Nanshan Zhong

    2009-01-01

    This study was to prepare the chitosan-pDer p 2 nanoparticles and to investigate the effect of chitosan-DNA nanoparticles on immune response in mice by oral delivery of chitosan-DNA nanoparticles. The nanoparticles were synthesized by complexing chitosan with plasmid DNA. The DNA was fully complexed into chitosan-DNA nanoparticles, suggesting a 100% encapsulation efficiency. Chitosan-DNA complex renders a significant protection of the plasmid. No effect on cell viability was observed in both cell types and average cell viability over 100% was obtained. Oral gene delivery with chitosan-DNA nanoparticles can generate a higher level expression of gene in vivo. Oral chitosan-pDer p 2 nanoparticles in BALB/c mice can induce IFN-γ in serum and prevent subsequent sensitization of Th2 cell-regulated specific IgE responses. The data indicate that the oral administration of chitosan-pDer p 2 nanoparticles results in the expression of Der p 2 in the epithelial cells of both stomach and small intestine and the induction of Th1-type immune response.

  4. DNA extraction from benthic Cyanobacteria: comparative assessment and optimization.

    Science.gov (United States)

    Gaget, V; Keulen, A; Lau, M; Monis, P; Brookes, J D

    2017-01-01

    Benthic Cyanobacteria produce toxic and odorous compounds similar to their planktonic counterparts, challenging the quality of drinking water supplies. The biofilm that benthic algae and other micro-organisms produce is a complex and protective matrix. Monitoring to determine the abundance and identification of Cyanobacteria, therefore, relies on molecular techniques, with the choice of DNA isolation technique critical. This study investigated which DNA extraction method is optimal for DNA recovery in order to guarantee the best DNA yield for PCR-based analysis of benthic Cyanobacteria. The conventional phenol-chloroform extraction method was compared with five commercial kits, with the addition of chemical and physical cell-lysis steps also trialled. The efficacy of the various methods was evaluated by measuring the quantity and quality of DNA by UV spectrophotometry and by quantitative PCR (qPCR) using Cyanobacteria-specific primers. The yield and quality of DNA retrieved with the commercial kits was significantly higher than that of DNA obtained with the phenol-chloroform protocol. Kits including a physical cell-lysis step, such as the MO BIO Power Soil and Biofilm kits, were the most efficient for DNA isolation from benthic Cyanobacteria. These commercial kits allow greater recovery and the elimination of dangerous chemicals for DNA extraction, making them the method of choice for the isolation of DNA from benthic mats. They also facilitate the extraction of DNA from benthic Cyanobacteria, which can help to improve the characterization of Cyanobacteria in environmental studies using qPCRs or population composition analysis using next-generation sequencing. © 2016 The Society for Applied Microbiology.

  5. Residual soil DNA extraction increases the discriminatory power between samples.

    Science.gov (United States)

    Young, Jennifer M; Weyrich, Laura S; Clarke, Laurence J; Cooper, Alan

    2015-06-01

    Forensic soil analysis relies on capturing an accurate and reproducible representation of the diversity from limited quantities of soil; however, inefficient DNA extraction can markedly alter the taxonomic abundance. The performance of a standard commercial DNA extraction kit (MOBIO PowerSoil DNA Isolation kit) and three modified protocols of this kit: soil pellet re-extraction (RE); an additional 24-h lysis incubation step at room temperature (RT); and 24-h lysis incubation step at 55°C (55) were compared using high-throughput sequencing of the internal transcribed spacer I ribosomal DNA. DNA yield was not correlated with fungal diversity and the four DNA extraction methods displayed distinct fungal community profiles for individual samples, with some phyla detected exclusively using the modified methods. Application of a 24 h lysis step will provide a more complete inventory of fungal biodiversity, and re-extraction of the residual soil pellet offers a novel tool for increasing discriminatory power between forensic soil samples.

  6. Evaluation of DNA extraction methods for freshwater eukaryotic microalgae.

    Science.gov (United States)

    Eland, Lucy E; Davenport, Russell; Mota, Cesar R

    2012-10-15

    The use of molecular methods to investigate microalgal communities of natural and engineered freshwater resources is in its infancy, with the majority of previous studies carried out by microscopy. Inefficient or differential DNA extraction of microalgal community members can lead to bias in downstream community analysis. Three commercially available DNA extraction kits have been tested on a range of pure culture freshwater algal species with diverse cell walls and mixed algal cultures taken from eutrophic waste stabilization ponds (WSP). DNA yield and quality were evaluated, along with DNA suitability for amplification of 18S rRNA gene fragments by polymerase chain reaction (PCR). QiagenDNeasy(®) Blood and Tissue kit (QBT), was found to give the highest DNA yields and quality. Denaturant Gradient Gel Electrophoresis (DGGE) was used to assess the diversity of communities from which DNA was extracted. No significant differences were found among kits when assessing diversity. QBT is recommended for use with WSP samples, a conclusion confirmed by further testing on communities from two tropical WSP systems. The fixation of microalgal samples with ethanol prior to DNA extraction was found to reduce yields as well as diversity and is not recommended.

  7. Automated genomic DNA extraction from saliva using the QIAxtractor.

    Science.gov (United States)

    Keijzer, Henry; Endenburg, Silvia C; Smits, Marcel G; Koopmann, Miriam

    2010-05-01

    Venipuncture is an invasive procedure to obtain whole blood in order to obtain high quality and sufficient amounts of genomic DNA. Obtaining DNA from non-invasive sources is preferred by patients, medical doctors and researchers. Saliva collected with cotton swabs (Salivette) is increasingly being used to study chemical compounds, and it can also be a source of DNA. However, extracting DNA from Salivettes is very laborious and time consuming. Therefore, we developed a protocol for automated genomic DNA extraction from saliva collected in Salivette using the QIAxtractor. Saliva (0.1-2.0 mL) was collected by chewing on a Salivette for 1-2 min. A total of 70 samples, collected from healthy volunteers, were extracted with the QIAxtractor robot and a Qiagen DX reagent pack. Quantity and quality was assessed using UV spectrometry and real-time polymerase chain reaction (PCR) (substitution at position -729 in the CYP1A2 gene). The average DNA concentration from the saliva samples was 6.0 microg/mL (95% CI 5.4-6.6 microg/mL). In 100% of the saliva samples, PCR products were detected with an average cycle threshold of 23.1 (95% CI 22.6-23.6). DNA can be extracted in sufficient amounts from Salivette with a fully automated system with a short turnaround time. Real-time PCR can be performed with these samples.

  8. DNA extraction protocol for rapid PCR detection of pathogenic bacteria.

    Science.gov (United States)

    Brewster, Jeffrey D; Paoli, George C

    2013-11-01

    Twelve reagents were evaluated to develop a direct DNA extraction method suitable for PCR detection of foodborne bacterial pathogens. Many reagents exhibited strong PCR inhibition, requiring significant dilution of the extract with a corresponding reduction in sensitivity. Most reagents also exhibited much lower recovery of DNA from the gram-positive test organism (Listeria monocytogenes) than from the gram-negative organism (Escherichia coli O157:H7), preventing unbiased detection and quantitation of both organisms. The 5× HotSHOT+Tween reagent exhibited minimal inhibition and high extraction efficiency for both test organisms, providing a 15-min single-tube DNA-extraction protocol suitable for highly sensitive quantitative PCR assays. Published by Elsevier Inc.

  9. Functional activity of plasmid DNA after entry into the atmosphere of earth investigated by a new biomarker stability assay for ballistic spaceflight experiments.

    Directory of Open Access Journals (Sweden)

    Cora S Thiel

    Full Text Available Sounding rockets represent an excellent platform for testing the influence of space conditions during the passage of Earth's atmosphere and re-entry on biological, physical and chemical experiments for astrobiological purposes. We designed a robust functionality biomarker assay to analyze the biological effects of suborbital spaceflights prevailing during ballistic rocket flights. During the TEXUS-49 rocket mission in March 2011, artificial plasmid DNA carrying a fluorescent marker (enhanced green fluorescent protein: EGFP and an antibiotic resistance cassette (kanamycin/neomycin was attached on different positions of rocket exterior; (i circular every 90 degree on the outer surface concentrical of the payload, (ii in the grooves of screw heads located in between the surface application sites, and (iii on the surface of the bottom side of the payload. Temperature measurements showed two major peaks at 118 and 130 °C during the 780 seconds lasting flight on the inside of the recovery module, while outer gas temperatures of more than 1000 °C were estimated on the sample application locations. Directly after retrieval and return transport of the payload, the plasmid DNA samples were recovered. Subsequent analyses showed that DNA could be recovered from all application sites with a maximum of 53% in the grooves of the screw heads. We could further show that up to 35% of DNA retained its full biological function, i.e., mediating antibiotic resistance in bacteria and fluorescent marker expression in eukaryotic cells. These experiments show that our plasmid DNA biomarker assay is suitable to characterize the environmental conditions affecting DNA during an atmospheric transit and the re-entry and constitute the first report of the stability of DNA during hypervelocity atmospheric transit indicating that sounding rocket flights can be used to model the high-speed atmospheric entry of organics-laden artificial meteorites.

  10. High-throughput DNA extraction of forensic adhesive tapes.

    Science.gov (United States)

    Forsberg, Christina; Jansson, Linda; Ansell, Ricky; Hedman, Johannes

    2016-09-01

    Tape-lifting has since its introduction in the early 2000's become a well-established sampling method in forensic DNA analysis. Sampling is quick and straightforward while the following DNA extraction is more challenging due to the "stickiness", rigidity and size of the tape. We have developed, validated and implemented a simple and efficient direct lysis DNA extraction protocol for adhesive tapes that requires limited manual labour. The method uses Chelex beads and is applied with SceneSafe FAST tape. This direct lysis protocol provided higher mean DNA yields than PrepFiler Express BTA on Automate Express, although the differences were not significant when using clothes worn in a controlled fashion as reference material (p=0.13 and p=0.34 for T-shirts and button-down shirts, respectively). Through in-house validation we show that the method is fit-for-purpose for application in casework, as it provides high DNA yields and amplifiability, as well as good reproducibility and DNA extract stability. After implementation in casework, the proportion of extracts with DNA concentrations above 0.01ng/μL increased from 71% to 76%. Apart from providing higher DNA yields compared with the previous method, the introduction of the developed direct lysis protocol also reduced the amount of manual labour by half and doubled the potential throughput for tapes at the laboratory. Generally, simplified manual protocols can serve as a cost-effective alternative to sophisticated automation solutions when the aim is to enable high-throughput DNA extraction of complex crime scene samples. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  11. Mechanisms of plasmid segregation: have multicopy plasmids been overlooked?

    Science.gov (United States)

    Million-Weaver, Samuel; Camps, Manel

    2014-09-01

    Plasmids are self-replicating pieces of DNA typically bearing non-essential genes. Given that plasmids represent a metabolic burden to the host, mechanisms ensuring plasmid transmission to daughter cells are critical for their stable maintenance in the population. Here we review these mechanisms, focusing on two active partition strategies common to low-copy plasmids: par systems type I and type II. Both involve three components: an adaptor protein, a motor protein, and a centromere, which is a sequence area in the plasmid that is recognized by the adaptor protein. The centromere-bound adaptor nucleates polymerization of the motor, leading to filament formation, which can pull plasmids apart (par I) or push them towards opposite poles of the cell (par II). No such active partition mechanisms are known to occur in high copy number plasmids. In this case, vertical transmission is generally considered stochastic, due to the random distribution of plasmids in the cytoplasm. We discuss conceptual and experimental lines of evidence questioning the random distribution model and posit the existence of a mechanism for segregation in high copy number plasmids that moves plasmids to cell poles to facilitate transmission to daughter cells. This mechanism would involve chromosomally-encoded proteins and the plasmid origin of replication. Modulation of this proposed mechanism of segregation could provide new ways to enhance plasmid stability in the context of recombinant gene expression, which is limiting for large-scale protein production and for bioremediation.

  12. Interaction of a C-terminal Truncated Hepatitis C Virus Core Protein with Plasmid DNA Vaccine Leads to in vitro Assembly of Heterogeneous Virus-like Particles

    Directory of Open Access Journals (Sweden)

    Nelson Acosta-Rivero

    2005-01-01

    Full Text Available Recently, it has been shown that HCV core proteins (HCcAg with C-terminal deletions assemble in vitro into virus-like particles (VLPs in the presence of structured RNA molecules. Results presented in this work showed that a truncated HCcAg variant covering the first 120 aa (HCcAg.120 with a 32 aa N-terminal fusion peptide (6xHistag-XpressTMepitope interacts with plasmid DNA vaccine. Interestingly, the buoyant density of VLPs containing HCcAg.120 in CsCl gradients changed from 1.15-1,17 g mLˉ1 to 1.30-1.34 g mLˉ1 after addition of plasmid DNA to assembly reactions. In addition, a delay in electrophoretic mobility of HCcAg.120-plasmid samples on agarose gels was observed indicating a direct interaction between VLPs and nucleic acids. Remarkably, addition of either plasmid DNA or tRNA to assembly reactions leaded to heterogeneous and larger VLPs formation than those observed in HCcAg.120 assembly reactions. VLPs containing HCcAg.120 induced a specific IgG antibodies in mice that reacted with hepatocytes from HCV-infected patients. VLPs obtained in this work would be important to elucidate the mechanisms behind the ability of HCcAg to assemble into a nucleocapsid structure. Besides, the capacity of particles containing HCcAg.120 to interact with nucleic acids could be used in the development of DNA vaccines and viral vectors based on these particles.

  13. Evaluation on the efficacy and immunogenicity of recombinant DNA plasmids expressing spike genes from porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus.

    Science.gov (United States)

    Meng, Fandan; Ren, Yudong; Suo, Siqingaowa; Sun, Xuejiao; Li, Xunliang; Li, Pengchong; Yang, Wei; Li, Guangxing; Li, Lu; Schwegmann-Wessels, Christel; Herrler, Georg; Ren, Xiaofeng

    2013-01-01

    Porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PDEV) can cause severe diarrhea in pigs. Development of effective vaccines against TGEV and PEDV is one of important prevention measures. The spike (S) protein is the surface glycoprotein of TGEV and PEDV, which can induce specific neutralization antibodies and is a candidate antigen for vaccination attempts. In this study, the open reading frames of the TGEV S1 protein and in addition of the S or S1 proteins of PEDV were inserted into the eukaryotic expression vector, pIRES, resulting in recombinant plasmids, pIRES-(TGEV-S1-PEDV-S1) and pIRES-(TGEV-S1-PEDV-S). Subsequently, 6-8 weeks old Kunming mice were inoculated with both DNA plasmids. Lymphocyte proliferation assay, virus neutralization assay, IFN-γ assay and CTL activity assay were performed. TGEV/PEDV specific antibody responses as well as kinetic changes of T lymphocyte subgroups of the immunized mice were analyzed. The results showed that the recombinant DNA plasmids increased the proliferation of T lymphocytes and the number of CD4+ and CD8+ T lymphocyte subgroups. In addition, the DNA vaccines induced a high level of IFN-γ in the immunized mice. The specific CTL activity in the pIRES-(TGEV-S1-PEDV-S) group became significant at 42 days post-immunization. At 35 days post-immunization, the recombinant DNA plasmids bearing full-length S genes of TGEV and PEDV stimulated higher levels of specific antibodies and neutralizing antibodies in immunized mice.

  14. Protective effect of extract of Crataegus pinnatifida pollen on DNA damage response to oxidative stress.

    Science.gov (United States)

    Cheng, Ni; Wang, Yuan; Gao, Hui; Yuan, Jialing; Feng, Fan; Cao, Wei; Zheng, Jianbin

    2013-09-01

    The protective effect of extract of Crataegus pinnatifida (Rosaceae) pollen (ECPP) on the DNA damage response to oxidative stress was investigated and assessed with an alkaline single-cell gel electrophoresis (SCGE) assay and pBR322 plasmid DNA breaks in site-specific and non-site-specific systems. Total phenolic content, total flavonoid content, individual phenolic compounds, antioxidant activities (1,1-diphenyl-2-picrylhydrazyl (DPPH), radical scavenging activity, FRAP, and chelating activity) were also determined. The results showed that ECPP possessed a strong ability to protect DNA from being damaged by hydroxyl radicals in both the site-specific system and the non-site-specific system. It also exhibited a cytoprotection effect in mouse lymphocytes against H₂O₂-induced DNA damage. These protective effects may be related to its high total phenolic content (17.65±0.97 mg GAE/g), total flavonoid content (8.04±0.97 mg rutin/g), strong free radical scavenging activity and considerable ferrous ion chelating ability (14.48±0.21 mg Na₂EDTA/g). Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Extraction DNA from Activated Sludge-Comparing with Soil Sample

    Institute of Scientific and Technical Information of China (English)

    谢冰; 奚旦立; 陈季华

    2003-01-01

    DNA directly extraction from activated sludge and soil sample with enzyme lyses methods was investigated in this paper. DNA yield from activated sludge was 3.0 mg/g. VLSS, and 28.2-43.8 μg/g soil respectively. The resulting DNA is suitable for PCR.By studied methods, higher quality and quantity of sludge DNA could be obtained rapidly and inexpensively from large number of samples, and the PCR product obtained from this protocol was not affected by contaminated higher concentration of heavy metals.

  16. The intracellular delivery of plasmid DNA using cationic reducible carbon nanotube - Disulfide conjugates of polyethylenimine.

    Science.gov (United States)

    Nia, Azadeh Hashem; Eshghi, Hossein; Abnous, Kalil; Ramezani, Mohammad

    2017-03-30

    A series of polyethylenimine conjugates of single-walled carbon nanotube (PEI-SWNT) containing bioreducible disulfide bonds was synthesized and evaluated for their transfection efficiency. Different molecular weights of polyethylenimine (PEI) were thiolated with different mole ratio of 2-iminothiolane (2-IT). Single-walled carbon nanotube (SWNT) was first carboxylated and then three different cysteine-functionalized SWNT formulations were synthesized via introduced linkers: a) carbonyl group b) spermidine c) 1,8-diamino 3,6-dioxo octane. The final nanocarriers were fabricated upon conjugation of thiolated PEIs and thiolated SWNT via oxidative disulfide bond formation. All PEI-disulfide-SWNT conjugates were capable of DNA condensation and showed improved viability and transfection efficiency compared to PEI itself. Transfection efficiencies were up to 1500 times greater than PEI 25kDa (C/P=0.8). The results of this study suggest that the synthesized formulations based on SWNT-CO-Cysteine and PEI 1.8kDa were the most efficient carriers. Considering the decreased cytotoxicity and higher transfection levels, the conjugates bear the potential for effective delivery of genetic materials. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Highly efficient DNA extraction method from skeletal remains

    Directory of Open Access Journals (Sweden)

    Irena Zupanič Pajnič

    2011-03-01

    Full Text Available Background: This paper precisely describes the method of DNA extraction developed to acquire high quality DNA from the Second World War skeletal remains. The same method is also used for molecular genetic identification of unknown decomposed bodies in routine forensic casework where only bones and teeth are suitable for DNA typing. We analysed 109 bones and two teeth from WWII mass graves in Slovenia. Methods: We cleaned the bones and teeth, removed surface contaminants and ground the bones into powder, using liquid nitrogen . Prior to isolating the DNA in parallel using the BioRobot EZ1 (Qiagen, the powder was decalcified for three days. The nuclear DNA of the samples were quantified by real-time PCR method. We acquired autosomal genetic profiles and Y-chromosome haplotypes of the bones and teeth with PCR amplification of microsatellites, and mtDNA haplotypes 99. For the purpose of traceability in the event of contamination, we prepared elimination data bases including genetic profiles of the nuclear and mtDNA of all persons who have been in touch with the skeletal remains in any way. Results: We extracted up to 55 ng DNA/g of the teeth, up to 100 ng DNA/g of the femurs, up to 30 ng DNA/g of the tibias and up to 0.5 ng DNA/g of the humerus. The typing of autosomal and YSTR loci was successful in all of the teeth, in 98 % dekalof the femurs, and in 75 % to 81 % of the tibias and humerus. The typing of mtDNA was successful in all of the teeth, and in 96 % to 98 % of the bones. Conclusions: We managed to obtain nuclear DNA for successful STR typing from skeletal remains that were over 60 years old . The method of DNA extraction described here has proved to be highly efficient. We obtained 0.8 to 100 ng DNA/g of teeth or bones and complete genetic profiles of autosomal DNA, Y-STR haplotypes, and mtDNA haplotypes from only 0.5g bone and teeth samples.

  18. Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize.

    Science.gov (United States)

    Abdel-Latif, Amani; Osman, Gamal

    2017-01-01

    The world's top three cereals, based on their monetary value, are rice, wheat, and corn. In cereal crops, DNA extraction is difficult owing to rigid non-cellulose components in the cell wall of leaves and high starch and protein content in grains. The advanced techniques in molecular biology require pure and quick extraction of DNA. The majority of existing DNA extraction methods rely on long incubation and multiple precipitations or commercially available kits to produce contaminant-free high molecular weight DNA. In this study, we compared three different methods used for the isolation of high-quality genomic DNA from the grains of cereal crop, Zea mays, with minor modifications. The DNA from the grains of two maize hybrids, M10 and M321, was extracted using extraction methods DNeasy Qiagen Plant Mini Kit, CTAB-method (with/without 1% PVP) and modified Mericon extraction. Genes coding for 45S ribosomal RNA are organized in tandem arrays of up to several thousand copies and contain codes for 18S, 5.8S and 26S rRNA units separated by internal transcribed spacers ITS1 and ITS2. While the rRNA units are evolutionary conserved, ITS regions show high level of interspecific divergence and have been used frequently in genetic diversity and phylogenetic studies. In this study, the genomic DNA was then amplified with PCR using primers specific for ITS gene. PCR products were then visualized on agarose gel. The modified Mericon extraction method was found to be the most efficient DNA extraction method, capable to provide high DNA yields with better quality, affordable cost and less time.

  19. Modification of gelatin-DNA interaction for optimised DNA extraction from gelatin and gelatin capsule.

    Science.gov (United States)

    Mohamad, Nurhidayatul Asma; Mustafa, Shuhaimi; El Sheikha, Aly Farag; Khairil Mokhtar, Nur Fadhilah; Ismail, Amin; Ali, Md Eaqub

    2016-05-01

    Poor quality and quantity of DNA extracted from gelatin and gelatin capsules often causes failure in the determination of animal species using PCR. Gelatin, which is mainly derived from porcine and bovine, has been a matter of concern among customers in order to fulfill religious obligation and safety precaution against several transmissible infectious diseases associated with bovine species. Thus, optimised DNA extraction from gelatin is very important for successful real-time PCR detection of gelatin species. In this work, the DNA extraction method was optimised in terms of lysis incubation period and inclusion of pre-treatment pH modification of samples. The yield of DNA extracted from porcine gelatin was significantly increased when the pH of the samples was adjusted to pH 8.5 prior to DNA precipitation with isopropanol. The optimal pH for DNA precipitation from bovine gelatin solution was then determined at the original pH range of solution: pH 7.6 to 8. A DNA fragment of approximately 300 base pairs was available for PCR amplification. DNA extracted from gelatin and commercially available capsules has been successfully utilised for species detection using real-time PCR assay. However, significant adulterations of porcine and bovine in pure gelatin and capsules have been detected, which require further analytical techniques for validation. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  20. Evaluation of the Genotoxic Potential against H2O2-Radical-Mediated DNA Damage and Acute Oral Toxicity of Standardized Extract of Polyalthia longifolia Leaf

    Directory of Open Access Journals (Sweden)

    Subramanion L. Jothy

    2013-01-01

    Full Text Available Medicinal plants have been used in medicoculturally diverse countries around the world, where it is a part of a time-honoured tradition that is respected even today. Polyalthia longifolia leaf extract has been previously reported as an efficient antioxidant in vitro. Hence, the genotoxic effects of P. longifolia leaf were investigated by using plasmid relation, comet, and Allium cepa assay. In the presence of  ∙OH radicals, the DNA in supercoil was start nicked into open circular form, which is the product of the single-stranded cleavage of supercoil DNA and quantified as fragmented separate bands on agarose gel in plasmid relation assay. In the plasmid relation and comet assay, the P. longifolia leaf extract exhibited strong inhibitory effects against H2O2-mediated DNA damage. A dose-dependent increase of chromosome aberrations was also observed in the Allium cepa assay. The abnormalities scored were stickiness, c-mitosis, bridges, and vagrant chromosomes. Micronucleated cells were also observed at the interphase. The results of Allium cepa assay confirmed that the methanol extracts of P. longifolia exerted no significant genotoxic or mitodepressive effects at 100 μg/mL. Thus, this study demonstrated that P. longifolia leaf extract has a beneficial effect against oxidative DNA damage. This experiment is the first report for the protective effect of P. longifolia on DNA damage-induced by hydroxyl radicals. Additionally in acute oral toxicity study, female rats were treated at 5000 mg/kg body weight of P. longifolia leaf extract and observed for signs of toxicity for 14 days. P. longifolia leaf extract did not produce any treatment-related toxic effects in rats.

  1. Evaluation of the Genotoxic Potential against H2O2-Radical-Mediated DNA Damage and Acute Oral Toxicity of Standardized Extract of Polyalthia longifolia Leaf

    Science.gov (United States)

    Jothy, Subramanion L.; Chen, Yeng; Kanwar, Jagat R.; Sasidharan, Sreenivasan

    2013-01-01

    Medicinal plants have been used in medicoculturally diverse countries around the world, where it is a part of a time-honoured tradition that is respected even today. Polyalthia longifolia leaf extract has been previously reported as an efficient antioxidant in vitro. Hence, the genotoxic effects of P. longifolia leaf were investigated by using plasmid relation, comet, and Allium cepa assay. In the presence of  ∙OH radicals, the DNA in supercoil was start nicked into open circular form, which is the product of the single-stranded cleavage of supercoil DNA and quantified as fragmented separate bands on agarose gel in plasmid relation assay. In the plasmid relation and comet assay, the P. longifolia leaf extract exhibited strong inhibitory effects against H2O2-mediated DNA damage. A dose-dependent increase of chromosome aberrations was also observed in the Allium cepa assay. The abnormalities scored were stickiness, c-mitosis, bridges, and vagrant chromosomes. Micronucleated cells were also observed at the interphase. The results of Allium cepa assay confirmed that the methanol extracts of P. longifolia exerted no significant genotoxic or mitodepressive effects at 100 μg/mL. Thus, this study demonstrated that P. longifolia leaf extract has a beneficial effect against oxidative DNA damage. This experiment is the first report for the protective effect of P. longifolia on DNA damage-induced by hydroxyl radicals. Additionally in acute oral toxicity study, female rats were treated at 5000 mg/kg body weight of P. longifolia leaf extract and observed for signs of toxicity for 14 days. P. longifolia leaf extract did not produce any treatment-related toxic effects in rats. PMID:23878610

  2. [Effect of plasmid pKM101 on the expression of bacterial genes not related to DNa metabolism].

    Science.gov (United States)

    Skavronskaya, A G; Tiganova, I G; Andreeva, I V; Rusina, O Iu

    1999-02-01

    An experimental system ensuring fusion of bacterial genes to the lac operon of the Mu dl(Aplac) phage was used. Fusion operons in which the lac operon was under the control of promoters of the elt gene, responsible for synthesis of the LT toxin, of the tetracyclin-resistance tet gene, and sfiA gene encoding filament production, was studied. Using this experimental system, plasmid pKM101 was shown to be capable of activating the expression of the above Escherichia coli and Salmonella typhimurium genes, which is manifested as the activation of beta-galactosidase synthesis. The activation of the elt gene expression by the pKM101 plasmid was also confirmed in experiments on detecting the LT toxin synthesized by bacteria carrying this plasmid. Effect of the plasmid on the activation of elt operon expression, unlike the effect of this plasmid on mutability, does not depend on the functioning of the lexA and recA genes, i.e., this is not a SOS-regulated process. The mutant plasmid pGW12, a derivative of pKM101, deficient in the mucAB genes responsible for mutagenesis, causes a more pronounced activation of the elt gene than plasmid pKM101.

  3. DNA sequence analysis of the composite plasmid pTC conferring virulence and antimicrobial resistance for porcine enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Fekete, Péter Z; Brzuszkiewicz, Elzbieta; Blum-Oehler, Gabriele; Olasz, Ferenc; Szabó, Mónika; Gottschalk, Gerhard; Hacker, Jörg; Nagy, Béla

    2012-01-01

    In this study the plasmid pTC, a 90 kb self-conjugative virulence plasmid of the porcine enterotoxigenic Escherichia coli (ETEC) strain EC2173 encoding the STa and STb heat-stable enterotoxins and tetracycline resistance, has been sequenced in two steps. As a result we identified five main distinct regions of pTC: (i) the maintenance region responsible for the extreme stability of the plasmid, (ii) the TSL (toxin-specific locus comprising the estA and estB genes) which is unique and characteristic for pTC, (iii) a Tn10 transposon, encoding tetracycline resistance, (iv) the tra (plasmid transfer) region, and (v) the colE1-like origin of replication. It is concluded that pTC is a self-transmissible composite plasmid harbouring antibiotic resistance and virulence genes. pTC belongs to a group of large conjugative E. coli plasmids represented by NR1 with a widespread tra backbone which might have evolved from a common ancestor. This is the first report of a completely sequenced animal ETEC virulence plasmid containing an antimicrobial resistance locus, thereby representing a selection advantage for spread of pathogenicity in the presence of antimicrobials leading to increased disease potential. Copyright © 2011. Published by Elsevier GmbH.

  4. Overcoming DNA extraction problems from carnivorous plants

    OpenAIRE

    Fleischmann, Andreas; Heubl, Günther

    2009-01-01

    We tested previously published protocols for DNA isolation from plants with high contents of polyphenols and polysaccharides for several taxa of carnivorous plants. However, we did not get satisfying results with fresh or silica dried leaf tissue obtained from field collected or greenhouse grown plants, nor from herbarium specimens. Therefore, we have developed a simple modified protocol of the commercially available Macherey- Nagel NucleoSpin® Plant kit for rapid, effective and reproducible ...

  5. Automated DNA extraction of single dog hairs without roots for mitochondrial DNA analysis.

    Science.gov (United States)

    Bekaert, Bram; Larmuseau, Maarten H D; Vanhove, Maarten P M; Opdekamp, Anouschka; Decorte, Ronny

    2012-03-01

    Dogs are intensely integrated in human social life and their shed hairs can play a major role in forensic investigations. The overall aim of this study was to validate a semi-automated extraction method for mitochondrial DNA analysis of telogenic dog hairs. Extracted DNA was amplified with a 95% success rate from 43 samples using two new experimental designs in which the mitochondrial control region was amplified as a single large (± 1260 bp) amplicon or as two individual amplicons (HV1 and HV2; ± 650 and 350 bp) with tailed-primers. The results prove that the extraction of dog hair mitochondrial DNA can easily be automated to provide sufficient DNA yield for the amplification of a forensically useful long mitochondrial DNA fragment or alternatively two short fragments with minimal loss of sequence in case of degraded samples.

  6. Proboscidean DNA from museum and fossil specimens: an assessment of ancient DNA extraction and amplification techniques.

    Science.gov (United States)

    Yang, H; Golenberg, E M; Shoshani, J

    1997-06-01

    Applications of reliable DNA extraction and amplification techniques to postmortem samples are critical to ancient DNA research. Commonly used methods for isolating DNA from ancient material were tested and compared using both soft tissue and bones from fossil and contemporary museum proboscideans. DNAs isolated using three principal methods served as templates in subsequent PCR amplifications, and the PCR products were directly sequenced. Authentication of the ancient origin of obtained nucleotide sequences was established by demonstrating reproducibility under a blind testing system and by phylogenetic analysis. Our results indicate that ancient samples may respond differently to extraction buffers or purification procedures, and no single method was universally successful. A CTAB buffer method, modified from plant DNA extraction protocols, was found to have the highest success rate. Nested PCR was shown to be a reliable approach to amplify ancient DNA templates that failed in primary amplification.

  7. Delivery of plasmid DNA expression vector for keratinocyte growth factor-1 using electroporation to improve cutaneous wound healing in a septic rat model.

    Science.gov (United States)

    Lin, Michael P; Marti, Guy P; Dieb, Rami; Wang, Jiaai; Ferguson, Mark; Qaiser, Rabia; Bonde, Pramod; Duncan, Mark D; Harmon, John W

    2006-01-01

    We have previously shown that wound healing was improved in a diabetic mouse model of impaired wound healing following transfection with keratinocyte growth factor-1 (KGF-1) cDNA. We now extend these findings to the characterization of the effects of DNA plasmid vectors delivered to rats using electroporation (EP) in vivo in a sepsis-based model of impaired wound healing. To assess plasmid transfection and wound healing, gWIZ luciferase and PCDNA3.1/KGF-1 expression vectors were used, respectively. Cutaneous wounds were produced using an 8 mm-punch biopsy in Sprague-Dawley rats in which healing was impaired by cecal ligation-induced sepsis. We used National Institutes of Health image analysis software and histologic assessment to analyze wound closure and found that EP increased expression of gWIZ luciferase vector up to 53-fold compared with transfection without EP (p < 0.001). EP-assisted plasmid transfection was found to be localized to skin. Septic rats had a 4.7 times larger average wound area on day 9 compared with control (p < 0.001). Rats that underwent PCDNA3.1/KGF-1 transfection with EP had 60% smaller wounds on day 12 compared with vector without EP (p < 0.009). Quality of healing with KGF-1 vector plus EP scored 3.0 +/- 0.3 and was significantly better than that of 1.8 +/- 0.3 for treatment with vector alone (p < 0.05). We conclude that both the rate and quality of healing were improved with DNA plasmid expression vector for growth factor delivered with EP to septic rats.

  8. Highly Effective Non-Viral Antitumor Gene Therapy System Comprised of Biocompatible Small Plasmid Complex Particles Consisting of pDNA, Anionic Polysaccharide, and Fully Deprotected Linear Polyethylenimine

    Directory of Open Access Journals (Sweden)

    Yoshiyuki Koyama

    2015-07-01

    Full Text Available We have reported that ternary complexes of plasmid DNA with conventional linear polyethylenimine (l-PEI and certain polyanions were very stably dispersed, and, with no cryoprotectant, they could be freeze-dried and re-hydrated without the loss of transfection ability. These properties enabled the preparation of a concentrated suspension of very small pDNA complex, by preparing the complexes at highly diluted conditions, followed by condensation via lyophilization-and-rehydration procedure. Recently, a high potency linear polyethylenimine having no residual protective groups, i.e., Polyethylenimine “Max” (PEI “Max”, is available, which has been reported to induce much higher gene expression than conventional l-PEI. We tried to prepare the small DNA/PEI “Max”/polyanion complexes by a similar freeze-drying method. Small complex particles could be obtained without apparent aggregation, but transfection activity of the rehydrated complexes was severely reduced. Complex-preparation conditions were investigated in details to achieve the freeze-dried DNA/PEI “Max”/polyanion small ternary complexes with high transfection efficiency. DNA/PEI “Max”/polyanion complexes containing cytokine-coding plasmids were then prepared, and their anti-tumor therapeutic efficacy was examined in tumor-bearing mice.

  9. Highly Effective Non-Viral Antitumor Gene Therapy System Comprised of Biocompatible Small Plasmid Complex Particles Consisting of pDNA, Anionic Polysaccharide, and Fully Deprotected Linear Polyethylenimine.

    Science.gov (United States)

    Koyama, Yoshiyuki; Sugiura, Kikuya; Yoshihara, Chieko; Inaba, Toshio; Ito, Tomoko

    2015-07-23

    We have reported that ternary complexes of plasmid DNA with conventional linear polyethylenimine (l-PEI) and certain polyanions were very stably dispersed, and, with no cryoprotectant, they could be freeze-dried and re-hydrated without the loss of transfection ability. These properties enabled the preparation of a concentrated suspension of very small pDNA complex, by preparing the complexes at highly diluted conditions, followed by condensation via lyophilization-and-rehydration procedure. Recently, a high potency linear polyethylenimine having no residual protective groups, i.e., Polyethylenimine "Max" (PEI "Max"), is available, which has been reported to induce much higher gene expression than conventional l-PEI. We tried to prepare the small DNA/PEI "Max"/polyanion complexes by a similar freeze-drying method. Small complex particles could be obtained without apparent aggregation, but transfection activity of the rehydrated complexes was severely reduced. Complex-preparation conditions were investigated in details to achieve the freeze-dried DNA/PEI "Max"/polyanion small ternary complexes with high transfection efficiency. DNA/PEI "Max"/polyanion complexes containing cytokine-coding plasmids were then prepared, and their anti-tumor therapeutic efficacy was examined in tumor-bearing mice.

  10. Sodium citrate and potassium phosphate as alternative adsorption buffers in hydrophobic and aromatic thiophilic chromatographic purification of plasmid DNA from neutralized lysate.

    Science.gov (United States)

    Bonturi, Nemailla; Radke, Vanessa Soraia Cortez Oliveira; Bueno, Sônia Maria Alves; Freitas, Sindélia; Azzoni, Adriano Rodrigues; Miranda, Everson Alves

    2013-03-01

    The number of studies on gene therapy using plasmid vectors (pDNA) has increased in recent years. As a result, the demand for preparations of pDNA in compliance with recommendations of regulatory agencies (EMEA, FDA) has also increased. Plasmid DNA is often obtained through fermentation of transformed Escherichia coli and purification by a series of unit operations, including chromatography. Hydrophobic interaction chromatography (HIC) and thiophilic aromatic chromatography (TAC), both using ammonium sulfate buffers, are commonly employed with success. This work was aimed at studying the feasibility of utilizing alternative salts in the purification of pDNA from neutralized lysate with phenyl-agarose (HIC) and mercaptopyrimidine-agarose (TAC) adsorbents. Their selectivity toward sc pDNA was evaluated through adsorption studies using 1.5 mol/L sodium citrate and 2.0 mol/L potassium phosphate as adsorption buffers. Chromatography with mercaptopyrimidine-agarose adsorbent and 1.5 mol/L sodium citrate was able to recover 91.1% of the pDNA with over 99.0% removal of gDNA and endotoxin. This represents a potential alternative for the primary recovery of sc pDNA. However, the most promising result was obtained using 2.0 mol/L potassium phosphate buffer and a mercaptopyrimidine-agarose column. In a single chromatographic step, this latter buffer/adsorbent system recovered 68.5% of the pDNA with 98.8% purity in accordance with the recommendations of regulatory agencies with regard to RNA and endotoxin impurity.

  11. Plasmid Rolling-Circle Replication.

    Science.gov (United States)

    Ruiz-Masó, J A; MachóN, C; Bordanaba-Ruiseco, L; Espinosa, M; Coll, M; Del Solar, G

    2015-02-01

    Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

  12. Nonhomologous DNA End Joining in Cell-Free Extracts

    OpenAIRE

    Sheetal Sharma; Raghavan, Sathees C.

    2010-01-01

    Among various DNA damages, double-strand breaks (DSBs) are considered as most deleterious, as they may lead to chromosomal rearrangements and cancer when unrepaired. Nonhomologous DNA end joining (NHEJ) is one of the major DSB repair pathways in higher organisms. A large number of studies on NHEJ are based on in vitro systems using cell-free extracts. In this paper, we summarize the studies on NHEJ performed by various groups in different cell-free repair systems.

  13. Nonhomologous DNA End Joining in Cell-Free Extracts

    Directory of Open Access Journals (Sweden)

    Sheetal Sharma

    2010-01-01

    Full Text Available Among various DNA damages, double-strand breaks (DSBs are considered as most deleterious, as they may lead to chromosomal rearrangements and cancer when unrepaired. Nonhomologous DNA end joining (NHEJ is one of the major DSB repair pathways in higher organisms. A large number of studies on NHEJ are based on in vitro systems using cell-free extracts. In this paper, we summarize the studies on NHEJ performed by various groups in different cell-free repair systems.

  14. DNA Extraction from Eriocaulon Plants and Construction of RAPD System

    Institute of Scientific and Technical Information of China (English)

    Xue Xian; Lin Shanzhi; Zhang Zhixiang

    2004-01-01

    There have been many arguments on the classification of Eriocaulon Linn. by morphology so far, and little is known about the use of molecular marker for genetic for genetic diversity of Eriocaulon plants. To apply the technique of molecular marker to the research of genetic diversity of Eriocaulon plants, the study of the extraction method of DNA from the Eriocaulon plants and the RAPD system are essential for researchers. In this paper, the extraction of genome DNA from the silica-gel-dried leaves of several species of Eriocaulon distributed in China was studied, and the best RAPD analysis technique condition of Eriocaulon plants was analyzed.

  15. An improved electroelution method for separation of DNA from humic substances in marine sediment DNA extracts.

    Science.gov (United States)

    Kallmeyer, Jens; Smith, David C

    2009-07-01

    We present a method for the rapid and simple extraction of DNA from marine sediments using electroelution. It effectively separates DNA from compounds, including humic substances, that interfere with subsequent DNA quantification and amplification. After extraction of the DNA from the sediment into an aqueous solution, the crude sample is encased in 2% agarose gel and exposed to an electrical current, which draws the DNA out of the gel into a centrifugal filter vial. After electroelution, the sample is centrifuged to remove contaminants method is quantitative and does not discriminate on the basis of size, as determined using DNA standards and DNA extracts from environmental samples. Amplification of DNA is considerably improved due to removal of PCR inhibitors. For Archaea, only these purified extracts yielded PCR products. This method allows for the use of relatively large volumes of sediment and is particularly useful for sediments containing low biomass such as deeply buried marine sediments. It works with both organic-rich and -poor sediment, as well as with sediment where calcium carbonate is abundant and sediment where it is limited; consequently, adjustment of protocols is unnecessary for samples with very different organic and mineral contents.

  16. Plant and metagenomic DNA extraction of mucilaginous seeds.

    Science.gov (United States)

    Ramos, Simone N M; Salazar, Marcela M; Pereira, Gonçalo A G; Efraim, Priscilla

    2014-01-01

    The pulp surrounding the seeds of some fruits is rich in mucilage, carbohydrates, etc. Some seeds are rich in proteins and polyphenols. Fruit seeds, like cacao (Theobroma cacao) and cupuassu (Theobroma grandiflorum), are subjected to fermentation to develop flavor. During fermentation, ethanol is produced [2-6]. All of these compounds are considered as interfering substances that hinder the DNA extraction [4-8]. Protocols commonly used in the DNA extraction in samples of plant origin were used, but without success. Thus, a protocol for DNA samples under different conditions that can be used for similar samples was developed and applied with success. The protocol initially described for RNA samples by Zeng et al. [9] and with changes proposed by Provost et al. [5] was adapted for extracting DNA samples from those described. However, several modifications have been proposed:•Samples were initially washed with petroleum ether for fat phase removal.•RNAse was added to the extraction buffer, while spermidin was removed.•Additional steps of extraction with 5 M NaCl, saturated NaCl and CTAB (10%) were included and precipitation was carried out with isopropanol, followed by washing with ethanol.

  17. Comparison of DNA and RNA extraction methods for mummified tissues.

    Science.gov (United States)

    Konomi, Nami; Lebwohl, Eve; Zhang, David

    2002-12-01

    Nucleic acids extracted from mummified tissues are valuable materials for the study of ancient human beings. Significant difficulty in extracting nucleic acids from mummified tissues has been reported due to chemical modification and degradation. The goal of this study was to determine a method that is more efficient for DNA and RNA extraction from mummified tissues. Twelve mummy specimens were analyzed with 9 different nucleic acid extraction methods, including guanidium thiocyanate (GTC) and proteinase K/detergent based methods prepared in our laboratory or purchased. Glyceraldehyde 3-phosphate dehydrogenase DNA and beta-actin RNA were used as markers for the presence of adequate DNA and RNA, respectively, for PCR and RT-PCR amplification. Our results show that 5 M GTC is more efficient of releasing nucleic acids from mummified tissue than proteinase K/detergent, and phenol/chloroform extraction with an additional chloroform step is more efficient than phenol/chloroform along. We were able to isolate DNAs from all 12 specimens and RNAs from 8 of 12 specimens, and the nucleic acids were sufficient for PCR and RT-PCR analysis. We further tested hepatitis viruses including hepatitis B virus, hepatitis C virus, hepatitis G virus, and TT virus DNA, and fail to detect these viruses in all 12 specimens.

  18. Evaluation of Four Automated Protocols for Extraction of DNA from FTA Cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura

    2013-01-01

    Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction...... protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA...... from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore...

  19. Evaluation of Four Automated Protocols for Extraction of DNA from FTA Cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura;

    2013-01-01

    protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA......Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction...... from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore...

  20. Construction of Recombinant Plasmids by Random Ligation of Blunt-Ended DNA%平末端DNA随机连接构建重组质粒

    Institute of Scientific and Technical Information of China (English)

    查向东; 刘杨; 吴敌; 卢颖虎; 梁琳; 陈青峰

    2012-01-01

    目的:拼接DNA片段并克隆.方法:用T4 DNA连接酶将DNA片段以平末端随机连接,随后用限制性内切酶切割,琼脂糖电泳分离酶切产物,挑选特定片段纯化回收,与线性化的载体质粒连接,转化大肠杆菌感受态细胞.结果:通过以上步骤,成功拼接了不同DNA片段,构建了含有目的拼接片段的重组质粒.结论:该方法简便、易行、可靠,可作为拼接、克隆DNA的备选方案,在分子生物学研究和基因工程中应用.%Objective: To join different DNA fragments and clone the joint DNA. Methods: We randomly ligated the blunt-ended DNA fragments with T4 DNA ligase,cleaved the ligated molecules with the restriction endonucleas-es,then separated the hydrolysed products by agarose gel electrophoresis. We identified the specific band on the agarose gel,from which the DNA molecules were recovered,integrated into the linearized vector plasmid and trans-formed into the competent E.coli cells. Results: In this way we successfully joined the different DNA fragments and constructed the recombinant plasmids haboring the joint DNA. Conclusion: The stragety is simple,convenient and reliable,providing an alternative procedure for joining and cloning DNA which can be used in molecular biology research and gene engenering.

  1. Combinations of various CpG motifs cloned into plasmid backbone modulate and enhance protective immunity of viral replicon DNA anthrax vaccines.

    Science.gov (United States)

    Yu, Yun-Zhou; Ma, Yao; Xu, Wen-Hui; Wang, Shuang; Sun, Zhi-Wei

    2015-08-01

    DNA vaccines are generally weak stimulators of the immune system. Fortunately, their efficacy can be improved using a viral replicon vector or by the addition of immunostimulatory CpG motifs, although the design of these engineered DNA vectors requires optimization. Our results clearly suggest that multiple copies of three types of CpG motifs or combinations of various types of CpG motifs cloned into a viral replicon vector backbone with strong immunostimulatory activities on human PBMC are efficient adjuvants for these DNA vaccines to modulate and enhance protective immunity against anthrax, although modifications with these different CpG forms in vivo elicited inconsistent immune response profiles. Modification with more copies of CpG motifs elicited more potent adjuvant effects leading to the generation of enhanced immunity, which indicated a CpG motif dose-dependent enhancement of antigen-specific immune responses. Notably, the enhanced and/or synchronous adjuvant effects were observed in modification with combinations of two different types of CpG motifs, which provides not only a contribution to the knowledge base on the adjuvant activities of CpG motifs combinations but also implications for the rational design of optimal DNA vaccines with combinations of CpG motifs as "built-in" adjuvants. We describe an efficient strategy to design and optimize DNA vaccines by the addition of combined immunostimulatory CpG motifs in a viral replicon DNA plasmid to produce strong immune responses, which indicates that the CpG-modified viral replicon DNA plasmid may be desirable for use as vector of DNA vaccines.

  2. Chitosan-plasmid DNA nanoparticles encoding small hairpin RNA targeting MMP-3 and -13 to inhibit the expression of dedifferentiation related genes in expanded chondrocytes.

    Science.gov (United States)

    Zhao, Jingxin; Fan, Xiangli; Zhang, Qiang; Sun, Fangfei; Li, Xiaojian; Xiong, Chuan; Zhang, Chunli; Fan, Hongbin

    2014-02-01

    Overexpression of matrix metalloproteinase (MMP)-3 and -13 can lead to the dedifferentiation of expanded chondrocytes. After implanting dedifferentiated cells for cartilage defect repair, graft failure may occur. Short hairpin RNA (shRNA) is a powerful genetic tool to reduce the expression of target genes. This study investigated the effects of chitosan-plasmid DNA (pDNA) nanoparticles encoding shRNA targeting MMP-3 and -13 on the dedifferentiation of expanded chondrocytes. The objective was to optimize the parameters of chitosan-pDNA formulation for achieving higher efficiency of pDNA delivery and gene silencing. The chitosan-pDNA nanoparticles were prepared using a complex coacervation process. Then the characteristics including size, shape, stability, and transfection efficiency were compared in different groups. The results indicated that chitosan of 800 kDa at N/P ratio of 4 and pH 7.0 was optimal to prepare chitosan-pDNA nanoparticles. These nanoparticles showed high DNA loading efficiency (95.8 ± 1.5%) and high gene transfection efficiency (24.5 ± 1.6%). After the expanded chondrocytes were transfected by chitosan-pDNA nanoparticles, MMP-3-610 and MMP-13-2024 groups showed greater suppression in mRNA and protein levels. The results indicated that chitosan-pDNA nanoparticles encoding shRNA targeting MMP-3 and -13 had great potential in silencing the dedifferentiation-related genes for regenerating prolonged and endurable cartilage.

  3. The application of polymer-mediated plasmid DNA transit systems in bone tissue engineering%聚合物介导质粒DNA转运系统在骨组织工程中的应用

    Institute of Scientific and Technical Information of China (English)

    夏伦果; 蒋欣泉; 张志愿

    2009-01-01

    Gene therapy can further promote osteogenesis in bone tissue engineering. By protecting DNA from degradation and maintaining the concentration of DNA effectively, polymer-mediated plasmid DNA transit systems could extend its endocytosis opportunities and enhance the efficiency of gene transfer. At present, polymer-mediated plasmid DNA transit systems used for bone tissue engineering mainly include plasmid DNA and collagen protein composite transit system, plasmid DNA and polyethylene glycol hyaluronic acid hydroge composite transit system, plasmid DNA and liposome composite transit system, plasmid DNA and cationic polymer composite transit systems. This review focuses on the present status of application of polymer-mediated plasmid DNA transit systems.%基因治疗技术应用于骨组织工程,可以进一步促进成骨.聚合物介导的质粒DNA转运系统通过保护DNA免受降解并维持DNA在效应浓度,延长其内吞的机会,从而提高基因转染效率.目前用于骨组织工程研究的聚合物介导的质粒DNA转运系统主要有质粒DNA与胶原蛋白复合转运系统、质粒DNA与聚乙二醇-透明质酸水凝胶复合转运系统、质粒DNA与脂质体复合转运系统、质粒DNA与阳离子聚合物复合转运系统等.本文对近年来聚合物介导的质粒DNA复合转运系统在骨组织工程中的应用进展做一综述.

  4. Direct RAPD evaluation of bacteria without conventional DNA extraction

    Directory of Open Access Journals (Sweden)

    Welington Luiz Araújo

    2004-07-01

    Full Text Available The present work reports successful DNA amplification of Pantoea agglomerans and Bacillus pumilus through Random Amplified Polymorphic DNA (RAPD. For this, template DNA was obtained without conventional DNA extraction. The procedure was as follows: cultures grown for 20 hours in 5 mL LB medium were centrifuged and the resulting preparation was suspended in TE buffer. After boiling, the cell suspension was diluted and 2.0 µl were used in reactions of 15 µl. The results showed no significant differences among the RAPD profile of the PCR reactions derived from the boiling and phenol extraction methods, suggesting the utilization of this method for genetic population analysis.O presente trabalho mostra a amplificação de DNA das bactérias Pantoea agglomerans e Bacillus pumilus por meio da técnica de RAPD (Amplificação ao acaso de DNA polimórfico. Para esta análise, o DNA molde foi obtido sem a utilização de técnicas de extração convencional, ou seja, sem a purificação do DNA. Bactérias foram cultivadas por 20 horas em 5 mL de meio LB, centrifugado e ressuspendido em tampão TE. A suspensão resultante foi fervida por 5 min., diluída e 2,0 µL foram usados em reações de 15 µL. Os resultados mostraram que os padrões observados com o DNA obtido pela fervura das células não apresentou diferenças significativas daquele obtido com DNA extraído e purificado com fenol, sugerindo a possibilidade da utilização deste método para o estudo da variabilidade genética de populações microbianas.

  5. AutoMate Express™ forensic DNA extraction system for the extraction of genomic DNA from biological samples.

    Science.gov (United States)

    Liu, Jason Y; Zhong, Chang; Holt, Allison; Lagace, Robert; Harrold, Michael; Dixon, Alan B; Brevnov, Maxim G; Shewale, Jaiprakash G; Hennessy, Lori K

    2012-07-01

    The AutoMate Express™ Forensic DNA Extraction System was developed for automatic isolation of DNA from a variety of forensic biological samples. The performance of the system was investigated using a wide range of biological samples. Depending on the sample type, either PrepFiler™ lysis buffer or PrepFiler BTA™ lysis buffer was used to lyse the samples. After lysis and removal of the substrate using LySep™ column, the lysate in the sample tubes were loaded onto AutoMate Express™ instrument and DNA was extracted using one of the two instrument extraction protocols. Our study showed that DNA was recovered from as little as 0.025 μL of blood. DNA extracted from casework-type samples was free of detectable PCR inhibitors and the short tandem repeat profiles were complete, conclusive, and devoid of any PCR artifacts. The system also showed consistent performance from day-to-day operation. 2012 American Academy of Forensic Sciences. Published 2012. This article is a U.S. Government work and is in the public domain in the U.S.A.

  6. Rapid DNA extraction methods and new primers for randomly amplified polymorphic DNA analysis of Giardia duodenalis.

    Science.gov (United States)

    Deng, M Q; Cliver, D O

    1999-08-01

    A randomly amplified polymorphic DNA (RAPD) procedure using simple genomic DNA preparation methods and newly designed primers was optimized for analyzing Giardia duodenalis strains. Genomic DNA was extracted from in vitro cultivated trophozoites by five freezing-thawing cycles or by sonic treatment. Compared to a conventional method involving proteinase K digestion and phenol extraction, both freezing-thawing and sonication were equally efficient, yet with the advantage of being much less time- and labor-intensive. Five of the 10 tested RAPD primers produced reproducible polymorphisms among five human origin G. duodenalis strains, and grouping of these strains based on RAPD profiles was in agreement among these primers. The consistent classification of two standard laboratory reference strains, Portland-1 and WB, in the same group confirmed previous results using other fingerprinting methods, indicating that the reported simple DNA extraction methods and the selected primers are useful in RAPD for molecular characterization of G. duodenalis strains.

  7. Comparison of different methodologies for DNA extraction from Aegla longirostri

    Directory of Open Access Journals (Sweden)

    João Vitor Trindade Bitencourt

    2007-11-01

    Full Text Available The aim of this study was to compare some DNA extraction methodologies for Aegla longirostri. The protocols were based on the traditional phenol-chloroform DNA extraction methodology and using a commercial kit for DNA extraction. They differed in tissues used, the addition - or not - of beta-mercaptoethanol to the lysis buffer, times and methods for the animal's conservation (frozen, in ethanol or fresh. Individuals stored at -20°C for a long time supplied lower molecular weight DNA than those stored for a short time. The best yield for the specimens preserved in ethanol was obtained for 15 days storage in 95% ethanol. The kit resulted in a low quantity of high molecular weight DNA. The best protocol for DNA extraction from Aeglidae, and probably for other crustaceans should, therefore, utilize fresh specimens, with addition of beta-mercaptoethanol to the lysis buffer.Marcadores moleculares são ferramentas úteis para esclarecer dúvidas a respeito dos Aeglidae, único grupo de crustáceos Anomura de água doce. Essas técnicas dependem da obtenção de DNA de boa qualidade e sem contaminantes. O objetivo deste estudo foi comparar algumas metodologias de extração de DNA de Aegla longirostri. Quatorze protocolos foram analisados, baseados na metodologia tradicional de extração de DNA com fenol-clorofórmio, exceto o protocolo K no qual se utilizou um Kit. Os procedimentos diferiram quanto aos tecidos utilizados e a adição de beta-mercaptoetanol ao tampão de lise. Avaliaram-se também diferentes tempos e maneiras de conservação. Indivíduos congelados apresentaram maior degradação do material obtido conforme o tempo em que ficaram congelados. Para os indivíduos conservados em álcool, aqueles mantidos em etanol 95% forneceram material de melhor qualidade. A utilização do Mini Kit resultou em uma quantidade muito pequena de DNA de alto peso molecular. O melhor protocolo para extração de DNA de Aeglidae utilizou músculos e br

  8. A PCR amplification method without DNA extraction.

    Science.gov (United States)

    Li, Hongwei; Xu, Haiyue; Zhao, Chunjiang; Sulaiman, Yiming; Wu, Changxin

    2011-02-01

    To develop a simple and inexpensive method for direct PCR amplification of animal DNA from tissues, we optimized different components and their concentration in lysis buffer systems. Finally, we acquired the optimized buffer system composed of 10 mmol tris(hydroxymethyl)aminomethane (Tris)-Cl (pH 8.0), 2 mmol ethylene diamine tetraacetic (EDTA) (pH 8.0), 0.2 mol NaCl and 200 μg/mL Proteinase K. Interestingly, the optimized buffer is also very effective when working with common human sample types, including blood, buccal cells and hair. The direct PCR method requires fewer reagents (Tris-Cl, EDTA, Protease K and NaCl) and less incubation time (only 35 min). The cost of treating every sample is less than $0.02, and all steps can be completed on a thermal cycler in a 96-well format. So, the proposed method will significantly improve high-throughput PCR-based molecular assays in animal systems and in common human sample types.

  9. A method suitable for DNA extraction from humus-rich soil.

    Science.gov (United States)

    Miao, Tianjin; Gao, Song; Jiang, Shengwei; Kan, Guoshi; Liu, Pengju; Wu, Xianming; An, Yingfeng; Yao, Shuo

    2014-11-01

    A rapid and convenient method for extracting DNA from soil is presented. Soil DNA is extracted by direct cell lysis in the presence of EDTA, SDS, phenol, chloroform and isoamyl alcohol (3-methyl-1-butanol) followed by precipitation with 2-propanol. The extracted DNA is purified by modified DNA purification kit and DNA gel extraction kit. With this method, DNA extracted from humus-rich dark brown forest soil was free from humic substances and, therefore, could be used for efficient PCR amplification and restriction digestion. In contrast, DNA sample extracted with the traditional CTAB-based method had lower yield and purity, and no DNA could be extracted from the same soil sample with a commonly-used commercial soil DNA isolation kit. In addition, this method is time-saving and convenient, providing an efficient choice especially for DNA extraction from humus-rich soils.

  10. Direct Extraction and Amplification of DNA from Soil.

    Science.gov (United States)

    Trevors, Jack T.; Leung, K.

    1998-01-01

    Presents an exercise that describes the direct extraction and purification of DNA from a small soil sample. Also discusses the subsequent amplification of a 343-bp Tn7 transposate A gene fragment (tnsA) from a strain of Pseudomonas aureofaciens 3732RNL11. Contains 21 references. (DDR)

  11. Direct Extraction and Amplification of DNA from Soil.

    Science.gov (United States)

    Trevors, Jack T.; Leung, K.

    1998-01-01

    Presents an exercise that describes the direct extraction and purification of DNA from a small soil sample. Also discusses the subsequent amplification of a 343-bp Tn7 transposate A gene fragment (tnsA) from a strain of Pseudomonas aureofaciens 3732RNL11. Contains 21 references. (DDR)

  12. Repeated intrathecal administration of plasmid DNA complexed with polyethylene glycol-grafted polyethylenimine led to prolonged transgene expression in the spinal cord.

    Science.gov (United States)

    Shi, L; Tang, G P; Gao, S J; Ma, Y X; Liu, B H; Li, Y; Zeng, J M; Ng, Y K; Leong, K W; Wang, S

    2003-07-01

    Gene delivery into the spinal cord provides a potential approach to the treatment of spinal cord traumatic injury, amyotrophic lateral sclerosis, and spinal muscular atrophy. These disorders progress over long periods of time, necessitating a stable expression of functional genes at therapeutic levels for months or years. We investigated in this study the feasibility of achieving prolonged transgene expression in the rat spinal cord through repeated intrathecal administration of plasmid DNA complexed with 25 kDa polyethylenimine (PEI) into the lumbar subarachnoid space. With a single injection, DNA/PEI complexes could provide transgene expression in the spinal cord 40-fold higher than naked plasmid DNA. The transgene expression at the initial level persisted for about 5 days, with a low-level expression being detectable for at least 8 weeks. When repeated dosing was tested, a 70% attenuation of gene expression was observed following reinjection at a 2-week interval. This attenuation was associated with apoptotic cell death and detected even using complexes containing a noncoding DNA that did not mediate any gene expression. When each component of the complexes, PEI polymer or naked DNA alone, were tested in the first dosing, no reduction was found. Using polyethylene glycol (PEG)-grafted PEI for DNA complexes, no attenuation of gene expression was detected after repeated intrathecal injections, even in those rats receiving three doses, administered 2 weeks apart. Lumbar puncture is a routine and relatively nontraumatic clinical procedure. Repeated administration of DNA complexed with PEG-grafted PEI through this less invasive route may prolong the time span of transgene expression when needed, providing a viable strategy for the gene therapy of spinal cord disorders.

  13. DNA extraction and barcode identification of development stages of forensically important flies in the Czech Republic.

    Science.gov (United States)

    Olekšáková, Tereza; Žurovcová, Martina; Klimešová, Vanda; Barták, Miroslav; Šuláková, Hana

    2017-03-21

    Several methods of DNA extraction, coupled with 'DNA barcoding' species identification, were compared using specimens from early developmental stages of forensically important flies from the Calliphoridae and Sarcophagidae families. DNA was extracted at three immature stages - eggs, the first instar larvae, and empty pupal cases (puparia) - using four different extraction methods, namely, one simple 'homemade' extraction buffer protocol and three commercial kits. The extraction conditions, including the amount of proteinase K and incubation times, were optimized. The simple extraction buffer method was successful for half of the eggs and for the first instar larval samples. The DNA Lego Kit and DEP-25 DNA Extraction Kit were useful for DNA extractions from the first instar larvae samples, and the DNA Lego Kit was also successful regarding the extraction from eggs. The QIAamp DNA mini kit was the most effective; the extraction was successful with regard to all sample types - eggs, larvae, and pupari.

  14. Comparison of DNA extraction methods for meat analysis.

    Science.gov (United States)

    Yalçınkaya, Burhanettin; Yumbul, Eylem; Mozioğlu, Erkan; Akgoz, Muslum

    2017-04-15

    Preventing adulteration of meat and meat products with less desirable or objectionable meat species is important not only for economical, religious and health reasons, but also, it is important for fair trade practices, therefore, several methods for identification of meat and meat products have been developed. In the present study, ten different DNA extraction methods, including Tris-EDTA Method, a modified Cetyltrimethylammonium Bromide (CTAB) Method, Alkaline Method, Urea Method, Salt Method, Guanidinium Isothiocyanate (GuSCN) Method, Wizard Method, Qiagen Method, Zymogen Method and Genespin Method were examined to determine their relative effectiveness for extracting DNA from meat samples. The results show that the salt method is easy to perform, inexpensive and environmentally friendly. Additionally, it has the highest yield among all the isolation methods tested. We suggest this method as an alternative method for DNA isolation from meat and meat products. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

    Directory of Open Access Journals (Sweden)

    Alena V Makarova

    Full Text Available Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+ ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA". We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

  16. Evaluation of DNA extraction methods of rumen microbial populations.

    Science.gov (United States)

    Villegas-Rivera, Gabriela; Vargas-Cabrera, Yevani; González-Silva, Napoleón; Aguilera-García, Florentino; Gutiérrez-Vázquez, Ernestina; Bravo-Patiño, Alejandro; Cajero-Juárez, Marcos; Baizabal-Aguirre, Víctor Manuel; Valdez-Alarcón, Juan José

    2013-02-01

    The dynamism of microbial populations in the rumen has been studied with molecular methods that analyze single nucleotide polymorphisms of ribosomal RNA gene fragments (rDNA). Therefore DNA of good quality is needed for this kind of analysis. In this work we report the evaluation of four DNA extraction protocols (mechanical lysis or chemical lysis with CTAB, ethylxanthogenate or DNAzol(®)) from ruminal fluid. The suitability of two of these protocols (mechanical lysis and DNAzol(®)) was tested on single-strand conformation polymorphism (SSCP) of rDNA of rumen microbial populations. DNAzol(®) was a simple method that rendered good integrity, yield and purity. With this method, subtle changes in protozoan populations were detected in young bulls fed with slightly different formulations of a supplement of multinutritional blocks of molasses and urea. Sequences related to Eudiplodinium maggi and a non-cultured Entodiniomorphid similar to Entodinium caudatum, were related to major fluctuating populations in an SSCP assay.

  17. Increased B and T Cell Responses in M. bovis Bacille Calmette-Guerin Vaccinated Pigs Co-Immunized with Plasmid DNA Encoding a Prototype Tuberculosis Antigen.

    Directory of Open Access Journals (Sweden)

    Nicolas Bruffaerts

    Full Text Available The only tuberculosis vaccine currently available, bacille Calmette-Guérin (BCG is a poor inducer of CD8(+ T cells, which are particularly important for the control of latent tuberculosis and protection against reactivation. As the induction of strong CD8(+ T cell responses is a hallmark of DNA vaccines, a combination of BCG with plasmid DNA encoding a prototype TB antigen (Ag85A was tested. As an alternative animal model, pigs were primed with BCG mixed with empty vector or codon-optimized pAg85A by the intradermal route and boosted with plasmid delivered by intramuscular electroporation. Control pigs received unformulated BCG. The BCG-pAg85A combination stimulated robust and sustained Ag85A specific antibody, lymphoproliferative, IL-6, IL-10 and IFN-γ responses. IgG1/IgG2 antibody isotype ratio reflected the Th1 helper type biased response. T lymphocyte responses against purified protein derivative of tuberculin (PPD were induced in all (BCG vaccinated animals, but responses were much stronger in BCG-pAg85A vaccinated pigs. Finally, Ag85A-specific IFN-γ producing CD8(+ T cells were detected by intracellular cytokine staining and a synthetic peptide, spanning Ag85A131-150 and encompassing two regions with strong predicted SLA-1*0401/SLA-1*0801 binding affinity, was promiscuously recognized by 6/6 animals vaccinated with the BCG-pAg85A combination. Our study provides a proof of concept in a large mammalian species, for a new Th1 and CD8(+ targeting tuberculosis vaccine, based on BCG-plasmid DNA co-administration.

  18. Reduction of Intimal Hyperplasia in Injured Rat Arteries Promoted by Catheter Balloons Coated with Polyelectrolyte Multilayers that Contain Plasmid DNA Encoding PKCδ

    Science.gov (United States)

    Bechler, Shane L.; Si, Yi; Yu, Yan; Ren, Jun; Liu, Bo; Lynn, David M.

    2012-01-01

    New therapeutic approaches that eliminate or reduce the occurrence of intimal hyperplasia following balloon angioplasty could improve the efficacy of vascular interventions and improve the quality of life of patients suffering from vascular diseases. Here, we report that treatment of arteries using catheter balloons coated with thin polyelectrolyte-based films (‘polyelectrolyte multilayers’, PEMs) can substantially reduce intimal hyperplasia in an in vivo rat model of vascular injury. We used a layer-by-layer (LbL) process to coat the surfaces of inflatable catheter balloons with PEMs composed of nanolayers of a cationic poly(β-amino ester) (polymer 1) and plasmid DNA (pPKCδ) encoding the δ isoform of protein kinase C (PKCδ), a regulator of apoptosis and other cell processes that has been demonstrated to reduce intimal hyperplasia in injured arterial tissue when administered via perfusion using viral vectors. Insertion of balloons coated with polymer 1/pPKCδ multilayers into injured arteries for 20 min resulted in local transfer of DNA and elevated levels of PKCδ expression in the media of treated tissue 3 days after delivery. IFC and IHC analysis revealed these levels of expression to promote downstream cellular processes associated with up-regulation of apoptosis. Analysis of arterial tissue 14 days after treatment revealed polymer 1/pPKCδ-coated balloons to reduce the occurrence of intimal hyperplasia by ~60% compared to balloons coated with films containing empty plasmid vectors. Our results demonstrate the potential therapeutic value of this nanotechnology-based approach to local gene delivery in the clinically important context of balloon-mediated vascular interventions. These PEM-based methods could also prove useful for other in vivo applications that require short-term, surface-mediated transfer of plasmid DNA. PMID:23069712

  19. Additives and Protein-DNA Combinations Modulate the Humoral Immune Response Elicited by a Hepatitis C Virus Core-encoding Plasmid in Mice

    Directory of Open Access Journals (Sweden)

    Alvarez-Lajonchere Liz

    2002-01-01

    Full Text Available Humoral and cellular immune responses are currently induced against hepatitis C virus (HCV core following vaccination with core-encoding plasmids. However, the anti-core antibody response is frequently weak or transient. In this paper, we evaluated the effect of different additives and DNA-protein combinations on the anti-core antibody response. BALB/c mice were intramuscularly injected with an expression plasmid (pIDKCo, encoding a C-terminal truncated variant of the HCV core protein, alone or combined with CaCl2, PEG 6000, Freund's adjuvant, sonicated calf thymus DNA and a recombinant core protein (Co.120. Mixture of pIDKCo with PEG 6000 and Freund's adjuvant accelerated the development of the anti-core Ab response. Combination with PEG 6000 also induced a bias to IgG2a subclass predominance among anti-core antibodies. The kinetics, IgG2a/IgG1 ratio and epitope specificity of the anti-core antibody response elicited by Co.120 alone or combined with pIDKCo was different regarding that induced by the pIDKCo alone. Our data indicate that the antibody response induced following DNA immunization can be modified by formulation strategies.

  20. Protective effect of a prime-boost strategy with plasmid DNA followed by recombinant adenovirus expressing TgAMA1 as vaccines against Toxoplasma gondii infection in mice.

    Science.gov (United States)

    Yu, Longzheng; Yamagishi, Junya; Zhang, Shoufa; Jin, Chunmei; Aboge, Gabriel Oluga; Zhang, Houshuang; Zhang, Guohong; Tanaka, Tetsuya; Fujisaki, Kozo; Nishikawa, Yoshifumi; Xuan, Xuenan

    2012-09-01

    A heterologous prime-boost strategy with priming plasmid DNA followed by recombinant virus expressing relevant antigens is known to stimulate protective immunity against intracellular parasites. In this study, we have evaluated a heterologous prime-boost strategy for immunizing mice against Toxoplasma gondii infection. Our results revealed that the prime-boost strategy using both plasmid DNA and adenoviral vector encoding TgAMA1 may stimulate both humoral and Th1/Th2 cellular immune responses specific for TgAMA1. Moreover, C57BL/6 mice immunized with the pAMA1/Ad5Null, pNull/Ad5AMA1, and pAMA1/Ad5AMA1 constructs showed survival rates of 12.5%, 37.5%, and 50%, respectively. In contrast, all the pNull/Ad5Null immunized mice died after infection with the PLK-GFP strain of T. gondii. Brain cyst burden was reduced by 23% in mice immunized with pAMA1/Ad5AMA1 compared with the pNull/Ad5AMA1 immunized mice. These results demonstrate that the heterologous DNA priming and recombinant adenovirus boost strategy may provide protective immunity against T. gondii infection.

  1. Persistence Mechanisms of Conjugative Plasmids

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Hansen, Lars H.; Sørensen, Søren Johannes

    2009-01-01

    Are plasmids selfish parasitic DNA molecules or an integrated part of the bacterial genome? This chapter reviews the current understanding of the persistence mechanisms of conjugative plasmids harbored by bacterial cells and populations. The diversity and intricacy of mechanisms affecting...... the successful propagation and long-term continued existence of these extra-chromosomal elements is extensive. Apart from the accessory genetic elements that may provide plasmid-harboring cells a selective advantage, special focus is placed on the mechanisms conjugative plasmids employ to ensure their stable...... maintenance in the host cell. These importantly include the ability to self-mobilize in a process termed conjugative transfer, which may occur across species barriers. Other plasmid stabilizing mechanisms include the multimer resolution system, active partitioning, and post-segregational-killing of plasmid...

  2. PCR-based typing of DNA extracted from cigarette butts.

    Science.gov (United States)

    Hochmeister, M N; Budowle, B; Jung, J; Borer, U V; Comey, C T; Dirnhofer, R

    1991-01-01

    Limited genetic marker information can be obtained from saliva by typing by conventional serological means. Thus, the application of PCR-based DNA typing methods was investigated as a potential approach for typing genetic markers in saliva. DNA was isolated from 200 cigarettes smoked by 10 different individuals (20 cigarettes per individual) and from 3 cigarette butts recovered from 2 crime scenes (adjudicated cases) using a Chelex 100 extraction procedure. The amount of recovered human DNA was quantified by slot-blot analysis and ranged from approximately less than 2-160 ng DNA per cigarette butt for the 200 samples, and 8 ng, 50 ng, and 100 ng for the cigarette butts from the adjudicated cases. The DNA was successfully amplified by the polymerase chain reaction (PCR) for the HLA-DQ alpha locus (99 out of 100 samples) as well as for the variable number of tandem repeat (VNTR) locus D1S80 (99 out of 100 samples). Amplification and typing of DNA was successful on all samples recovered from the crime scenes. The results suggest that PCR-based typing of DNA offers a potential method for genetically characterizing traces of saliva on cigarette butts.

  3. Rapid methods to extract DNA and RNA from Cryptococcus neoformans.

    Science.gov (United States)

    Bolano, A; Stinchi, S; Preziosi, R; Bistoni, F; Allegrucci, M; Baldelli, F; Martini, A; Cardinali, G

    2001-12-01

    Extraction of nucleic acids from the pathogenic yeast Cryptococcus neoformans is normally hampered by a thick and resistant capsule, accounting for at least 70% of the whole cellular volume. This paper presents procedures based on mechanical cell breakage to extract DNA and RNA from C. neoformans and other capsulated species. The proposed system for DNA extraction involves capsule relaxation by means of a short urea treatment and bead beating. These two steps allow a consistent extraction even from strains resistant to other procedures. Yield and quality of DNA obtained with the proposed method were higher than those obtained with two earlier described methods. This protocol can be extended to every yeast species and particularly to those difficult to handle for the presence of a capsule. RNA purification is accomplished using an original lysing matrix and the FastPrep System (Bio101) after a preliminary bead beating treatment. Yields range around 1 mg RNA from 15 ml overnight culture (10(9) cells), RNA appears undegraded, making it suitable for molecular manipulations.

  4. An E3-14.7K peptide that promotes microtubules-mediated transport of plasmid DNA increases polyplexes transfection efficiency.

    Science.gov (United States)

    Pigeon, Lucie; Gonçalves, Cristine; Gosset, David; Pichon, Chantal; Midoux, Patrick

    2013-11-25

    Chemical vectors as cationic polymers and cationic lipids are promising alternatives to viral vectors for gene therapy. Beside endosome escape and nuclear import, plasmid DNA (pDNA) migration in the cytosol toward the nuclear envelope is also regarded as a limiting step for efficient DNA transfection with non-viral vectors. Here, the interaction between E3-14.7K and FIP-1 to favor migration of pDNA along microtubules is exploited. E3-14.7K is an early protein of human adenoviruses that interacts via FIP-1 (Fourteen.7K Interacting Protein 1) protein with the light-chain components of the human microtubule motor protein dynein (TCTEL1). This peptide is conjugated with pDNA and mediates interaction of pDNA in vitro with isolated microtubules as well as with microtubules in cellulo. Videomicroscopy and tracking treatment of images clearly demonstrate that P79-98/pDNA conjugate exhibits a linear transport with large amplitude along microtubules upon 2 h transfection with polyplexes whereas control pDNA conjugate exhibits small non-directional movements in the cytoplasm. Remarkably, P79-98/peGFP polyplexes enhance by a factor 2.5 (up to 76%) the number of transfected cells. The results demonstrate, for the first time, that the transfection efficiency of polyplexes can be drastically increased when the microtubules migration of pDNA is facilitated by a peptide allowing pDNA docking to TCTEL1. This is a real breakthrough in the non viral gene delivery field that opens hope to build artificial viruses.

  5. Single -and double-strand breaks induced in plasmid DNA irradiated by ultra -soft X-ray; Cassures simple- et double-brin induites par X ultra-mous dans l`ADN plasmidique

    Energy Technology Data Exchange (ETDEWEB)

    Fayard, B.; Touati, A.; Abel, F.; Champion, C.; Chetoui, A. [Paris-6 Univ., 75 (France)]|[Paris-7 Univ., 75 (France); Sage, E. [Institut Curie, UMR 218 CNRS, 75 - Paris (France). Section de Recherche

    1999-01-01

    In order to investigate the molecular consequences of a carbon K photo-ionization located on DNA, dry pBS plasmid samples were irradiated with ultra-soft X-rays at energies below and above the carbon K-threshold (E{sub k}=278 eV). Single- and double-strand breaks (ssb and dsb) were quantified after resolution of the three plasmid forms (supercoiled, relaxed circular, linear) by gel electrophoresis. A factor of 1.2 was found between the doses required at 250 eV and 380 eV to induce the same number of dsb per plasmid. (authors) 6 refs.

  6. Chamomile flower extract-directed CuO nanoparticle formation for its antioxidant and DNA cleavage properties

    Energy Technology Data Exchange (ETDEWEB)

    Duman, Fatih, E-mail: fduman@erciyes.edu.tr [Erciyes University, Science Faculty, Biology Department, Kayseri 38039, Kayseri (Turkey); Ocsoy, Ismail [Department of Analytical Chemistry, Faculty of Pharmacy, Erciyes University, 38039, Kayseri (Turkey); Erciyes University, Nanotechnology Research Center, 38039, Kayseri (Turkey); Kup, Fatma Ozturk [Erciyes University, Science Faculty, Biology Department, Kayseri 38039, Kayseri (Turkey)

    2016-03-01

    In this study, we report the synthesis of copper oxide nanoparticles (CuO NPs) using a medicinal plant (Matricaria chamomilla) flower extract as both reducing and capping agent and investigate their antioxidant activity and interaction with plasmid DNA (pBR322).The CuO NPs were characterized using Uv–Vis spectroscopy, FT-IR (Fourier transform infrared spectroscopy), DLS (dynamic light scattering), XRD (X-ray diffraction), EDX (energy-dispersive X-ray) spectroscopy and SEM (scanning electron microscopy). The CuO NPs exhibited nearly mono-distributed and spherical shapes with diameters of 140 nm size. UV–Vis absorption spectrum of CuO NPs gave a broad peak around 285 and 320 nm. The existence of functional groups on the surface of CuO NPs was characterized with FT-IR analysis. XRD pattern showed that the NPs are in the form of a face-centered cubic crystal. Zeta potential value was measured as − 20 mV due to the presence of negatively charged functional groups in plant extract. Additionally, we demonstrated concentration-dependent antioxidant activity of CuO NPs and their interaction with plasmid DNA. We assumed that the CuO NPs both cleave and break DNA double helix structure. - Highlights: • The synthesis of microwave assisted green synthesis of CuO nanoparticles • The synthesized nanoparticles were analyzed by FT-IR, DLS, XRD, EDX and SEM. • Concentration-dependent antioxidant activity of CuO NPs was determined. • CuO NPs cause both cleavage in the DNA double helix structure and breaks as well.

  7. DNA Everywhere. A Guide for Simplified Environmental Genomic DNA Extraction Suitable for Use in Remote Areas

    Energy Technology Data Exchange (ETDEWEB)

    Pecora, Gabrielle N. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Reid, Francine C. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Tom, Lauren M. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Piceno, Yvette M. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Andersen, Gary L. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2016-05-01

    Collecting field samples from remote or geographically distant areas can be a financially and logistically challenging. With participation of a local organization where the samples are originated from, gDNA samples can be extracted from the field and shipped to a research institution for further processing and analysis. The ability to set up gDNA extraction capabilities in the field can drastically reduce cost and time when running long-term microbial studies with a large sample set. The method outlined here has developed a compact and affordable method for setting up a “laboratory” and extracting and shipping gDNA samples from anywhere in the world. This white paper explains the process of setting up the “laboratory”, choosing and training individuals with no prior scientific experience how to perform gDNA extractions and safe methods for shipping extracts to any research institution. All methods have been validated by the Andersen group at Lawrence Berkeley National Laboratory using the Berkeley Lab PhyloChip.

  8. Preparation of Ag/AgBr/TiO2 as Catalyst Carriers and Its Damage to Plasmid DNA andTetrahymena

    Institute of Scientific and Technical Information of China (English)

    LIU Liwei; ZHANG Yinlong

    2015-01-01

    The composites based on the TiO2 are potentially used in wetland pollution control. In this work, the biological effect of the Ag/AgBr/TiO2/Active carbon (AC) composites was studied on the plasmid DNA andTetrahymena membrane. The atomic force micrograph (AFM) images showed that, in the presence of the composites under illumination, most pUC18 DNA molecules showed quite different topography and were opened and relaxed circle shapes. After DNA was catalyzed for 40 min, all supercoiled and circular DNA were changed into the linear DNA molecules. The gel electrophoresis experiment confirmed the results and demonstrated the dynamic process of DNA degradation. ATR-FTIR spectra revealed that amide groups and PO2− of the phospho-lipid phospho-diester onTetrahymena surface were oxidized in the presence of the composites under illumination. An increase in the lfuorescence polarization of DPH was observed, relfecting a signiifcant decrease in membrane lfuidity ofTetrahymena.

  9. Effect of Polyethylene Glycol Multi-walled Carbon Nanotubes to Plasmid DNA%聚乙二醇修饰多壁碳纳米管对质粒DNA的影响

    Institute of Scientific and Technical Information of China (English)

    劳文艳; 商迎辉; 焦正; 劳凤学

    2011-01-01

    利用体外评价方法(plasmid DNA assay)和原子力显微镜(atomic force microscopy,AFM)直接观测相结合,研究原始多壁碳纳米管(multi-walled carbon nanotubes,MWNTs)、纯化后的MWNTs以及聚乙二醇(polyethylene glycol,PEG)修饰的MWNTs与DNA的相互作用.结果表明:原始MWNTs对质粒的损伤较为严重;纯化后的MWNTs对质粒的损伤有所减弱;而经PEG修饰的MWNTs对质粒的损伤非常小,表现出良好的生物相容性.%The biocompatibility of pristine multi-walled carbon nanotubes (MWNTs), purified MWNTs and polyethylene glycol MWNTs (PEG-MWNTs) was studied using the method of plasmid DNA assay and atomic force microscopy (AFM).The results show that pristine MWNTs can cause serious damage to plasmid DNA.After purification, MWNTs may cause less damage to plasmid DNA, while at the same concentration level, PEG-MWNTs cause the smallest damage to plasmid DNA.

  10. Comparison of eight methods of genomic DNA extraction from babassu.

    Science.gov (United States)

    Viana, J P G; Borges, A N C; Lopes, A C A; Gomes, R L F; Britto, F B; Lima, P S C; Valente, S E S

    2015-12-22

    Babassu (Orbignya phalerata Martius) is one of the most important palms in Brazil because of the largest morphological variation, wide geographic distribution, and high socio-economic importance. The diversity present in babassu germplasm should be protected against loss to ensure their use with high productivity. Study of the available variability in populations of babassu is necessary to develop conservation strategies. The study of genetic variability can be conducted using molecular markers and many of these studies require significant quantity of high-quality DNA. The present study aimed to effect comparison among eight DNA extraction methods in case of O. phalerata. The quality and concentration of nucleic acids were analyzed by spectrophotometry and integrity of DNA was ascertained by agarose gel electrophoresis. The spectrophotometry revealed that some methods resulted in high levels of concentration of nucleic acids, in which values of the ratio A260/280 and A260/230 were outside the range of purity. The agarose gel electrophoresis established the concentration and integrity of DNA. The methods of Murray and Thompson (1980) and Ferreira and Grattapaglia (1998) did not result in satisfactory quantities of DNA. Conversely, the method proposed by Khanuja et al. (1999) resulted in DNA of adequate quality and quantity that could be satisfactorily used for amplification reactions performed with two ISSR primers.

  11. Co-administration of a plasmid DNA encoding IL-15 improves long-term protection of a genetic vaccine against Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Christopher S Eickhoff

    Full Text Available BACKGROUND: Immunization of mice with the Trypanosoma cruzi trans-sialidase (TS gene using plasmid DNA, adenoviral vector, and CpG-adjuvanted protein delivery has proven highly immunogenic and provides protection against acute lethal challenge. However, long-term protection induced by TS DNA vaccines has not been reported. The goal of the present work was to test whether the co-administration of a plasmid encoding IL-15 (pIL-15 could improve the duration of protection achieved through genetic vaccination with plasmid encoding TS (pTS alone. METHODOLOGY: We immunized BALB/c mice with pTS in the presence or absence of pIL-15 and studied immune responses [with TS-specific IFN-γ ELISPOT, serum IgG ELISAs, intracellular cytokine staining (IFN-γ, TNF-α, and IL-2, tetramer staining, and CFSE dilution assays] and protection against lethal systemic challenge at 1 to 6 months post vaccination. Mice receiving pTS alone developed robust TS-specific IFN-γ responses and survived a lethal challenge given within the first 3 months following immunization. The addition of pIL-15 to pTS vaccination did not significantly alter T cell responses or protection during this early post-vaccination period. However, mice vaccinated with both pTS and pIL-15 challenged 6 months post-vaccination were significantly more protected against lethal T. cruzi challenges than mice vaccinated with pTS alone (P6 months post immunization. Also, these TS-specific T cells were better able to expand after in vitro re-stimulation. CONCLUSION: Addition of pIL-15 during genetic vaccination greatly improved long-term T cell survival, memory T cell expansion, and long-term protection against the important human parasite, T. cruzi.

  12. Detection of Streptococcus mutans Genomic DNA in Human DNA Samples Extracted from Saliva and Blood

    Science.gov (United States)

    Vieira, Alexandre R.; Deeley, Kathleen B.; Callahan, Nicholas F.; Noel, Jacqueline B.; Anjomshoaa, Ida; Carricato, Wendy M.; Schulhof, Louise P.; DeSensi, Rebecca S.; Gandhi, Pooja; Resick, Judith M.; Brandon, Carla A.; Rozhon, Christopher; Patir, Asli; Yildirim, Mine; Poletta, Fernando A.; Mereb, Juan C.; Letra, Ariadne; Menezes, Renato; Wendell, Steven; Lopez-Camelo, Jorge S.; Castilla, Eduardo E.; Orioli, Iêda M.; Seymen, Figen; Weyant, Robert J.; Crout, Richard; McNeil, Daniel W.; Modesto, Adriana; Marazita, Mary L.

    2011-01-01

    Caries is a multifactorial disease, and studies aiming to unravel the factors modulating its etiology must consider all known predisposing factors. One major factor is bacterial colonization, and Streptococcus mutans is the main microorganism associated with the initiation of the disease. In our studies, we have access to DNA samples extracted from human saliva and blood. In this report, we tested a real-time PCR assay developed to detect copies of genomic DNA from Streptococcus mutans in 1,424 DNA samples from humans. Our results suggest that we can determine the presence of genomic DNA copies of Streptococcus mutans in both DNA samples from caries-free and caries-affected individuals. However, we were not able to detect the presence of genomic DNA copies of Streptococcus mutans in any DNA samples extracted from peripheral blood, which suggests the assay may not be sensitive enough for this goal. Values of the threshold cycle of the real-time PCR reaction correlate with higher levels of caries experience in children, but this correlation could not be detected for adults. PMID:21731912

  13. CD44-Targeted Hyaluronic Acid-Coated Redox-Responsive Hyperbranched Poly(amido amine)/Plasmid DNA Ternary Nanoassemblies for Efficient Gene Delivery.

    Science.gov (United States)

    Gu, Jijin; Chen, Xinyi; Ren, Xiaoqing; Zhang, Xiulei; Fang, Xiaoling; Sha, Xianyi

    2016-07-20

    Hyaluronic acid (HA), which can specifically bind to CD44 receptor, is a specific ligand for targeting to CD44-overexpressing cancer cells. The current study aimed to develop ternary nanoassemblies based on HA-coating for targeted gene delivery to CD44-positive tumors. A novel reducible hyperbranched poly(amido amine) (RHB) was assembled with plasmid DNA (pDNA) to form RHB/pDNA nanoassemblies. HA/RHB/pDNA nanoassemblies were fabricated by coating HA on the surface of the RHB/pDNA nanoassembly core through electrostatic interaction. After optimization, HA/RHB/pDNA nanoassemblies were spherical, core-shell nanoparticles with nanosize (187.6 ± 11.4 nm) and negative charge (-9.1 ± 0.3 mV). The ternary nanoassemblies could efficiently protect the condensed pDNA from enzymatic degradation by DNase I, and HA could significantly improve the stability of nanoassemblies in the sodium heparin solution or serum in vitro. As expected, HA significantly decreased the cytotoxicity of RHB/pDNA nanoassemblies due to the negative surface charges. Moreover, it revealed that HA/RHB/pDNA nanoassemblies showed higher transfection activity than RHB/pDNA nanoassemblies in B16F10 cells, especially in the presence of serum in vitro. Because of the active recognition between HA and CD44 receptor, there was significantly different transfection efficiency between B16F10 (CD44+) and NIH3T3 (CD44-) cells after treatment with HA/RHB/pDNA nanoassemblies. In addition, the cellular targeting and transfection activity of HA/RHB/pDNA nanoassemblies were further evaluated in vivo. The results indicated that the interaction between HA and CD44 receptor dramatically improved the accumulation of HA/RHB/pDNA nanoassemblies in CD44-positive tumor, leading to higher gene expression than RHB/pDNA nanoassemblies. Therefore, HA/RHB/pDNA ternary nanoassemblies may be a potential gene vector for delivery of therapeutic genes to treat CD44-overexpressing tumors in vivo.

  14. Optimizing Fungal DNA Extraction Methods from Aerosol Filters

    Science.gov (United States)

    Jimenez, G.; Mescioglu, E.; Paytan, A.

    2016-12-01

    Fungi and fungal spores can be picked up from terrestrial ecosystems, transported long distances, and deposited into marine ecosystems. It is important to study dust-borne fungal communities, because they can stay viable and effect the ambient microbial populations, which are key players in biogeochemical cycles. One of the challenges of studying dust-borne fungal populations is that aerosol samples contain low biomass, making extracting good quality DNA very difficult. The aim of this project was to increase DNA yield by optimizing DNA extraction methods. We tested aerosol samples collected from Haifa, Israel (polycarbonate filter), Monterey Bay, CA (quartz filter) and Bermuda (quartz filter). Using the Qiagen DNeasy Plant Kit, we tested the effect of altering bead beating times and incubation times, adding three freeze and thaw steps, initially washing the filters with buffers for various lengths of time before using the kit, and adding a step with 30 minutes of sonication in 65C water. Adding three freeze/thaw steps, adding a sonication step, washing with a phosphate buffered saline overnight, and increasing incubation time to two hours, in that order, resulted in the highest increase in DNA for samples from Israel (polycarbonate). DNA yield of samples from Monterey (quart filter) increased about 5 times when washing with buffers overnight (phosphate buffered saline and potassium phophate buffer), adding a sonication step, and adding three freeze and thaw steps. Samples collected in Bermuda (quartz filter) had the highest increase in DNA yield from increasing incubation to 2 hours, increasing bead beating time to 6 minutes, and washing with buffers overnight (phosphate buffered saline and potassium phophate buffer). Our results show that DNA yield can be increased by altering various steps of the Qiagen DNeasy Plant Kit protocol, but different types of filters collected at different sites respond differently to alterations. These results can be used as

  15. Arduino-based automation of a DNA extraction system.

    Science.gov (United States)

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile.

  16. Calcium phosphate nanoparticles carrying BMP-7 plasmid DNA induce an osteogenic response in MC3T3-E1 pre-osteoblasts.

    Science.gov (United States)

    Hadjicharalambous, Chrystalleni; Kozlova, Diana; Sokolova, Viktoriya; Epple, Matthias; Chatzinikolaidou, Maria

    2015-12-01

    Functionalized calcium phosphate nanoparticles with osteogenic activity were prepared. Polyethyleneimine-stabilized calcium phosphate nanoparticles were coated with a shell of silica and covalently functionalized by silanization with thiol groups. Between the calcium phosphate surface and the outer silica shell, plasmid DNA which encoded either for bone morphogenetic protein 7 (BMP-7) or for enhanced green fluorescent protein was incorporated as cargo. The plasmid DNA-loaded calcium phosphate nanoparticles were used for the transfection of the pre-osteoblastic MC3T3-E1 cells. The cationic nanoparticles showed high transfection efficiency together with a low cytotoxicity. Their potential to induce an osteogenic response by transfection was demonstrated by measuring the alkaline phosphatase (ALP) activity and calcium deposition with alizarin red staining. The expression of the osteogenic markers Alp, Runx2, ColIa1 and Bsp was investigated by means of real-time quantitative polymerase chain reaction. It was shown that phBMP-7-loaded nanoparticles can provide a means of transient transfection and localized production of BMP-7 in MC3T3-E1 cells, with a subsequent increase of two osteogenic markers, specifically ALP activity and calcium accumulation in the extracellular matrix. Future strategies to stimulate bone regeneration focus into enhancing transfection efficiency and achieving higher levels of BMP-7 produced by the transfected cells.

  17. Optimization of DNA Extractions from Iron-rich Microbial Mats

    Science.gov (United States)

    Fullerton, H.; Hilton, T. S.; Moyer, C. L.

    2013-12-01

    Iron is the fourth most abundant element in the Earth's crust and is potentially one of the most abundant energy sources on the earth as an electron donor for chemolithoautotrophicgrowth coupled to Fe(II) oxidation. Many microbes have adapted to this energy source. One such bacterial class are the Zetaproteobacteria, which dominate Iron-rich microbial mats at Loihi seamount. Although cell counts are very high (up to 5.3x108 cells/ml), efficient DNA yields are low in comparison. In this study we compared extraction efficiency across different methods and with the addition of various buffers. Regardless of protocol (i.e., kit), the addition of sodium citrate drastically increased the DNA yield. The addition of sodium citrate did not alter community structure as determined by T-RFLP and qPCR. Citrate is a well-known ferric iron chelator and will bind ferrous as well. The chelated iron is then unable to participate in the Fenton reaction and this stops the generation of hydroxyl radicals which in turn can react and degrade the extracted DNA. We have utilized this relationship to allow us to obtain nearly an order of magnitude more microbial community DNA per sample, which should also have implications when processing low biomass samples, e.g., from the deep subsurface.

  18. DNA extraction from Ascaris suum muscle tissue

    OpenAIRE

    Di Mito, Carmela; Betschart, Bruno

    2009-01-01

    A new method for the extraction of DNA from Ascaris suum muscle has been developed. It combines a standard SDS-based extraction with a plant DNA extraction procedure. The use of SDS and proteinase K allows the elimination of proteins, while CTAB and polyclar AT eliminate glycogen and polyphenols. The DNA thus obtained can easily be digested by endonucleases and amplified by PCR.

  19. Isolation of bacterial plasmids by density gradient centrifugation in cesium trifluoroacetate (CsTFA) without the use of ethidium bromide.

    Science.gov (United States)

    Andersson, K; Hjorth, R

    1985-01-01

    Plasmids extracted from bacterial cells by alkaline extraction can easily be isolated from linear DNA by isopycnic centrifugation in CsTFA. This is a fast and simple method which circumvents the use of the intercalating dye, ethidium bromide, and consequently the problems associated with its removal. The buoyant densities for covalently closed circular DNA and linear DNA in CsTFA are 1.60 g/ml and 1.65 g/ml, respectively. The isolation is achieved regardless of plasmid size and can be accomplished at temperatures of between 4 and 30 degrees C. Plasmid DNA isolated in gradients of CsTFA are of a high purity and have been found to be intact when cleaved with restriction enzymes and ligated with T4 DNA ligase.

  20. Bacterial community analysis of activated sludge: an evaluation of four commonly used DNA extraction methods

    NARCIS (Netherlands)

    Vanysacker, L.; Declerck, S.A.J.; Hellemans, B.; De Meester, L.; Vankelecom, I.; Declerck, P.

    2010-01-01

    The effectiveness of three commercially available direct DNA isolation kits (Mobio, Fast, Qiagen) and one published direct DNA extraction protocol (Bead) for extracting bacterial DNA from different types of activated sludge was investigated and mutually compared. The DNA quantity and purity were

  1. Comparison of different protocols for the extraction of microbial DNA from reef corals

    NARCIS (Netherlands)

    Santos, H. F.; Carmo, F. L.; Leite, D. C. A.; Jesus, H. E.; De Carvalho Maalouf, P.; Almeida, C.; Soriano, A. U.; Altomari, D.; Suhett, L.; Volaro, V.; Valoni, E.; Francisco, M.; Vieira, J.; Rocha, R.; Sardinha, B. L.; Mendes, L. B.; Joao, R. R.; Lacava, B.; Jesus, R. F.; Sebastian, G.; Pessoa, A.; van Elsas, J. D.; Rezende, R. P.; Pires, D. O.; Duarte, G.; Castro, C. B.; Rosado, A. S.; Peixoto, R. S.

    2012-01-01

    This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR

  2. COMPARISON OF DIFFERENT PROTOCOLS FOR THE EXTRACTION OF MICROBIAL DNA FROM REEF CORALS

    NARCIS (Netherlands)

    Santos, H. F.; Carmo, F. L.; Leite, D. C. A.; Jesus, H. E.; De Carvalho Maalouf, P.; Almeida, C.; Soriano, A. U.; Altomari, D.; Suhett, L.; Volaro, V.; Valoni, E.; Francisco, M.; Vieira, J.; Rocha, R.; Sardinha, B. L.; Mendes, L. B.; Joao, R. R.; Lacava, B.; Jesus, R. F.; Sebastian, G.; Pessoa, A.; van Elsas, J. D.; Rezende, R. P.; Pires, D. O.; Duarte, G.; Castro, C. B.; Rosado, A. S.; Peixoto, R. S.

    2012-01-01

    This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR

  3. Comparison of different protocols for the extraction of microbial DNA from reef corals

    NARCIS (Netherlands)

    Santos, H. F.; Carmo, F. L.; Leite, D. C. A.; Jesus, H. E.; De Carvalho Maalouf, P.; Almeida, C.; Soriano, A. U.; Altomari, D.; Suhett, L.; Volaro, V.; Valoni, E.; Francisco, M.; Vieira, J.; Rocha, R.; Sardinha, B. L.; Mendes, L. B.; Joao, R. R.; Lacava, B.; Jesus, R. F.; Sebastian, G.; Pessoa, A.; van Elsas, J. D.; Rezende, R. P.; Pires, D. O.; Duarte, G.; Castro, C. B.; Rosado, A. S.; Peixoto, R. S.

    2012-01-01

    This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR

  4. Bacterial community analysis of activated sludge: an evaluation of four commonly used DNA extraction methods

    NARCIS (Netherlands)

    Vanysacker, L.; Declerck, S.A.J.; Hellemans, B.; De Meester, L.; Vankelecom, I.; Declerck, P.

    2010-01-01

    The effectiveness of three commercially available direct DNA isolation kits (Mobio, Fast, Qiagen) and one published direct DNA extraction protocol (Bead) for extracting bacterial DNA from different types of activated sludge was investigated and mutually compared. The DNA quantity and purity were det

  5. Assessing the Utility of Soil DNA Extraction Kits for Increasing DNA Yields and Eliminating PCR Inhibitors from Buried Skeletal Remains.

    Science.gov (United States)

    Hebda, Lisa M; Foran, David R

    2015-09-01

    DNA identification of human remains is often necessary when decedents are skeletonized; however, poor DNA recovery and polymerase chain reaction (PCR) inhibition are frequently encountered, a situation exacerbated by burial. In this research, the utility of integrating soil DNA isolation kits into buried skeletal DNA analysis was evaluated and compared to a standard human DNA extraction kit and organic extraction. The soil kits successfully extracted skeletal DNA at quantities similar to standard methods, although the two kits tested, which differ mechanistically, were not equivalent. Further, the PCR inhibitors calcium and humic acid were effectively removed using the soil kits, whereas collagen was less so. Finally, concordant control region sequences were obtained from human skeletal remains using all four methods. Based on these comparisons, soil DNA isolation kits, which quickened the extraction process, proved to be a viable extraction technique for skeletal remains that resulted in positive identification of a decedent. © 2015 American Academy of Forensic Sciences.

  6. Plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.

    1982-01-01

    DNA recombination in exponential phase and competent Haemophilus influenzae was measured by an electron microscopic assay that relies on the conversion of plasmid RSF0885 monomers into multimeric forms. Dimer circles were present at a frequency of 2% in plasmid preparations from competent Rd (wild-type) cells; multimers were present at a frequency of 0.2% in preparations from exponential phase cells. Thus, plasmid recombination was stimulated in competent cells. Multimer formation occurred efficiently in cells of the transformation defective mutant rec2, implying that the rec2 gene product is not required for plasmid recombination. However, the absence of multimer plasmids in preparations from competent cells of the transformation defective mutant rec1 suggests that the rec1 gene product is required. Digestion of purified plasmids with restriction endonuclease PvuII, which makes a single cut in the monomer, revealed the presence of recombination intermediates composed of two linear plasmids joined to form two pairs of arms resembling the Greek letter chi. Length measurements of these arms taken from a population of recombination intermediates gave evidence that the plasmids were joined at sites of homology. The distributions of individual DNA strands, at the intersections of the four arms, could be resolved in some recombination intermediates and were of two types. The first type of junction appeared as a single-stranded arm appended to each corner. The second type of junction consisted of a single strand of DNA linking the two linear plasmids at a site of homology. The single-stranded linker was frequently situated at the edge of a short gap on one of the plasmids in the pair. The fine structures of the recombinational joints have been interpreted in terms of previously proposed models of recombination.

  7. Properties and nucleotide se- quence of linear plasmid-like DNA pC4 from mitochondria of Cucumis sativus

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Four kinds of mitochondrial plasmid-like DNAs, designated pC1, pC2, pC3 and pC4, were detected in Cucumis sativus Jinyan No. 4. The electron microscopy ob- servation showed that pC4 was linear conformation. Complete sequence of pC4 was cloned into pUC19 with E. coli JM109 as host. Sequence analysis revealed that pC4 was 370 bp long, the shortest one among all the reported mitochondrial plasmid-like DNAs. pC4 was AT rich. It contained terminal direct repeat sequence (35 bp in length) as well as many short direct and inverted repeats. ORFs in pC4 were short. pC4 was found to be homologous to nuclear DNAs, but lack homology with main mitochondrial and chloroplast DNAs. pC4-homologous sequence also occurred in nuclear genome of Jinyan No. 7 which contained no mito- chondrial plasmid-like DNAs. The hybridization pattern of Jinyan No. 7 was slightly different from that of Jinyan No. 4. This suggested that pC4 occurred at the forepart of Cucumis sativus species divergence and integrated into the nuclear genome, and the pC4-homologous sequence in nucleus varied during species diverging.

  8. Plasmid profile in oral Fusobacterium nucleatum from humans and Cebus apella monkeys

    Directory of Open Access Journals (Sweden)

    Paula Marcia O.

    2003-01-01

    Full Text Available Fusobacterium nucleatum is a strict anaerobe and is indigenous of the human oral cavity. This organism is commonly recovered from different monomicrobial and mixed infections in humans and animals. In this study, the plasmid profile, the plasmid stability and the penicillin-resistance association in oral F. nucleatum isolated from periodontal patients, healthy subjects and Cebus apella monkeys were evaluated. Forty-five F. nucleatum strains from patients, 38 from healthy subjects and seven from C. apella were identified and analyzed. Plasmid extraction was performed in all the isolated strains. These elements were found in 26.7% strains from patients and one strain from C. apella. Strains from healthy subjects did not show any plasmid. Most of strains showed two plasmid bands ranging from 4 to 16 Kb, but digestions with endonucleases showed that they belonged to a single plasmid. The plasmid profile was similar and stable in human and monkey strains. Also, plasmids were classified into three groups according to size. Two strains were positive to beta-lactamase production and no plasmid DNA-hybridization with a beta-lactamase gene probe was observed, suggesting a chromosomal resistance.

  9. Phase IIb trial of in vivo electroporation mediated dual-plasmid hepatitis B virus DNA vaccine in chronic hepatitis B patients under lamivudine therapy

    Science.gov (United States)

    Yang, Fu-Qiang; Rao, Gui-Rong; Wang, Gui-Qiang; Li, Yue-Qi; Xie, Yao; Zhang, Zhan-Qing; Deng, Cun-Liang; Mao, Qing; Li, Jun; Zhao, Wei; Wang, Mao-Rong; Han, Tao; Chen, Shi-Jun; Pan, Chen; Tan, De-Ming; Shang, Jia; Zhang, Ming-Xiang; Zhang, Yue-Xin; Yang, Ji-Ming; Chen, Guang-Ming

    2017-01-01

    AIM To assess the efficacy and safety of in vivo electroporation (EP)-mediated dual-plasmid hepatitis B virus (HBV) DNA vaccine vs placebo for sequential combination therapy with lamivudine (LAM) in patients with chronic hepatitis B. METHODS Two hundred and twenty-five patients were randomized to receive either LAM + vaccine (vaccine group, n = 109) or LAM + placebo (control group, n = 116). LAM treatment lasted 72 wk. Patients received the DNA vaccine or placebo by intramuscular injection mediated by EP at weeks 12 (start of treatment with vaccine or placebo, SOT), 16, 24, and 36 (end of treatment with vaccine or placebo, EOT). RESULTS In the modified intent-to-treat population, more patients had a decrease in HBV DNA > 2 log10 IU/mL in the vaccine group at week 12 after EOT compared with the control group. A trend toward a difference in the number of patients with undetectable HBV DNA at week 28 after EOT was obtained. Adverse events were similar. In the dynamic per-protocol set, which excluded adefovir (ADV) add-on cases at each time point instantly after ADV administration due to LAM antiviral failure, more patients had a decrease in HBV DNA > 2 log10 IU/mL in the vaccine group at week 12 and 28 after EOT compared with the control group. More patients with undetectable HBV DNA at week 28 after EOT in the vaccine group were also observed. Among patients with a viral load < 1000 copies/mL at week 12, more patients achieved HBeAg seroconversion in the vaccine group than among controls at week 36 after EOT, as well as less virological breakthrough and YMDD mutations. CONCLUSION The primary endpoint was not achieved using the HBV DNA vaccine. The HBV DNA vaccine could only be beneficial in subjects that have achieved initial virological response under LAM chemotherapy. PMID:28127204

  10. Correlation between plasmid DNA damage induced by PM10 and trace metals in inhalable particulate matters in Beijing air

    Institute of Scientific and Technical Information of China (English)

    Lü; Senlin

    2006-01-01

    2.5 in Beijing Air:Physicochemistry and Bioreactivy.Dissertation of China Univ.of Mining and Tech.(Beijing),2003[1]Lü SL.PM10 in Beijing Air:Mineralogy and Plasmid DNA assay.Dissertation of China Univ.of Mining and Tech.(Beijing),2003[2]See footnote 1) on page 1324.[1]See footnote 1) on page 1324[2]See footnote 1) on page 1326[1]Wang W,Zhang J,Tang D.Source Apportionment of inhalable particles,Chinese Academy of Environmental Science (in Chinese),1999[1]See footnote 1) on page 2.

  11. An efficient and cost-effective method for DNA extraction from athalassohaline soil using a newly formulated cell extraction buffer.

    Science.gov (United States)

    Narayan, Avinash; Jain, Kunal; Shah, Amita R; Madamwar, Datta

    2016-06-01

    The present study describes the rapid and efficient indirect lysis method for environmental DNA extraction from athalassohaline soil by newly formulated cell extraction buffer. The available methods are mostly based on direct lysis which leads to DNA shearing and co-extraction of extra cellular DNA that influences the community and functional analysis. Moreover, during extraction of DNA by direct lysis from athalassohaline soil, it was observed that, upon addition of poly ethylene glycol (PEG), isopropanol or absolute ethanol for precipitation of DNA, salt precipitates out and affecting DNA yield significantly. Therefore, indirect lysis method was optimized for extraction of environmental DNA from such soil containing high salts and low microbial biomass (CFU 4.3 × 10(4) per gram soil) using newly formulated cell extraction buffer in combination with low and high speed centrifugation. The cell extraction buffer composition and its concentration were optimized and PEG 8000 (1 %; w/v) and 1 M NaCl gave maximum cell mass for DNA extraction. The cell extraction efficiency was assessed with acridine orange staining of soil samples before and after cell extraction. The efficiency, reproducibility and purity of extracted DNA by newly developed procedure were compared with previously recognized methods and kits having different protocols including indirect lysis. The extracted environmental DNA showed better yield (5.6 ± 0.7 μg g(-1)) along with high purity ratios. The purity of DNA was validated by assessing its usability in various molecular techniques like restriction enzyme digestion, amplification of 16S rRNA gene using PCR and UV-Visible spectroscopy analysis.

  12. Evaluation of four automated protocols for extraction of DNA from FTA cards.

    Science.gov (United States)

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura; Frank-Hansen, Rune; Poulsen, Lena; Hansen, Anders J; Morling, Niels

    2013-10-01

    Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore, we demonstrated that it was possible to successfully extract sufficient DNA for STR profiling from previously processed FTA card pieces that had been stored at 4 °C for up to 1 year. This showed that rare or precious FTA card samples may be saved for future analyses even though some DNA was already extracted from the FTA cards.

  13. Safety and immunogenicity of an HIV-1 gag DNA vaccine with or without IL-12 and/or IL-15 plasmid cytokine adjuvant in healthy, HIV-1 uninfected adults.

    Directory of Open Access Journals (Sweden)

    Spyros A Kalams

    Full Text Available BACKGROUND: DNA vaccines are a promising approach to vaccination since they circumvent the problem of vector-induced immunity. DNA plasmid cytokine adjuvants have been shown to augment immune responses in small animals and in macaques. METHODOLOGY/PRINCIPAL FINDINGS: We performed two first in human HIV vaccine trials in the US, Brazil and Thailand of an RNA-optimized truncated HIV-1 gag gene (p37 DNA derived from strain HXB2 administered either alone or in combination with dose-escalation of IL-12 or IL-15 plasmid cytokine adjuvants. Vaccinations with both the HIV immunogen and cytokine adjuvant were generally well-tolerated and no significant vaccine-related adverse events were identified. A small number of subjects developed asymptomatic low titer antibodies to IL-12 or IL-15. Cellular immunogenicity following 3 and 4 vaccinations was poor, with response rates to gag of 4.9%/8.7% among vaccinees receiving gag DNA alone, 0%/11.5% among those receiving gag DNA+IL-15, and no responders among those receiving DNA+high dose (1500 ug IL-12 DNA. However, after three doses, 44.4% (4/9 of vaccinees receiving gag DNA and intermediate dose (500 ug of IL-12 DNA demonstrated a detectable cellular immune response. CONCLUSIONS/SIGNIFICANCE: This combination of HIV gag DNA with plasmid cytokine adjuvants was well tolerated. There were minimal responses to HIV gag DNA alone, and no apparent augmentation with either IL-12 or IL-15 plasmid cytokine adjuvants. Despite the promise of DNA vaccines, newer formulations or methods of delivery will be required to increase their immunogenicity. TRIAL REGISTRATION: Clinicaltrials.gov NCT00115960 NCT00111605.

  14. Comparison of four commercial DNA extraction kits for the recovery of Bacillus spp. spore DNA from spiked powder samples.

    Science.gov (United States)

    Mölsä, Markos; Kalin-Mänttäri, Laura; Tonteri, Elina; Hemmilä, Heidi; Nikkari, Simo

    2016-09-01

    Bacillus spp. include human pathogens such as Bacillus anthracis, the causative agent of anthrax and a biothreat agent. Bacillus spp. form spores that are physically highly resistant and may remain active over sample handling. We tested four commercial DNA extraction kits (QIAamp DNA Mini Kit, RTP Pathogen Kit, ZR Fungal/Bacterial DNA MiniPrep, and genesig Easy DNA/RNA Extraction kit) for sample inactivation and DNA recovery from two powders (icing sugar and potato flour) spiked with Bacillus thuringiensis spores. The DNA was analysed using a B. thuringiensis-specific real-time PCR assay. The detection limit was 3×10(1)CFU of spiked B. thuringiensis spores with the QIAamp DNA Mini, RTP Pathogen, and genesig Easy DNA/RNA Extraction kits, and 3×10(3)CFU with the ZR Fungal/Bacterial DNA MiniPrep kit. The results showed that manual extraction kits are effective and safe for fast and easy DNA extraction from powder samples even in field conditions. Adding a DNA filtration step to the extraction protocol ensures the removal of Bacillus spp. spores from DNA samples without affecting sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. A single protocol for extraction of gDNA from bacteria and yeast.

    Science.gov (United States)

    Vingataramin, Laurie; Frost, Eric H

    2015-03-01

    Guanidine thiocyanate breakage of microorganisms has been the standard initial step in genomic DNA (gDNA) extraction of microbial DNA for two decades, despite the requirement for pretreatments to extract DNA from microorganisms other than Gram-negative bacteria. We report a quick and low-cost gDNA extraction protocol called EtNa that is efficient for bacteria and yeast over a broad range of concentrations. EtNa is based on a hot alkaline ethanol lysis. The solution can be immediately centrifuged to yield a crude gDNA extract suitable for PCR, or it can be directly applied to a silica column for purification.

  16. Isolation and characterization of yeast DNA repair genes. II. Isolation of plasmids that complement the mutations rad50-1, rad51-1, rad54-3, and rad55-3

    Energy Technology Data Exchange (ETDEWEB)

    Calderon, I.L.; Contopoulou, C.R.; Mortimer, R.K.

    1983-01-01

    Plasmids that complement the yeast mutations rad50-1, rad51-1, rad54-3, and rad55-3 were obtained by transforming strains that carried a leu2 marker and the particular rad mutation, with YEp 13 plasmids containing near random yeast DNA inserts. Integration of these plasmids or of fragments of these plasmids was accomplished. Genetic studies using the integrants established the presence of the genes RAD50, RAD54 and RAD55 in the respective plasmids. However, a BamHI subclone of the rad50-1 complementing plasmid failed to integrate at the RAD50 locus, indicating that no homology exists between this fragment and the RAD50 gene. A BamHI fragment for the RAD54 plasmid was shown to be internal to the RAD54 gene: its integration within a wild type copy of RAD54 causes the cell to become Rad/sup -/; its excision is X-ray inducible and restores the Rad/sup -/ phenotype. Since cells bearing a disrupted copy of RAD54 are able to survive, the author concludes that this is not essential.

  17. Gene delivery nanocarriers of bioactive glass with unique potential to load BMP2 plasmid DNA and to internalize into mesenchymal stem cells for osteogenesis and bone regeneration

    Science.gov (United States)

    Kim, Tae-Hyun; Singh, Rajendra K.; Kang, Min Sil; Kim, Joong-Hyun; Kim, Hae-Won

    2016-04-01

    The recent development of bioactive glasses with nanoscale morphologies has spurred their specific applications in bone regeneration, for example as drug and gene delivery carriers. Bone engineering with stem cells genetically modified with this unique class of nanocarriers thus holds great promise in this avenue. Here we report the potential of the bioactive glass nanoparticle (BGN) system for the gene delivery of mesenchymal stem cells (MSCs) targeting bone. The composition of 15% Ca-added silica, proven to be bone-bioactive, was formulated into surface aminated mesoporous nanospheres with enlarged pore sizes, to effectively load and deliver bone morphogenetic protein-2 (BMP2) plasmid DNA. The enlarged mesopores were highly effective in loading BMP2-pDNA with an efficiency as high as 3.5 wt% (pDNA w.r.t. BGN), a level more than twice than for small-sized mesopores. The BGN nanocarriers released the genetic molecules in a highly sustained manner (for as long as 2 weeks). The BMP2-pDNA/BGN complexes were effectively internalized to rat MSCs with a cell uptake level of ~73%, and the majority of cells were transfected to express the BMP2 protein. Subsequent osteogenesis of the transfected MSCs was demonstrated by the expression of bone-related genes, including bone sialoprotein, osteopontin, and osteocalcin. The MSCs transfected with BMP2-pDNA/BGN were locally delivered inside a collagen gel to the target calvarium defects. The results showed significantly improved bone regeneration, as evidenced by the micro-computed tomographic, histomorphometric and immunohistochemical analyses. This study supports the excellent capacity of the BGN system as a pDNA-delivery nanocarrier in MSCs, and the engineered system, BMP2-pDNA/BGN with MSCs, may be considered a new promising candidate to advance the therapeutic potential of stem cells through genetic modification, targeting bone defects and diseases.The recent development of bioactive glasses with nanoscale morphologies has

  18. DNA extraction on bio-chip: history and preeminence over conventional and solid-phase extraction methods.

    Science.gov (United States)

    Ayoib, Adilah; Hashim, Uda; Gopinath, Subash C B; Md Arshad, M K

    2017-09-23

    This review covers a developmental progression on early to modern taxonomy at cellular level following the advent of electron microscopy and the advancement in deoxyribonucleic acid (DNA) extraction for expatiation of biological classification at DNA level. Here, we discuss the fundamental values of conventional chemical methods of DNA extraction using liquid/liquid extraction (LLE) followed by development of solid-phase extraction (SPE) methods, as well as recent advances in microfluidics device-based system for DNA extraction on-chip. We also discuss the importance of DNA extraction as well as the advantages over conventional chemical methods, and how Lab-on-a-Chip (LOC) system plays a crucial role for the future achievements.

  19. Designing easy DNA extraction: Teaching creativity through laboratory practice.

    Science.gov (United States)

    Susantini, Endang; Lisdiana, Lisa; Isnawati; Tanzih Al Haq, Aushia; Trimulyono, Guntur

    2017-05-01

    Subject material concerning Deoxyribose Nucleic Acid (DNA) structure in the format of creativity-driven laboratory practice offers meaningful learning experience to the students. Therefore, a laboratory practice in which utilizes simple procedures and easy-safe-affordable household materials should be promoted to students to develop their creativity. This study aimed to examine whether designing and conducting DNA extraction with household materials could foster students' creative thinking. We also described how this laboratory practice affected students' knowledge and views. A total of 47 students participated in this study. These students were grouped and asked to utilize available household materials and modify procedures using hands-on worksheet. Result showed that this approach encouraged creative thinking as well as improved subject-related knowledge. Students also demonstrated positive views about content knowledge, social skills, and creative thinking skills. This study implies that extracting DNA with household materials is able to develop content knowledge, social skills, and creative thinking of the students. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(3):216-225, 2017. © 2016 The International Union of Biochemistry and Molecular Biology.

  20. Co-Delivery of Imiquimod and Plasmid DNA via an Amphiphilic pH-Responsive Star Polymer that Forms Unimolecular Micelles in Water

    Directory of Open Access Journals (Sweden)

    Wenjing Lin

    2016-11-01

    Full Text Available Dual functional unimolecular micelles based on a pH-responsive amphiphilic star polymer β-CD-(PLA-b-PDMAEMA-b-PEtOxMA21 have been developed for the co-delivery of imiquimod and plasmid DNA to dendritic cells. The star polymer with well-defined triblock arms was synthesized by combining activator regenerated by electron-transfer atom-transfer radical polymerization with ring-opening polymerization. Dissipative particle dynamics simulation showed that core-mesophere-shell-type unimolecular micelles could be formed. Imiquimod-loaded micelles had a drug loading of 1.6 wt % and a larger average size (28 nm than blank micelles (19 nm. The release of imiquimod in vitro was accelerated at the mildly acidic endolysosomal pH (5.0 in comparison to physiologic pH (7.4. Compared with blank micelles, a higher N:P ratio was required for imiquimod-loaded micelles to fully condense DNA into micelleplexes averaging 200–400 nm in size. In comparison to blank micelleplexes, imiquimod-loaded micelleplexes of the same N:P ratio displayed similar or slightly higher efficiency of gene transfection in a mouse dendritic cell line (DC2.4 without cytotoxicity. These results suggest that such pH-responsive unimolecular micelles formed by the well-defined amphiphilic star polymer may serve as promising nano-scale carriers for combined delivery of hydrophobic immunostimulatory drugs (such as imiquimod and plasmid DNA with potential application in gene-based immunotherapy.

  1. Radioprotection against DNA damage by an extract of Indian green mussel, Perna viridis (L).

    Science.gov (United States)

    Kumaran, Sreekumar P; Kutty, Binoj C; Chatterji, Anil; Subrayan, Parameswaran P; Mishra, Kaushala Prasad

    2007-01-01

    This study describes the radioprotective ability of a hydrolysate prepared using an enzyme-acid hydrolysis method from the green mussel Perna viridis in terms of its ability to prevent radiation-induced damage in plasmid DNA, cell death, reactive oxygen species (ROS) formation, and DNA damage in mice lymphocytes. The mussel hydrolysate (MH) present during irradiation showed significant protection from gamma-radiation-induced strand breaks in plasmid DNA as evaluated by gel electrophoresis. Viability studies by trypan blue dye exclusion and MTT assay showed that preincubation of mice splenic lymphocytes with MH protected them from gamma-radiation-mediated killing. Moreover, the presence of MH during irradiation of isolated mice lymphocytes significantly decreased the DNA damage, as measured by comet assay. Measurement of intracellular ROS by dichlorofluorescein fluorescence revealed that the presence of MH effectively reduced the ROS generated in lymphocytes by both chemical method and gamma-irradiation. Prevention of DNA damage both in plasmid and lymphocytes and cell death in lymphocytes appears correlated with reduction of oxidatively generated free radicals. It is concluded that protection against radiation-induced cell death and DNA damage by MH was attributable to reduction of reactive free radical species generated by gamma-radiation.

  2. In vivo Site-Specific Transfection of Naked Plasmid DNA and siRNAs in Mice by Using a Tissue Suction Device

    Science.gov (United States)

    Shimizu, Kazunori; Kawakami, Shigeru; Hayashi, Kouji; Kinoshita, Hideyuki; Kuwahara, Koichiro; Nakao, Kazuwa; Hashida, Mitsuru; Konishi, Satoshi

    2012-01-01

    We have developed an in vivo transfection method for naked plasmid DNA (pDNA) and siRNA in mice by using a tissue suction device. The target tissue was suctioned by a device made of polydimethylsiloxane (PDMS) following the intravenous injection of naked pDNA or siRNA. Transfection of pDNA encoding luciferase was achieved by the suction of the kidney, liver, spleen, and heart, but not the duodenum, skeletal muscle, or stomach. Luciferase expression was specifically observed at the suctioned region of the tissue, and the highest luciferase expression was detected at the surface of the tissue (0.12±0.03 ng/mg protein in mice liver). Luciferase expression levels in the whole liver increased linearly with an increase in the number of times the liver was suctioned. Transfection of siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene significantly suppressed the expression of GAPDH mRNA in the liver. Histological analysis shows that severe damage was not observed in the suctioned livers. Since the suction device can be mounted onto the head of the endoscope, this method is a minimally invasive. These results indicate that the in vivo transfection method developed in this study will be a viable approach for biological research and therapies using nucleic acids. PMID:22844458

  3. High levels of gene expression in the hepatocytes of adult mice, neonatal mice and tree shrews via retro-orbital sinus hydrodynamic injections of naked plasmid DNA.

    Science.gov (United States)

    Yan, Shaoduo; Fu, Qiuxia; Zhou, Yong; Wang, Jidong; Liu, Ying; Duan, Xiangguo; Jia, Shuaizheng; Peng, Jianchun; Gao, Bo; Du, Juan; Zhou, Qianqian; Li, Yuan; Wang, Xiaohui; Zhan, Linsheng

    2012-08-10

    Hydrodynamic-based gene delivery has emerged as an efficient and simple method for the intracellular transfection of naked plasmid DNA (pDNA) in vivo. In this system, a hydrodynamic injection via the tail vein is the most effective non-viral method of liver-targeted gene delivery. However, this injection is often technically challenging when used in animals whose tail veins are difficult to visualize or too small to operate on. To overcome this limitation, an alternative in vivo gene delivery method, the rapid injection of large volume of pDNA solution through retro-orbital sinus, was established. Using this technique, we successfully delivered pDNA to the tissue of adult mice, neonatal mice and tree shrews. The efficient expression of exogenous genes was specifically detected in the liver of test animals treated with this gene delivery method. This study demonstrates for the first time that the hydrodynamic gene delivery via the retro-orbital sinus can not only reach the same transgene efficiency as a tradition hydrodynamic-based intravascular injection but also be used in animals that are difficult to inject via the tail vein. This method could open up new areas in gene function studies and gene therapy disease treatment.

  4. [Genomic DNA extraction from hair sacs of pigs using modified phenol-chloroform method].

    Science.gov (United States)

    Wang, Ji-Ying; Yu, Ying; Feng, Li-Xia; Wang, Huai-Zhong; Zhang, Qin

    2010-07-01

    In referring to various methods for genomic DNA extraction from different tissues, we modified the classical phenol-chloroform procedure and reaction system for use in genomic DNA extraction from pig hair sacs. With the modified the phenol-chloroform method we successfully obtained high quality genomic DNA from pig hair sacs. Genomic DNA can be extracted from sacs of one to six pig hairs with satisfied quantity and quality for the need of PCR-based molecular ex-periment.

  5. Comparing two protocols of DNA extraction of Trypanosoma cruzi cultured in axenic medium

    OpenAIRE

    López, Mariela; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, Venezuela. Escuela de Bioanálisis, Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, Venezuela. Licenciada en Bioanálisis; Rivera, María G.; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, Venezuela. Licenciada en Bioanálisis.; Viettri, Mercedes; Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, Venezuela. Licenciada en Bioanálisis.; Lares, María; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, Venezuela. Técnico superior universitario en Química.; Morocoima, Antonio; Centro de Medicina Tropical de Oriente, Facultad de Medicina, Universidad de Oriente. Anzoátegui, Venezuela. Médico cirujano, magíster en Parasitología.; Herrera, Leidi; Instituto de Zoología y Ecología Tropical, Facultad de Ciencias, Universidad Central de Venezuela. Caracas, Venezuela. Biólogo, doctora en Ciencias.; Ferrer, Elizabeth; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, Venezuela. Departamento de Parasitología, Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, Venezuela. Licenciada en bioanálisis, doctora en Biología Molecular.

    2014-01-01

    Objectives. To compare two extraction protocols of Trypanosoma cruzi DNA for use in DNA amplification of kinetoplast minicircles (kDNA) through the technique of Polymerase Chain Reaction (PCR). Materials and methods. Epimastigotes of T. cruzi were cultured in axenic conditions and masses from 1.5 to 100 x 106 parasites were obtained. DNA extraction was performed using two protocols: extraction with organic solvents (phenol/chloroform), and with resin (Chelex®100), from different parasitic sed...

  6. PREPARATION OF Ag NANOPARTICLES BASED ON PHOTOIRRADIATION METHOD WITH PLASMID DNA-TEMPLATED%质粒DNA光化学反应制备Ag纳米颗粒

    Institute of Scientific and Technical Information of China (English)

    刘建华; 章锦丹; 张晓亮; 李松梅; 于美

    2012-01-01

    以碱基对数为7.5 kB的大肠杆菌环状质粒DNA为模板,通过紫外线辐射作用下的光化学反应方法制备了Ag纳米颗粒.采用紫外-可见光吸收光谱,TEM和EDS等方法研究了辐射时间、溶液浓度等工艺条件对Ag纳米颗粒组成、形貌和结构的影响.结果表明,获得的Ag纳米颗粒为fcc结构,颗粒直径为25-40 nm;一定条件下,Ag纳米颗粒为环状,中间孔洞直径为8-10 nm,Ag+与碱基对数摩尔比为4、辐射时间为40 min时,Ag纳米颗粒中环状结构最多.%Over the past two decades, interest has been focused on the synthesis of novel nano-materials with different sizes and different morphologies due to their intriguing chemical, electronic, biosensing, optical and catalytic properties. The biomacromolecules like nucleic acids, proteins, amino acids and peptides have been employed as template for nanomaterial design and synthesis. DNA, which could self-assemble into complex structures such as cubes and squares, is being actively explored for the synthesis of nanomaterials with novel structures and unexpected properties. In this work, the plasmid DNA with 7.5 kB base pairs separated from the bacillus was used as a template to prepare Ag nanoparticles based on the ultraviolet ray (UV) photoirradiation method. The effects of experimental conditions, such as the photoirradiation time and concentration of metallic Ag+ ions, on the composition, morphology, and structure of the obtained nanoparticles were detailed studied. The Ag nanoparticles were characterized by using UV-visible light absorption spectroscopy, TEM and EDS. It is found that Ag+ and UV are necessary in this preparation based on plasmid DNA. The resulted Ag nanoparticles are fcc structure, and the average diameter of obtained nanoparticles is 25-40 nm. The sizes of nanoparticles increased with the increasing of the photoirradiation time and the concentration of Ag+. When the mol ratio of Ag+ to base pairs is 4 :1 and the

  7. A Recombinant DNA Plasmid Encoding the sIL-4R-NAP Fusion Protein Suppress Airway Inflammation in an OVA-Induced Mouse Model of Asthma.

    Science.gov (United States)

    Liu, Xin; Fu, Guo; Ji, Zhenyu; Huang, Xiabing; Ding, Cong; Jiang, Hui; Wang, Xiaolong; Du, Mingxuan; Wang, Ting; Kang, Qiaozhen

    2016-08-01

    Asthma is a chronic inflammatory airway disease. It was prevalently perceived that Th2 cells played the crucial role in asthma pathogenesis, which has been identified as the important target for anti-asthma therapy. The soluble IL-4 receptor (sIL-4R), which is the decoy receptor for Th2 cytokine IL-4, has been reported to be effective in treating asthma in phase I/II clinical trail. To develop more efficacious anti-asthma agent, we attempt to test whether the Helicobacter pylori neutrophil-activating protein (HP-NAP), a novel TLR2 agonist, would enhance the efficacy of sIL-4R in anti-asthma therapy. In our work, we constructed a pcDNA3.1-sIL-4R-NAP plasmid, named PSN, encoding fusion protein of murine sIL-4R and HP-NAP. PSN significantly inhibited airway inflammation, decreased the serum OVA-specific IgE levels and remodeled the Th1/Th2 balance. Notably, PSN is more effective on anti-asthma therapy comparing with plasmid only expressing sIL-4R.

  8. Extraction of DNA from malaria-infected erythrocytes using isotachophoresis.

    Science.gov (United States)

    Marshall, Lewis A; Han, Crystal M; Santiago, Juan G

    2011-12-15

    We demonstrate a technique for purification of nucleic acids from malaria parasites infecting human erythrocytes using isotachophoresis (ITP). We release nucleic acids from malaria-infected erythrocytes by lysing with heat and proteinase K for 10 min and immediately, thereafter, load sample onto a capillary device. We study the effect of temperature on lysis efficiency. We also implement pressure-driven counterflow during ITP extraction to extend focusing time and increase nucleic acid yield. We show that the purified genomic DNA samples are compatible with polymerase chain reaction (PCR) and demonstrate a clinically relevant limit of detection of 0.5 parasites per nanoliter using quantitative PCR.

  9. The Expression of the Plasmid DNA Encoding TGF-β1 in Endothelium after Injection into the Anterior Chamber

    Institute of Scientific and Technical Information of China (English)

    胡燕华; 黄琼; 姜发纲; 陈宏

    2002-01-01

    Summary: The method of gene transfer into corneal endothelium was investigated to provide afoundation for the study of TGF-β1 gene transfer to inhibit corneal graft rejection. Two days afterdirect injection of pMAM TGF-β1 mediated by liposome into the anterior chamber of rabbits, onehalf of corneas were made into paraffin slides and the endothelial layer was carefully torn from theother half to make a single layer slide of endothelia. By means of immunohistochemical technique,the plasmid pMAM TGF-β1 expression product TGF-β1 in the endothelia was detected. SpecificTGF-β1 expression was positive in the endothelia on both the paraffin slide and the single layerslide. The results showed that by direct injection into the anterior chamber, foreign plasmid DNAcould be transferred into the endothelia and its expression was obtained. This may provide a foun-dation for further study on TGF-β1 participating in local induction of corneal immune tolerance.

  10. Determination of an efficient and reliable method for DNA extraction from ticks.

    Science.gov (United States)

    Halos, Lénaïg; Jamal, Taoufik; Vial, Laurence; Maillard, Renaud; Suau, Antonia; Le Menach, Arnaud; Boulouis, Henri-Jean; Vayssier-Taussat, Muriel

    2004-01-01

    Molecular detection of pathogenic microorganisms in ticks is based on DNA amplification of the target pathogen; therefore, extraction of DNA from the tick is a major step. In this study, we compared three different tick DNA extraction protocols based on an enzymatic digestion by proteinase K followed by DNA extraction by a commercial kit (method 1), or on mortar crushing, proteinase K digestion and phenol/chloroform DNA extraction (method 2) and fine crushing with a beads beater, proteinase K digestion and DNA extraction using a commercial kit (method 3). The absence of PCR inhibitors and the DNA quality were evaluated by PCR amplification of the tick mitochondrial 16S rRNA gene using tick-specific primers. With method 1, 23/30 (77%) of the samples were extracted; with method 2, 30/31 (97%) of the samples were extracted and with method 3, 30/30 (100%) of the samples were extracted. DNA extraction efficiency using method 3 is significantly higher than DNA extraction efficiency using method 1 (100% versus 77%, P DNA extraction and applicable to the treatment of small samples such as nymphs and soft ticks with 100% efficiency.

  11. THE OCCURRENCE OF PLASMID DNA IN CAMPYLOBACTER JEJUNI AND CAMPYLOBACTER COLI AND ANALYSIS OF RESTRICTION ENDONUCLEASE%空肠弯曲菌质粒的提取鉴定和限制性内切酶水解片段的分析

    Institute of Scientific and Technical Information of China (English)

    孙自捷; 宋后燕; 段恕诚

    1988-01-01

    Thirty-two strains of different serotypes and biotypes of Campylobaoter jejuni and Campylobacter coli isolated from the stool of children and domestic animals were examined for the presence of plasmid DNA Agarose gel electrophoresis of alkaline-extracted DNA showed the occurrence of plasmid bands in 12 strains.Ten of them showed only one plasmid band in>28Kb area of gel,and 7 strains contained this plasmid were further analyzed by restriction endonuclease Hind Ⅲ.All restriction fragments showed heterogeneity.Other 2 of the 12 strains from domestic animals were observed and 3 and 4 plasmid bands were demonstrated in agarose gel respectively.The incidenoe of plasmid in Campylobacter jejuni strains was similar to Campylobacter coli,while the incidence of plasmid in Campylobacter strains from the stool of domestic animals was higher than in children.%使用修改的Brruboim快速碱抽提法,对32株来源不同,和不同血清型和生物型空肠弯曲菌株进行了质粒DNA检测和分析.12株细菌的质粒抽提液在琼脂糖电泳上显示有质粒带,其中10株均仅含有一条>23Kb的质粒,同时7株用限制性内切酶Hind Ⅲ消化后水解片段分析发现这一条>23Kb的质粒为非同源性.其他2株细菌在琼脂糖电泳上显示3和4条质粒带.不同生物型空肠弯曲菌的质粒携带率基本相同,而从家禽分离的空肠弯曲菌株比从儿童分离的菌株质粒携带率要高.

  12. Effect of the head-group geometry of amino acid-based cationic surfactants on interaction with plasmid DNA.

    Science.gov (United States)

    Jadhav, Vaibhav; Maiti, Souvik; Dasgupta, Antara; Das, Prasanta Kumar; Dias, Rita S; Miguel, Maria G; Lindman, Björn

    2008-07-01

    The interaction between DNA and different types of amino acid-based cationic surfactants was investigated. Particular attention was directed to determine the extent of influence of surfactant head-group geometry toward tuning the interaction behavior of these surfactants with DNA. An overview is obtained by gel retardation assay, isothermal titration calorimetry, fluorescence spectroscopy, and circular dichroism at different mole ratios of surfactant/DNA; also, cell viability was assessed. The studies show that the surfactants with more complex/bulkier hydrophobic head group interact more strongly with DNA but exclude ethidium bromide less efficiently; th