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Sample records for dna electrophoretic mobility

  1. Coarse-grained model of conformation-dependent electrophoretic mobility and its influence on DNA dynamics.

    PubMed

    Pandey, Harsh; Underhill, Patrick T

    2015-11-01

    The electrophoretic mobility of molecules such as λ-DNA depends on the conformation of the molecule. It has been shown that electrohydrodynamic interactions between parts of the molecule lead to a mobility that depends on conformation and can explain some experimental observations. We have developed a new coarse-grained model that incorporates these changes of mobility into a bead-spring chain model. Brownian dynamics simulations have been performed using this model. The model reproduces the cross-stream migration that occurs in capillary electrophoresis when pressure-driven flow is applied parallel or antiparallel to the electric field. The model also reproduces the change of mobility when the molecule is stretched significantly in an extensional field. We find that the conformation-dependent mobility can lead to a new type of unraveling of the molecule in strong fields. This occurs when different parts of the molecule have different mobilities and the electric field is large. PMID:26651689

  2. Coarse-grained model of conformation-dependent electrophoretic mobility and its influence on DNA dynamics

    NASA Astrophysics Data System (ADS)

    Pandey, Harsh; Underhill, Patrick T.

    2015-11-01

    The electrophoretic mobility of molecules such as λ -DNA depends on the conformation of the molecule. It has been shown that electrohydrodynamic interactions between parts of the molecule lead to a mobility that depends on conformation and can explain some experimental observations. We have developed a new coarse-grained model that incorporates these changes of mobility into a bead-spring chain model. Brownian dynamics simulations have been performed using this model. The model reproduces the cross-stream migration that occurs in capillary electrophoresis when pressure-driven flow is applied parallel or antiparallel to the electric field. The model also reproduces the change of mobility when the molecule is stretched significantly in an extensional field. We find that the conformation-dependent mobility can lead to a new type of unraveling of the molecule in strong fields. This occurs when different parts of the molecule have different mobilities and the electric field is large.

  3. Using Electrophoretic Mobility Shift Assays to Measure Equilibrium Dissociation Constants: GAL4-p53 Binding DNA as a Model System

    ERIC Educational Resources Information Center

    Heffler, Michael A.; Walters, Ryan D.; Kugel, Jennifer F.

    2012-01-01

    An undergraduate biochemistry laboratory experiment is described that will teach students the practical and theoretical considerations for measuring the equilibrium dissociation constant (K[subscript D]) for a protein/DNA interaction using electrophoretic mobility shift assays (EMSAs). An EMSA monitors the migration of DNA through a native gel;…

  4. Predicting Electrophoretic Mobility of Protein-Ligand Complexes for Ligands from DNA-Encoded Libraries of Small Molecules.

    PubMed

    Bao, Jiayin; Krylova, Svetlana M; Cherney, Leonid T; Hale, Robert L; Belyanskaya, Svetlana L; Chiu, Cynthia H; Shaginian, Alex; Arico-Muendel, Christopher C; Krylov, Sergey N

    2016-05-17

    Selection of target-binding ligands from DNA-encoded libraries of small molecules (DELSMs) is a rapidly developing approach in drug-lead discovery. Methods of kinetic capillary electrophoresis (KCE) may facilitate highly efficient homogeneous selection of ligands from DELSMs. However, KCE methods require accurate prediction of electrophoretic mobilities of protein-ligand complexes. Such prediction, in turn, requires a theory that would be applicable to DNA tags of different structures used in different DELSMs. Here we present such a theory. It utilizes a model of a globular protein connected, through a single point (small molecule), to a linear DNA tag containing a combination of alternating double-stranded and single-stranded DNA (dsDNA and ssDNA) regions of varying lengths. The theory links the unknown electrophoretic mobility of protein-DNA complex with experimentally determined electrophoretic mobilities of the protein and DNA. Mobility prediction was initially tested by using a protein interacting with 18 ligands of various combinations of dsDNA and ssDNA regions, which mimicked different DELSMs. For all studied ligands, deviation of the predicted mobility from the experimentally determined value was within 11%. Finally, the prediction was tested for two proteins and two ligands with a DNA tag identical to those of DELSM manufactured by GlaxoSmithKline. Deviation between the predicted and experimentally determined mobilities did not exceed 5%. These results confirm the accuracy and robustness of our model, which makes KCE methods one step closer to their practical use in selection of drug leads, and diagnostic probes from DELSMs. PMID:27119259

  5. Mapping DNA Quantity into Electrophoretic Mobility through Quantum Dot Nanotethers for High Resolution Genetic and Epigenetic Analysis

    PubMed Central

    Zhang, Yi; Liu, Kelvin J.; Wang, Tian-Li; Shih, Ie-Ming; Wang, Tza-Huei

    2011-01-01

    Newly discovered nanoparticle properties have driven the development of novel applications and uses. We report a new observation where the electrophoretic mobility of a quantum dot-DNA nanoassembly can be precisely modulated by the degree of surface DNA conjugation. By using streptavidin-coated quantum dots (QD) as nanotethers to gather biotin-labeled DNA into electrophoretic nanoassemblies, the QD surface charge is modulated and transformed into electrophoretic mobility shifts using standard agarose gel electrophoresis. Typical fluorescent assays quantify based on relative intensity. However, this phenomenon uses a novel approach that accurately maps DNA quantity into shifts in relative band position. This property was applied in a quantum dot enabled nanoassay called Quantum Dot Electrophoretic Mobility Shift Assay (QEMSA) that enables accurate quantification of DNA targets down to 1.1-fold (9%) changes in quantity, beyond what is achievable in qPCR. In addition to these experimental findings, an analytical model is presented to explain this behavior. Finally, QEMSA was applied to both genetic and epigenetic analysis of cancer. First, it was used to analyze copy number variation (CNV) of the RSF1/HBXAP gene where conventional approaches for CNV analysis based on comparative genomic hybridization (CGH), microarrays, and qPCR are unable to reliably differentiate less than 2-fold changes in copy number. Then, QEMSA was used for DNA methylation analysis of the p16/CDK2A tumor suppressor gene where its ability to detect subtle changes in methylation was shown to be superior to that of qPCR. PMID:22136600

  6. Non-monotonic mobility vs. length dependence observed in electrophoretic separation of 25 bp DNA ladder in Pluronic gels.

    NASA Astrophysics Data System (ADS)

    You, Seungyong; van Winkle, David

    2009-03-01

    We electrophoresed a double-stranded DNA ladder first in an agarose gel, then in gels of Pluronic F-127 at room temperature. The DNA ladder consisted of 19 discrete fragments ranging in length from 25 to 450 bp at 25 bp increments plus 500 bp. The DNA fragments were first separated in agarose gel and stacked normally with 25 bp having the highest mobility. A single lane of the separated DNA ladder in the agarose gel was inserted at the edge of a Pluronic gel slab. The DNA was electrophoresed from the agarose into the Pluronic gels perpendicular to the original separation axis. Mobilities of DNA fragments increased from 25 bp to 175 bp and then decreased from 175 bp to 500 bp. The 25 bp and 500 bp bands of the ladder had approximately the same mobility in several different Pluronic gel concentrations. Both were slower than most bands in between. The highest mobility fragments with length of 175 bp have 59.5 nm contour length which is about 3.5 times the diameter of a micelle (17 nm). This result suggests a crossover from chromatographic separation to electrophoretic separation for these short DNAs. This research is supported by the state of Florida (Martech) and Research Corporation.

  7. Electrophoretic mobility of linear and star-branched DNA in semidilute polymer solutions.

    PubMed

    Saha, Sourav; Heuer, Daniel M; Archer, Lynden A

    2006-08-01

    Electrophoresis of large linear T2 (162 kbp) and 3-arm star-branched (N(Arm) = 48.5 kbp) DNA in linear polyacrylamide (LPA) solutions above the overlap concentration c* has been investigated using a fluorescence visualization technique that allows both the conformation and mobility mu of the DNA to be determined. LPA solutions of moderate polydispersity index (PI approximately 1.7-2.1) and variable polymer molecular weight Mw (0.59-2.05 MDa) are used as the sieving media. In unentangled semidilute solutions (c* < c < c(e)), we find that the conformational dynamics of linear and star-branched DNA in electric fields are strikingly different; the former migrating in predominantly U- or I-shaped conformations, depending on electric field strength E, and the latter migrating in a squid-like profile with the star-arms outstretched in the direction opposite to E and dragging the branch point through the sieving medium. Despite these visual differences, mu for linear and star-branched DNA of comparable size are found to be nearly identical in semidilute, unentangled LPA solutions. For LPA concentrations above the entanglement threshold (c > c(e)), the conformation of migrating linear and star-shaped DNA manifest only subtle changes from their unentangled solution features, but mu for the stars decreases strongly with increasing LPA concentration and molecular weight, while mu for linear DNA becomes nearly independent of c and Mw. These findings are discussed in the context of current theories for electrophoresis of large polyelectrolytes. PMID:16850503

  8. Photoaffinity electrophoretic mobility shift assay using photoreactive DNA bearing 3-trifluoromethyl-3-phenyldiazirine in its phosphate backbone.

    PubMed

    Sadakane, Yutaka; Hatanaka, Yasumaru

    2016-08-01

    Photoaffinity cross-linking enables the analysis of interactions between DNA and proteins even under denaturing conditions. We present a photoaffinity electrophoretic mobility shift assay (EMSA) in which two heterogeneous techniques-photoaffinity cross-linking using DNA bearing 3-trifluoromethyl-3-phenyldiazirine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis-are combined. To prepare the photoreactive DNA, which is an essential tool for photoaffinity EMSA, we first determined the optimal conditions for the integration of 4-(3-trifluoromethyl-3H-diazirin-3-yl)benzyl bromide to the specific site of oligonucleotide where phosphodiester linkage was replaced with phosphorothioate linkage. The photoaffinity EMSA was developed using the POU (initial letters of three genes: Pit-l, Oct-1,2, and unc-86) domain region of Oct-1 protein, which specifically bound to octamer DNA motif (ATGCAAAT). The affinity-purified recombinant POU domain proteins conjugated with glutathione-S-transferase (GST) contained three distinct proteins with molecular weights of 34, 36, and 45 kDa. The photoaffinity EMSA could clearly distinguish the individual binding abilities of three proteins on a single lane and showed that the whole POU domain protein specifically bound to octamer DNA motif by competition experiments. Using the nuclear extract of HeLa cells, the photoaffinity EMSA revealed that at least five specific proteins could bind to the octamer DNA motif. These results show that photoaffinity EMSA using 3-trifluoromethyl-3-phenyldiazirine can provide high-performance analysis of DNA-binding proteins. PMID:27156811

  9. Sedimentation of macroscopic rigid knots and its relation to gel electrophoretic mobility of DNA knots.

    PubMed

    Weber, Cédric; Carlen, Mathias; Dietler, Giovanni; Rawdon, Eric J; Stasiak, Andrzej

    2013-01-01

    We address the general question of the extent to which the hydrodynamic behaviour of microscopic freely fluctuating objects can be reproduced by macrosopic rigid objects. In particular, we compare the sedimentation speeds of knotted DNA molecules undergoing gel electrophoresis to the sedimentation speeds of rigid stereolithographic models of ideal knots in both water and silicon oil. We find that the sedimentation speeds grow roughly linearly with the average crossing number of the ideal knot configurations, and that the correlation is stronger within classes of knots. This is consistent with previous observations with DNA knots in gel electrophoresis. PMID:23346349

  10. Sedimentation of macroscopic rigid knots and its relation to gel electrophoretic mobility of DNA knots

    PubMed Central

    Weber, Cédric; Carlen, Mathias; Dietler, Giovanni; Rawdon, Eric J.; Stasiak, Andrzej

    2013-01-01

    We address the general question of the extent to which the hydrodynamic behaviour of microscopic freely fluctuating objects can be reproduced by macrosopic rigid objects. In particular, we compare the sedimentation speeds of knotted DNA molecules undergoing gel electrophoresis to the sedimentation speeds of rigid stereolithographic models of ideal knots in both water and silicon oil. We find that the sedimentation speeds grow roughly linearly with the average crossing number of the ideal knot configurations, and that the correlation is stronger within classes of knots. This is consistent with previous observations with DNA knots in gel electrophoresis. PMID:23346349

  11. Electrophoretic mobilities of erythrocytes in various buffers

    NASA Technical Reports Server (NTRS)

    Plank, L. D.; Kunze, M. E.; Todd, P. W.

    1985-01-01

    The calibration of space flight equipment depends on a source of standard test particles, this test particle of choice is the fixed erythrocyte. Erythrocytes from different species have different electrophoretic mobilities. Electrophoretic mobility depends upon zeta potential, which, in turn depends upon ionic strength. Zeta potential decreases with increasing ionic strength, so cells have high electrophoretic mobility in space electrophoresis buffers than in typical physiological buffers. The electrophoretic mobilities of fixed human, rat, and rabbit erythrocytes in 0.145 M salt and buffers of varying ionic strength, temperature, and composition, to assess the effects of some of the unique combinations used in space buffers were characterized. Several effects were assessed: glycerol or DMSO (dimethylsulfoxide) were considered for use as cryoprotectants. The effect of these substances on erythrocyte electrophoretic mobility was examined. The choice of buffer depended upon cell mobility. Primary experiments with kidney cells established the choice of buffer and cryoprotectant. A nonstandard temperature of EPM in the suitable buffer was determined. A loss of ionic strength control occurs in the course of preparing columns for flight, the effects of small increases in ionic strength over the expected low values need to be evaluated.

  12. ELECTROPHORETIC MOBILITY OF MYCOBACTERIUM AVIUM COMPLEX ORGANISMS

    EPA Science Inventory

    The electrophoretic mobilities (EPMs) of thirty Mycobacterium avium Complex (MAC) organisms isolated from clinical and environmental sources were measured in 9.15 mM KH2PO4 buffered water. The EPMs of fifteen clinical isolates ranged from -1.9 to -5.0 µm cm V-1 ...

  13. ELECTROPHORETIC MOBILITY OF MYCOBACTERIUM AVIUM COMPLEX ORGANISMS

    EPA Science Inventory

    The electrophoretic mobilities (EPMs) of thirty Mycobacterium avium Complex (MAC) organisms were measured. The EPMs of fifteen clinical isolates ranged from -1.9 to -5.0 µm cm V-1s-1, and the EPMs of fifteen environmental isolates ranged from -1...

  14. Electrophoretic mobility of semi-flexible double-stranded DNA in defect-controlled polymer networks: Mechanism investigation and role of structural parameters

    NASA Astrophysics Data System (ADS)

    Khairulina, Kateryna; Li, Xiang; Nishi, Kengo; Shibayama, Mitsuhiro; Chung, Ung-il; Sakai, Takamasa

    2015-06-01

    Our previous studies have reported an empirical model, which explains the electrophoretic mobility (μ) of double-stranded DNA (dsDNA) as a combination of a basic migration term (Rouse-like or reptation) and entropy loss term in polymer gels with ideal network structure. However, this case is of exception, considering a large amount of heterogeneity in the conventional polymer gels. In this study, we systematically tune the heterogeneity in the polymer gels and study the migration of dsDNA in these gels. Our experimental data well agree with the model found for ideal networks. The basic migration mechanism (Rouse-like or reptation) persists perfectly in the conventional heterogeneous polymer gel system, while the entropy loss term continuously changes with increase in the heterogeneity. Furthermore, we found that in the limit where dsDNA is shorter than dsDNA persistence length, the entropy loss term may be related to the collisional motions between DNA fragments and the cross-links.

  15. Sequence-specific nucleic acid mobility using a reversible block copolymer gel matrix and DNA amphiphiles (lipid-DNA) in capillary and microfluidic electrophoretic separations.

    PubMed

    Wagler, Patrick; Minero, Gabriel Antonio S; Tangen, Uwe; de Vries, Jan Willem; Prusty, Deepak; Kwak, Minseok; Herrmann, Andreas; McCaskill, John S

    2015-10-01

    Reversible noncovalent but sequence-dependent attachment of DNA to gels is shown to allow programmable mobility processing of DNA populations. The covalent attachment of DNA oligomers to polyacrylamide gels using acrydite-modified oligonucleotides has enabled sequence-specific mobility assays for DNA in gel electrophoresis: sequences binding to the immobilized DNA are delayed in their migration. Such a system has been used for example to construct complex DNA filters facilitating DNA computations. However, these gels are formed irreversibly and the choice of immobilized sequences is made once off during fabrication. In this work, we demonstrate the reversible self-assembly of gels combined with amphiphilic DNA molecules, which exhibit hydrophobic hydrocarbon chains attached to the nucleobase. This amphiphilic DNA, which we term lipid-DNA, is synthesized in advance and is blended into a block copolymer gel to induce sequence-dependent DNA retention during electrophoresis. Furthermore, we demonstrate and characterize the programmable mobility shift of matching DNA in such reversible gels both in thin films and microchannels using microelectrode arrays. Such sequence selective separation may be employed to select nucleic acid sequences of similar length from a mixture via local electronics, a basic functionality that can be employed in novel electronic chemical cell designs and other DNA information-processing systems. PMID:26095642

  16. Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA).

    PubMed

    Miller, Daniel E; Patel, Zubin H; Lu, Xiaoming; Lynch, Arthur T; Weirauch, Matthew T; Kottyan, Leah C

    2016-01-01

    Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype. PMID:27585267

  17. Electrophoretic mobility of particles in concentrated solutions of electrolytes

    SciTech Connect

    Deinega, Yu.F.; Polyakova, V.M.; Aleksandrova, L.N.

    1986-11-01

    The electrophoretic mobility of particles of phenol-formaldehyde and aniline-formaldehyde resins in zinc sulfate solutions has been investigated. It is shown that as the electrolyte concentration rises, the electrophoretic mobility falls, reaches a minimum, and then increases. A possible mechanism for the formation of an electric double layer on the surface of particles in concentrated solutions of electrolytes is proposed.

  18. Electrophoretic Migration of Branched DNA in Polymer Solutions

    NASA Astrophysics Data System (ADS)

    Lau, Henry; Archer, Lynden

    2009-03-01

    The electrophoretic migration of large, star-branched DNA molecules has previously been studied in both neutral polymer solutions and gels, and the results have provided insight into the local interactions between the analytes and the sieving matrix during electrophoresis (Electrophoresis, 2006, 27, 3128). This talk focuses on using rigid-rod DNA molecules of complex shapes as model analytes in studying the effects of analyte architecture on mobility in polymer solutions. Electrophoresis of a series of Y-shaped DNA molecules that mimick the shapes of antibodies, was performed in polymer solutions above the overlap concentration and at electric fields up to 300V/cm. The location of the branch point as well as the arm sizes are varied in order to examine their influence on mobility. Our results point to novel, topology-based fractionation strategies for separating biological molecules using capillary electrophoresis with polymer sieving media.

  19. Identification of tissue-specific DNA-protein binding sites by means of two-dimensional electrophoretic mobility shift assay display.

    PubMed

    Chernov, Igor P; Timchenko, Kira A; Akopov, Sergey B; Nikolaev, Lev G; Sverdlov, Eugene D

    2007-05-01

    We developed a technique of differential electrophoretic mobility shift assay (EMSA) display allowing identification of tissue-specific protein-binding sites within long genomic sequences. Using this approach, we identified 10 cell type-specific protein-binding sites (protein target sites [PTSs]) within a 137-kb human chromosome 19 region. In general, tissue-specific binding of proteins from different nuclear extracts by individual PTSs did not follow the all-or-nothing principle. Most often, PTS-protein complexes were formed in all cases, but they were different for different nuclear extracts used. PMID:17359930

  20. Simulations of electrophoretic collisions of DNA knots with gel obstacles

    NASA Astrophysics Data System (ADS)

    Weber, C.; DeLos Rios, P.; Dietler, G.; Stasiak, A.

    2006-04-01

    Gel electrophoresis can be used to separate nicked circular DNA molecules of equal length but forming different knot types. At low electric fields, complex knots drift faster than simpler knots. However, at high electric field the opposite is the case and simpler knots migrate faster than more complex knots. Using Monte Carlo simulations we investigate the reasons of this reversal of relative order of electrophoretic mobility of DNA molecules forming different knot types. We observe that at high electric fields the simulated knotted molecules tend to hang over the gel fibres and require passing over a substantial energy barrier to slip over the impeding gel fibre. At low electric field the interactions of drifting molecules with the gel fibres are weak and there are no significant energy barriers that oppose the detachment of knotted molecules from transverse gel fibres.

  1. The Electrophoretic Mobility of Proteins near Surfaces

    NASA Astrophysics Data System (ADS)

    Ramasamy, Perumal; Singh, Avtar; Rafailovich, Miriam; Sokolov, Jonathan

    2004-03-01

    We have attempted to apply the methods developed for surface DNA electrophoresis (1) for proteomics. Droplets of FITC stained Abumin, Poly- L-Lysine, or Casein purchased from Sigma were deposited on glass cover slips. The droplets were then place in contact with a TBE buffer solution contained in a cell molded from PDMS. Pt electrodes were inserted into the cell and a voltage was a applied. The motion of the protein was then imaged with a Leica Confocal microscope as a function of buffer concentration, distance from the surface, and applied voltage. The mobilities were then compared with those of uncharged one micron florescent Polystyrene beads. References: 1)Henzel WJ, Watanabe C, Stults JT., !0 Protein Identification: The Origins of Peptide Mass Fingerprinting. !1 J. American Society for Mass Spectrometry. 14 (September 2003): 931-942 2)Mathesius U, Imin N, Natera SH, Rolfe BG., !0 Proteomics as a functional genomics tool. !1 Methods of Molecular Biology 236: 395-414. *Work supported in part by the NSF-MRSEC program

  2. Electrophoretic mobility of oil droplets in electrolyte and surfactant solutions.

    PubMed

    Wuzhang, Jiachen; Song, Yongxin; Sun, Runzhe; Pan, Xinxiang; Li, Dongqing

    2015-10-01

    Electrophoretic mobility of oil droplets of micron sizes in PBS and ionic surfactant solutions was measured in this paper. The experimental results show that, in addition to the applied electric field, the speed and the direction of electrophoretic motion of oil droplets depend on the surfactant concentration and on if the droplet is in negatively charged SDS solutions or in positively charged hexadecyltrimethylammonium bromide (CTAB) solutions. The absolute value of the electrophoretic mobility increases with increased surfactant concentration before the surfactant concentration reaches to the CMC. It was also found that there are two vortices around the oil droplet under the applied electric field. The size of the vortices changes with the surfactant and with the electric field. The vortices around the droplet directly affect the drag of the flow field to the droplet motion and should be considered in the studies of electrophoretic mobility of oil droplets. The existence of the vortices will also influence the determination and the interpretation of the zeta potential of the oil droplets based on the measured mobility data. PMID:26140616

  3. Controlled method of reducing electrophoretic mobility of various substances

    NASA Technical Reports Server (NTRS)

    Vanalstine, James M. (Inventor)

    1989-01-01

    A method of reducing electrophoretic mobility of macromolecules, particles, cells, and the like is provided. The method comprises interacting the particles or cells with a polymer-linked affinity compound composed of: a hydrophilic neutral polymer such as polyethylene glycol, and an affinity component consisting of a hydrophobic compound such as a fatty acid ester, an immunocompound such as an antibody or active fragment thereof or simular macromolecule, or other ligands. The reduction of electrophoretic mobility achieved is directly proportional to the concentration of the polymer-linked affinity compound employed, and the mobility reduction obtainable is up to 100 percent for particular particles and cells. The present invention is advantageous in that analytical electrophoretic separation can not be achieved for macromolecules, particles, and cells whose native surface charge structure had prevented them from being separated by normal electrophoretic means. Depending on the affinity component utilized, separation can be achieved on the basis of specific/irreversible, specific/reversible, semi-specific/reversible, relatively nonspecific/reversible, or relatively nonspecific/irreversible ligand-substance interactions. The present method is also advantageous in that it can be used in a variety of standard laboratory electrophoresis equipment.

  4. Electrophoretic mobilities and migrating analytes: Part 1: Relationships.

    PubMed

    Cross, Reginald F; Wong, Margaret G

    2002-01-01

    The molecular radii (r) of a series of peptides have been determined by molecular modeling. With these data, it is shown that electrophoretic mobility (mu(ep)) is proportional to 1/r2, and that the dependence presented in textbooks (mu(ep) infinity 1/r) is wrong. Use of the approximately equivalent, mass-based Offord equation is discussed, and other relevant considerations are presented. PMID:12546161

  5. Electrophoretic dynamics of self-assembling branched DNA structures

    NASA Astrophysics Data System (ADS)

    Heuer, Daniel Milton

    This study advances our understanding of the electrophoretic dynamics of branched biopolymers and explores technologies designed to exploit their unique properties. New self-assembly techniques were developed to create branched DNA for visualization via fluorescence microscopy. Experiments in fixed gel networks reveal a distinct trapping behavior, in contrast with linear topologies. The finding that detection can be achieved by introducing a branch point contributes significantly to the field of separation science and can be exploited to develop new applications. Results obtained in polymer solutions point to identical mobilities for branched and linear topologies, despite large differences in their dynamics. This finding led to a new description of electrophoresis based on non-Newtonian viscoelastic effects in the electric double layer surrounding a charged object. This new theoretical framework presents a new outlook important not only to the electrophoretic physics of nucleic acids, but all charged objects including proteins, colloids, and nanoparticles. To study the behavior of smaller biopolymers, such as restriction fragments and recombination intermediates, a library of symmetrically branched DNA was synthesized followed by characterization in gels. The experimental results contribute a large body of information relating molecular architecture and the dynamics of rigid structures in an electric field. The findings allow us to create new separation technologies based on topology. These contributions can also be utilized in a number of different applications including the study of recombination intermediates and the separation of proteins according to structure. To demonstrate the importance of these findings, a sequence and mutation detection technique was envisioned and applied for genetic analysis. Restriction fragments from mutation "hotspots" in the p53 tumor suppressor gene, known to play a role in cancer development, were analyzed with this technique

  6. Electrophoretic mobility of spherical particles in bounded domain.

    PubMed

    Liu, Yu-Wei; Pennathur, Sumita; Meinhart, Carl D

    2016-01-01

    In this study, we improve on our 3D steady-state model of electrophoretic motion of spherical particles in bounded fluidic channels (Liu et al., 2014) to include the effect of nonsymmetric electrolytes, and further validate this improved model with detailed comparisons to experimental data. Specifically, we use the experimentally-measured particle mobilities from the work of Semenov et al. (2013), Napoli et al. (2011), and Wynne et al. (2012) to determine the corresponding particle zeta potentials using our model, and compare these results with classical theory. Incorporating the effects of nonsymmetric electrolytes, EDL polarization, and confinement, we show that our improved model is applicable to a wide range of practical experimental conditions, for example, particles that have high zeta potentials in a bounded channel filled with nonsymmetric electrolyte solutions, where classical theory is not applicable. In addition, we find that when electrolyte concentration is comparable to the concentration of hydronium or hydroxide ions, the complicated composition of ions increases the particle mobility. Finally, increased electrophoretic mobility can be observed when buffer solutions (phosphate or borate) were used as electrolyte solutions in experiments as opposed to simple symmetric electrolytes. PMID:26397906

  7. Electrophoretic mobilities of counterions and a polymer in cylindrical pores

    PubMed Central

    Singh, Sunil P.; Muthukumar, M.

    2014-01-01

    We have simulated the transport properties of a uniformly charged flexible polymer chain and its counterions confined inside cylindrical nanopores under an external electric field. The hydrodynamic interaction is treated by describing the solvent molecules explicitly with the multiparticle collision dynamics method. The chain consisting of charged monomers and the counterions interact electrostatically with themselves and with the external electric field. We find rich behavior of the counterions around the polymer under confinement in the presence of the external electric field. The mobility of the counterions is heterogeneous depending on their location relative to the polymer. The adsorption isotherm of the counterions on the polymer depends nonlinearly on the electric field. As a result, the effective charge of the polymer exhibits a sigmoidal dependence on the electric field. This in turn leads to a nascent nonlinearity in the chain stretching and electrophoretic mobility of the polymer in terms of their dependence on the electric field. The product of the electric field and the effective polymer charge is found to be the key variable to unify our simulation data for various polymer lengths. Chain extension and the electrophoretic mobility show sigmoidal dependence on the electric field, with crossovers from the linear response regime to the nonlinear regime and then to the saturation regime. The mobility of adsorbed counterions is nonmonotonic with the electric field. For weaker and moderate fields, the adsorbed counterions move with the polymer and at higher fields they move opposite to the polymer's direction. We find that the effective charge and the mobility of the polymer decrease with a decrease in the pore radius. PMID:25240366

  8. Electrophoretic mobility of cells in a vertical Ficoll gradiant

    NASA Technical Reports Server (NTRS)

    Plank, L. D.; Todd, P. W.; Kunze, M. E.; Gaines, R. A.

    1985-01-01

    The upward migration of living cells and test particles under the influence of a constant electric field in a low conductivity Ficoll gradient occurs at nearly constant velocity. Viscosity and neutral polymer concentration affect migration rate. Decreasing viscosity speeds up the particle migration, decreasing neutral polymer (Ficoll) concentration, slows particle migration, since electrophoretic mobility increases approximately linearly with neutral polymer concentration. Neutral polymers interact with the cell surface to effectively raise its zeta potential. An analytic function was developed from the known dependence of these physical variables on migration distance; the analysis expresses migration velocity as an explicit function of position in the density gradient. It predicts an almost linear increase in velocity of about 12 to 16% over the working region of the gradient. It was numerically integrated and correctly predicts cell migration distance vs time curves without the use of any fitted parameters. The resulting migration curves follow the expected slowly varying exponential form that closely resembles a straight line. The ability to determine standard electrophoretic mobilities from such curves depends on knowledge of the effect of Ficoll on the zeta potential of the cell type that is separated.

  9. Electrophoretic behavior of DNA-methyl-CpG-binding domain protein complexes revealed by capillary electrophoreses laser-induced fluorescence.

    PubMed

    Zhong, Shangwei; Zou, Dandan; Zhao, Bailin; Zhang, Dapeng; Li, Xiangjun; Wang, Hailin

    2015-12-01

    The free solution electrophoretic behavior of DNA-protein complexes depends on their charge and mass in a certain experimental condition, which are two fundamental properties of DNA-protein complexes in free solution. Here, we used CE LIF to study the free solution behavior of DNA-methyl-CpG-binding domain protein (MBD2b) complexes through exploring the relationship between the mobilities, charge, and mass of DNA-protein complexes. This method is based on the effective separation of free DNA and DNA-protein complexes because of their different electrophoretic mobility in a certain electric field. In order to avoid protein adsorption, a polyacrylamide-coated capillary was used. Based on the evaluation of the electrophoretic behavior of formed DNA-MBD2b complexes, we found that the values of (μ0 /μ)-1 were directly proportional to the charge-to-mass ratios of formed complexes, where the μ0 and μ are the mobility of free DNA probe and DNA-protein complex, respectively. The models were further validated by the complex mobilities of protein with various lengths of DNA probes. The deviation of experimental and calculated charge-to-mass ratios of formed complexes from the theoretical data was less than 10%, suggesting that our models are useful to analyze the DNA-binding properties of the purified MBD2b protein and help to analyze other DNA-protein complexes. Additionally, this study enhances the understanding of the influence of the charge-to-mass ratios of formed DNA-protein complexes on their separation and electrophoretic behaviors. PMID:26377303

  10. Electrophoretic mobility patterns of collagen following laser welding

    NASA Astrophysics Data System (ADS)

    Bass, Lawrence S.; Moazami, Nader; Pocsidio, Joanne O.; Oz, Mehmet C.; LoGerfo, Paul; Treat, Michael R.

    1991-06-01

    Clinical application of laser vascular anastomosis in inhibited by a lack of understanding of its mechanism. Whether tissue fusion results from covalent or non-covalent bonding of collagen and other structural proteins is unknown. We compared electrophoretic mobility of collagen in laser treated and untreated specimens of rat tail tendon (>90% type I collagen) and rabbit aorta. Welding was performed, using tissue shrinkage as the clinical endpoint, using the 808 nm diode laser (power density 14 watts/cm2) and topical indocyanine green dye (max absorption 805 nm). Collagen was extracted with 8 M urea (denaturing), 0.5 M acetic acid (non-denaturing) and acetic acid/pepsin (cleaves non- helical protein). Mobility patterns on gel electrophoresis (SDS-PAGE) after urea or acetic acid extraction were identical in the lasered and control tendon and vessel (confirmed by optical densitometry), revealing no evidence of formation of novel covalent bonds. Alpha and beta band intensity was diminished in pepsin incubated lasered specimens compared with controls (optical density ratio 0.00 +/- 9 tendon, 0.65 +/- 0.12 aorta), indicating the presence of denatured collagen. With the laser parameters used, collagen is denatured without formation of covalent bonds, suggesting that non-covalent interaction between denatured collagen molecules may be responsible for the weld. Based on this mechanism, welding parameters can be chosen which produce collagen denaturation without cell death.

  11. Electrophoretic mobilities of cultured human embryonic kidney cells in various buffers

    NASA Technical Reports Server (NTRS)

    1985-01-01

    Data on the electrophoretic mobility distributions of cells in the new D-1 buffer and the interlaboratory standardization of urokinase assay methods are presented. A table of cell strains and recent data on cell dispersal methods are also included. It was decided that glycerol in A-1 electrophoretic mobility data on cultured human embryonic kidney cells subjected to electrophoresis in this buffer. The buffer composition is presented.

  12. Effect of passage number on electrophoretic mobility distributions of cultured human embryonic kidney cells

    NASA Technical Reports Server (NTRS)

    Kunze, M. E.

    1985-01-01

    A systematic investigation was undertaken to characterize population shifts that occur in cultured human embryonic kidney cells as a function of passage number in vitro after original explantation. This approach to cell population shift analysis follows the suggestion of Mehreshi, Klein and Revesz that perturbed cell populations can be characterized by electrophoretic mobility distributions if they contain subpopulations with different electrophoretic mobilities. It was shown that this is the case with early passage cultured human embryo cells.

  13. Electrophoretic mobilities of neutral analytes and electroosmotic flow markers in aqueous solutions of Hofmeister salts.

    PubMed

    Křížek, Tomáš; Kubíčková, Anna; Hladílková, Jana; Coufal, Pavel; Heyda, Jan; Jungwirth, Pavel

    2014-03-01

    Small neutral organic compounds have traditionally the role of EOF markers in electrophoresis, as they are expected to have zero electrophoretic mobility in external electric fields. The BGE contains, however, ions that have unequal affinities to the neutral molecules, which in turn results in their mobilization. In this study we focused on two EOF markers-thiourea and DMSO, as well as on N-methyl acetamide (NMA) as a model of the peptide bond. By means of CE and all atom molecular dynamics simulations we explored mobilization of these neutral compounds in large set of Hofmeister salts. Employing a statistical mechanics approach, we were able to reproduce by simulations the experimental electrophoretic mobility coefficients. We also established the role of the chemical composition of marker and the BGE on the measured electrophoretic mobility coefficient. For NMA, we interpreted the results in terms of the relative affinities of cations versus anions to the peptide bond. PMID:24338984

  14. Capillary electrophoresis separation of vinpocetine and related compounds: prediction of electrophoretic mobilities in partly aqueous media.

    PubMed

    Mazák, K; Szakács, Z; Nemes, A; Noszál, B

    2000-07-01

    Offord's equation, a relationship between electrophoretic mobility and charge, size and shape of peptides, has been extended to quantitate the electrophoretic mobility of vinca alkaloids. Partly aqueous protonation constants and the derived theoretical mobilities have been proven to be able to predict experimental electrophoretic mobilities. In practice, seven vincamine derivatives of very low water-solubility were separated by capillary electrophoresis. Buffer total concentration, apparent pH and methanol content, the three most important parameters of the running buffer, were used in triangular resolution mapping to characterize separation. Even though electrophoresis is well known to slow down in partly aqueous media, under our optimized circumstances a baseline separation was achieved within 8 min in each case. PMID:10939454

  15. Electrophoretic mobility patterns of immunologically phenotyped cells in different hemopoietic malignancies.

    PubMed

    Babusíková, O; Koníková, E; Ujházy, P

    1986-01-01

    The electrophoretic mobility distribution along with the immunologic phenotype (rosette tests, surface membrane immunoglobulin determination and mainly the detection of differentiation and leukemia-associated antigens by monoclonal antibodies) have been studied in 120 patients with different hemopoietic cell malignancies and in a group of healthy donors. The aim of our study was to compare the electrophoretic mobility character of malignant cells with that of normal T and B lymphocytes and their immune phenotype. Normal peripheral blood lymphocytes showed the typical bimodal pattern of two clearly distinguishable populations of different electrophoretic mobilities, corresponding to T and B cells. In leukemia and lymphoma cells the sharp unimodal peak has appeared, which was attributed to monoclonal origin of these cells. Moreover, the electrophoretic mobility of cells in different hemopoietic cell malignancies reflected different cell lineages and maturation stages within the given lineage group. Utilizing of the cell electrophoretic mobility as an additional marker to the immunologic data for the characterization of leukemia and lymphoma cells is proposed. PMID:3808126

  16. The electrophoretic mobility of montmorillonite. Zeta potential and surface conductivity effects.

    PubMed

    Leroy, Philippe; Tournassat, Christophe; Bernard, Olivier; Devau, Nicolas; Azaroual, Mohamed

    2015-08-01

    Clay minerals have remarkable adsorption properties because of their high specific surface area and surface charge density, which give rise to high electrochemical properties. These electrochemical properties cannot be directly measured, and models must be developed to estimate the electrostatic potential at the vicinity of clay mineral surfaces. In this context, an important model prediction is the zeta potential, which is thought to be representative of the electrostatic potential at the plane of shear. The zeta potential is usually deduced from electrophoretic measurements but for clay minerals, high surface conductivity decreases their mobility, thereby impeding straightforward interpretation of these measurements. By combining a surface complexation, conductivity and electrophoretic mobility model, we were able to reconcile zeta potential predictions with electrophoretic measurements on montmorillonite immersed in NaCl aqueous solutions. The electrochemical properties of the Stern and diffuse layers of the basal surfaces were computed by a triple-layer model. Computed zeta potentials have considerably higher amplitudes than measured zeta potentials calculated with the Smoluchowski equation. Our model successfully reproduced measured electrophoretic mobilities. This confirmed our assumptions that surface conductivity may be responsible for montmorillonite's low electrophoretic mobility and that the zeta potential may be located at the beginning of the diffuse layer. PMID:25875489

  17. Alteration of the electrophoretic mobility of human peripheral blood mononuclear cells following treatment with dimethyl sulfoxide

    SciTech Connect

    Skrabut, E.M.; Catsimpoolas, N.; Kurtz, S.R.; Griffith, A.L.; Valeri, C.R.

    1983-12-01

    Studies have been conducted to determine the effects of DMSO and freezing on the electrophoretic distribution of peripheral blood mononuclear cells. Sodium (/sup 51/Cr)chromate was used to label the cells, and the distributions of cell number and cell-associated radioactivity were determined. Cells treated with DMSO had a narrower distribution of electrophoretic mobilities when compared with those not treated. DMSO-treated cells also demonstrated a more homogeneous distribution of radioactivity relative to the cell distribution than did the nontreated cells. The freezing of DMSO-treated cells did not result in any additional alteration of electrophoretic pattern compared to DMSO treatment alone. Analysis by linear categorization techniques indicated that the DMSO-treated and nontreated cells were completely distinguished by their electrophoretic behavior.

  18. Sterically stabilized liposomes. Reduction in electrophoretic mobility but not electrostatic surface potential.

    PubMed Central

    Woodle, M C; Collins, L R; Sponsler, E; Kossovsky, N; Papahadjopoulos, D; Martin, F J

    1992-01-01

    The electrophoretic mobility of liposomes containing a negatively charged derivative of phosphatidylethanolamine with a large headgroup composed of the hydrophilic polymer polyethylene glycol (PEG-PE) was determined by Doppler electrophoretic light scattering. The results show that this method is improved by the use of measurements at multiple angles to eliminate artifacts and that very small mobilities can be measured. The electrophoretic mobility of liposomes with 5 to 10 mol% PEG-PE is approximately -0.5 mu ms-1/Vcm-1 regardless of PEG-PE content compared with approximately -2 mu ms-1/Vcm-1 for similar liposomes but containing 7.5% phosphatidylglycerol (PG) instead of PEG-PE. Measurements of surface potential by distribution of an anionic fluorescent probe show that the PEG-PE imparts a negative charge identical to that by PG, consistent with the expectation of similar locations of the ionized phosphate responsible for the charge. The reduced mobility imparted by the surface bound PEG is attributed to a mechanism similar to that described for colloidal steric stabilization: hydrodynamic drag moves the hydrodynamic plane of shear, or the hydrodynamic radius, away from the charge-bearing plane, that of the phosphate moities. An extended length of approximately 50 A for the 2,000 molecular weight PEG is estimated from the reduction in electrophoretic mobility. PMID:1581503

  19. Enhancement of electrophoretic mobility of microparticles near a solid wall--experimental verification.

    PubMed

    Liang, Qian; Zhao, Cunlu; Yang, Chun

    2015-03-01

    Although the existing theories have predicted enhancement of electrophoretic mobility of microparticles near a solid wall, the relevant experimental studies are rare. This is mainly due to difficulties in experimentally controlling and measuring particle-wall separations under dynamic electrophoretic conditions. This paper reports an experimental verification of the enhancement of electrophoretic mobility of a microparticle moving near the wall of a microchannel. This is achieved by balancing dielectrophoretic and lift forces against gravitational force acting on the microparticle so as to control the gap of particle-wall separation. A simple experimental setup is configured and a fabrication method is developed to measure such separation gap. The experiments are conducted for various particle sizes under different electric field strengths. Our experimental results are compared against the available theoretical predictions in the literature. PMID:25421107

  20. Characterization of the Cell Surface Properties of Drinking Water Pathogens by Microbial Adhesion to Hydrocarbon and Electrophoretic Mobility Measurements

    EPA Science Inventory

    The surface characteristics of microbial cells directly influence their mobility and behavior within aqueous environments. The cell surface hydrophobicity (CSH) and electrophoretic mobility (EPM) of microbial cells impact a number of interactions and processes including aggregati...

  1. ELECTROPHORETIC MOBILITIES OF ESCHERICHIA COLI 0157:H7 AND WILD-TYPE ESCHERICHIA COLI STRAINS

    EPA Science Inventory

    The electrophoretic mobility (EPM) of a number of human-virulent and "wild-type" Escherichia coli strains in phosphate buffered water was measured. The impact of pH, ionic strength, cation type (valence) and concentration, and bacterial strain on the EPM was investigated. Resul...

  2. Controlled method of reducing electrophoretic mobility of macromolecules, particles, or cells

    NASA Technical Reports Server (NTRS)

    Vanalstine, James M. (Inventor)

    1992-01-01

    A method of reducing electrophoretic mobility of macromolecules, particles, cells, and other substances is provided which comprises interacting in a conventional electrophoretic separating procedure, the substances with a polymer-linked affinity compound comprised of a hydrophilic neutral polymer such as polyethylene glycol bound to a second component such as a hydrophobic compound, an immunocompound such as an antibody or antibody active fragment, or a ligand such as a hormone, drug, antigen, or a hapten. The reduction of electrophoretic mobility achieved is directly proportional to the concentration of the polymer-linked affinity compound employed, and such reduction can comprise up to 100 percent for particular particles and cells. The present invention is advantageous in that electrophoretic separation can now be achieved for substances whose native surface charge structure had prevented them from being separated by normal electrophoretic means. Depending on the affinity component utilized, separation can be achieved on the basis of the specific/irreversible, specific/reversible, semi-specific/reversible, relatively nonspecific/reversible, or relatively nonspecific/irreversible ligand-substance interactions.

  3. Size and DNA distributions of electrophoretically separated cultured human kidney cells

    NASA Technical Reports Server (NTRS)

    Kunze, M. E.; Plank, L. D.; Todd, P. W.

    1985-01-01

    Electrophoretic purification of purifying cultured cells according to function presumes that the size of cycle phase of a cell is not an overriding determinant of its electrophoretic velocity in an electrophoretic separator. The size distributions and DNA distributions of fractions of cells purified by density gradient electrophoresis were determined. No systematic dependence of electrophoretic migration upward in a density gradient column upon either size or DNA content were found. It was found that human leukemia cell populations, which are more uniform function and found in all phases of the cell cycle during exponential growth, separated on a vertical sensity gradient electrophoresis column according to their size, which is shown to be strictly cell cycle dependent.

  4. Electrophoretic Mobility of a Dilute, Highly Charged "Soft" Spherical Particle in a Charged Hydrogel.

    PubMed

    Allison, Stuart; Li, Fei; Le, Melinda

    2016-08-25

    In this paper, numerical modeling studies are carried out on the electrophoretic mobility of a dilute, highly charged "soft" spherical particle in a hard hydrogel subjected to a weak, constant, external electric field. The particle contains a solid core with either a uniform charge density or "zeta" potential on its surface. Outside of this lies a charged gel layer of uniform thickness, composition, and charge density. The present work extends previous studies by accounting for the "relaxation effect", or distortion of the charge distribution in the vicinity of the model particle due to the imposition of an external electric and/or flow field. The particle gel layer and ambient hydrogel are modeled as porous Brinkman media. The (steady state) electrodynamic problem is solved at the level of the Poisson equation. Applications emphasize the influence of the relaxation effect and hydrogel charge density on the electrophoretic mobility. PMID:26815300

  5. Electrophoretic Mobility of Poly(acrylic acid)-Coated Alumina Particles

    SciTech Connect

    Bhosale, Prasad S.; Chun, Jaehun; Berg, John C.

    2011-06-01

    The effect of poly (acrylic acid) (PAA) adsorption on the electrokinetic behavior of alumina dispersions under high pH conditions was investigated as a function of polymer concentration and molecular weight as well as the presence, concentration and ion type of background electrolyte. Systems of this type are relevant to nuclear waste treatment, in which PAA is known to be an effective rheology modifier. The presence of all but the lowest molecular weight PAA studied (1800) led to decreases in dynamic electrophoretic mobility at low polymer concentrations, attributable to bridging flocculation, as verified by measurements of particle size distribution. Bridging effects increased with polymer molecular weight, and decreased with polymer concentration. Increases in background electrolyte concentration enhanced dynamic electrophoretic mobility as the polymer layers were compressed and bridging was reduced. Such enhancements were reduced as the cation was changed from Na+ to K+ to Cs+.

  6. Cobalt ferrite nanoparticles with improved aqueous colloidal stability and electrophoretic mobility

    NASA Astrophysics Data System (ADS)

    Munjal, Sandeep; Khare, Neeraj

    2016-04-01

    We have synthesized CoFe2O4 (CFO) nanoparticles of size ˜ 12.2 nm by hydrothermal synthesis method. To control the size of these CFO nanoparticles, oleic acid was used as a surfactant. The inverse spinel phase of the synthesized nanoparticles was confirmed by X-ray diffraction method. As synthesized oleic acid coated CFO (OA@CFO) nanoparticles has very less electrophoretic mobility in the water and are not water dispersible. These OA@CFO nanoparticles were successfully turned into water soluble phase with a better colloidal aqueous stability, through a chemical treatment using citric acid. The modified citric acid coated CFO (CA@CFO) nanoparticles were dispersible in water and form a stable aqueous solution with high electrophoretic mobility.

  7. Fenton fragmentation for faster electrophoretic on chip purification of amplifiable genomic DNA.

    PubMed

    Hakenberg, S; Hügle, M; Meyer, P; Behrmann, O; Dame, G; Urban, G A

    2015-05-15

    With a rapid and simple actuation protocol electrophoretic nucleic acid extraction is easy automatable, requires no moving parts, is easy to miniaturize and furthermore possesses a size dependent cut-off filter adjustable by the pore size of the hydrogel. However electrophoretic nucleic acid extraction from bacteria has so far been applied mainly for short RNA targets. One of the reasons is that electrophoretic processing of unfragmented genomic DNA strands is time-consuming, because of the length. Here DNA fragmentation would accelerate extraction and isolation. We introduce on-chip lysis and non-enzymatic DNA cleavage directly followed by a purifying step for receiving amplifiable DNA fragments from bacteria in less than 25 min. In contrast to restriction enzymes the Fenton reaction is known to cleave DNA without nucleotide specificity. The reaction mix contains iron(II) EDTA, sodium ascorbate, hydrogen peroxide and lysozyme. The degree of fragmentation can be adjusted by the concentration of reagents. The results enable electrophoretic extraction methods to unspecifically process long genomic DNA in a short time frame, e.g. for pathogen detection in a lab-on-a-chip format. PMID:24970713

  8. Microflora on explanted silicone rubber voice prostheses: taxonomy, hydrophobicity and electrophoretic mobility.

    PubMed

    Neu, T R; Verkerke, G J; Herrmann, I F; Schutte, H K; Van der Mei, H C; Busscher, H J

    1994-05-01

    Silicone rubber voice prostheses are implants which are inserted in a non-sterile environment and therefore become quickly colonized by micro-organisms. The micro-organisms exist on the medical grade silicone rubber as mixed biofilms of bacteria and yeasts. A total of 79 bacterial and 39 yeast strains were isolated from these biofilms by soft ultrasonic treatment. Gram-positive/catalase-negative and Gram-positive/catalase-positive cocci represented the dominant bacterial strains. The yeasts were mainly Candida species. Further characterization of cell surface properties such as hydrophobicity by microbial adhesion to hexadecane and electrophoretic mobility showed a distinct difference when the bacterial strains were compared with the yeasts. The bacterial hydrophobicities ranged from 0 to 100% adhesion to hexadecane, whereas the yeast strains, especially the Candida albicans strains, all had markedly hydrophilic cell surfaces. A comparison of the electrophoretic mobilities showed also differences between bacteria and yeast. The values for the bacteria were found to be between -2.5 to -0.5 (10(-8) m2 V-1 s-1), whereas for the yeasts electrophoretic mobilities were more positive. Based on the adhesive properties of the isolated micro-organisms, strategies can now be developed to modify the properties of the silicone rubber to reduce biofilm formation on such prostheses. PMID:8005837

  9. Approximate Analytic Expression for the Electrophoretic Mobility of Moderately Charged Cylindrical Colloidal Particles.

    PubMed

    Ohshima, Hiroyuki

    2015-12-29

    An approximate analytic expression for the electrophoretic mobility of an infinitely long cylindrical colloidal particle in a symmetrical electrolyte solution in a transverse electric field is obtained. This mobility expression, which is correct to the order of the third power of the zeta potential ζ of the particle, considerably improves Henry's mobility formula correct to the order of the first power of ζ (Proc. R. Soc. London, Ser. A 1931, 133, 106). Comparison with the numerical calculations by Stigter (J. Phys. Chem. 1978, 82, 1417) shows that the obtained mobility formula is an excellent approximation for low-to-moderate zeta potential values at all values of κa (κ = Debye-Hückel parameter and a = cylinder radius). PMID:26639309

  10. High-Throughput Electrophoretic Mobility Shift Assays for Quantitative Analysis of Molecular Binding Reactions

    PubMed Central

    2015-01-01

    We describe a platform for high-throughput electrophoretic mobility shift assays (EMSAs) for identification and characterization of molecular binding reactions. A photopatterned free-standing polyacrylamide gel array comprised of 8 mm-scale polyacrylamide gel strips acts as a chassis for 96 concurrent EMSAs. The high-throughput EMSAs was employed to assess binding of the Vc2 cyclic-di-GMP riboswitch to its ligand. In optimizing the riboswitch EMSAs on the free-standing polyacrylamide gel array, three design considerations were made: minimizing sample injection dispersion, mitigating evaporation from the open free-standing polyacrylamide gel structures during electrophoresis, and controlling unit-to-unit variation across the large-format free-standing polyacrylamide gel array. Optimized electrophoretic mobility shift conditions allowed for 10% difference in mobility shift baseline resolution within 3 min. The powerful 96-plex EMSAs increased the throughput to ∼10 data/min, notably more efficient than either conventional slab EMSAs (∼0.01 data/min) or even microchannel based microfluidic EMSAs (∼0.3 data/min). The free-standing polyacrylamide gel EMSAs yielded reliable quantification of molecular binding and associated mobility shifts for a riboswitch–ligand interaction, thus demonstrating a screening assay platform suitable for riboswitches and potentially a wide range of RNA and other macromolecular targets. PMID:25233437

  11. DNA analysis on microfabricated electrophoretic devices with bubble cells.

    PubMed

    Tseng, Wei-Lung; Lin, Yang-Wei; Chen, Ko-Chun; Chang, Huan-Tsung

    2002-08-01

    Microfluidic devices with bubble cells have been fabricated on poly(methyl methacrylate) (PMMA) plates and have been employed for the analysis of DNA using polyethylene oxide (PEO) solutions. First, the separation channel was fabricated using a wire-imprinting method. Then, wires with greater sizes or a razor blade glued in a polycarbonate plate was used to fabricate bubble cells, with sizes of 190-650 microm. The improvements in resolution and sensitivity have been achieved for large DNA (> 603 base pair, bp) using such devices, which depend on the geometry of the bubble cell. The main contributor for optimal resolution is mainly due to DNA migration at lower electric field strengths inside the bubble cell. On the other hand, slight losses of resolution for small DNA fragments have been found mainly due to diffusion, supported by the loss of resolution when separating two small solutes. With a bubble cell of 75 microm (width) x 500 microm (depth), the sensitivity improvement up to 17-fold has been achieved for the 271 bp fragment in the separation of PhiX-174/HaeIII DNA restriction fragments. We have also found that a microfluidic device with a bubble cell of 360 microm x 360 microm is appropriate for DNA analysis. Such a device has been used for separating DNA ranging from 8 to 2176 bp and polymerase chain reaction (PCR) products amplified after 30 cycles, with rapidity and improvements in the sensitivity as well as resolution. PMID:12210206

  12. Passive trapping of rigid rods due to conformation-dependent electrophoretic mobility.

    PubMed

    Pandey, Harsh; Szafran, Sylvia A; Underhill, Patrick T

    2016-03-16

    We present computer simulations of a rigid rod in a combination of an extensional fluid flow and extensional electric field. The electrophoretic mobility of the rod is different parallel or perpendicular to the rod. The dependence of the mobility on the conformation (orientation) leads to a new phenomenon where the rods can be passively trapped in all directions at the stagnation point. This contrasts with the behavior in either fluid flow or electric field alone, in which an object can be pushed towards the stagnation point along some directions but is pushed away in others. We have determined the state space where trapping occurs and have developed a model that describes the strength of trapping when it does occur. This new phenomenon could be used in the future to separate objects based on a coupling between their mobility and ability to be oriented. PMID:26892384

  13. DNA fingerprinting and electrophoretic karyotype of environmental and clinical isolates of Candida parapsilosis.

    PubMed Central

    Carruba, G; Pontieri, E; De Bernardis, F; Martino, P; Cassone, A

    1991-01-01

    The endonuclease restriction pattern (DNA fingerprinting) and the electrophoretic karyotype of 16 Candida parapsilosis isolates from environmental and clinical sources were investigated. DNA from both whole cells and separated mitochondria was digested with enzymes, including EcoRI, BamHI, KpnI, BglII, HpaII, PvuII, and HindIII. Regardless of their source and pathogenic properties, all isolates showed a uniform, reproducible, and overlapping whole-cell DNA fingerprinting with each endonuclease digest. Mitochondrial DNA fragments were, in all cases, major contributors to the total cellular DNA restriction pattern. In contrast, the electrophoretic karyotype generated by rotating field gel electrophoresis (RFGE) or contour clamped homogeneous field electrophoresis (CHEF) showed a remarkable polymorphism among the isolates. This polymorphism concerned the smaller molecular size section of the karyotype (range, 1.8 to 0.7 Mb), where at least two to five chromosomal bands could be consistently detected by both RFGE and CHEF. Larger (greater than or equal to 3.0 to 1.9 Mb) chromosome-sized DNA bands (four in CHEF and three in RFGE) were quite distinct and common to all isolates. Thus, seven karyotype classes could be defined, on the basis of both the number and size of putative chromosomes. The three categories of isolates (soil, vaginal, and hematological) were not randomly distributed among the seven classes. In particular, the four hematological isolates had a karyotype pattern which was clearly distinct from that shown by the three environmental isolates, and of the nine vaginal isolates only one shared a class with isolates from another source (soil). Although tentative, the classification was totally consistent with the independent and reproducible results obtained by the two pulse-field electrophoretic techniques employed. It is suggested that the electrophoretic analysis of the karyotype might be particularly useful for epidemiological and pathogenicity studies on

  14. Electrophoretic mobility of silica particles in a mixture of toluene and ethanol at different particle concentrations.

    PubMed

    Medrano, M; Pérez, A T; Lobry, L; Peters, F

    2009-10-20

    In this paper we present measurements of the electrophoretic mobility of colloidal particles by using heterodyne detection of light scattering. The measurements have been made up to concentrations of 5.4% silica nanoparticles, with a diameter on the order of 80 nm, in a mixture of 70% toluene and 30% ethanol. To make possible the measurements at these concentrations, the liquid mixture is chosen so as to match the index of refraction of the particles, thus resulting in a transparent suspension. PMID:19754057

  15. The influence of hydrodynamic slip on the electrophoretic mobility of a spherical colloidal particle

    NASA Astrophysics Data System (ADS)

    Khair, Aditya S.; Squires, Todd M.

    2009-04-01

    Recent theoretical studies have suggested a significant enhancement in electro-osmotic flows over hydrodynamically slipping surfaces, and experiments have indeed measured O(1) enhancements. In this paper, we investigate whether an equivalent effect occurs in the electrophoretic motion of a colloidal particle whose surface exhibits hydrodynamic slip. To this end, we compute the electrophoretic mobility of a uniformly charged spherical particle with slip length λ as a function of the zeta (or surface) potential of the particle ζ and diffuse-layer thickness κ-1. In the case of a thick diffuse layer, κa ≪1 (where a is the particle size), simple arguments show that slip does lead to an O(1) enhancement in the mobility, owing to the reduced viscous drag on the particle. On the other hand, for a thin-diffuse layer κa ≫1, the situation is more complicated. A detailed asymptotic analysis, following the method of O'Brien [J. Colloid Interface Sci. 92, 204 (1983)], reveals that an O(κλ) increase in the mobility occurs at low-to-moderate zeta potentials (with ζ measured on the scale of thermal voltage kBT /e≈25 mV). However, as ζ is further increased, the mobility decreases and ultimately becomes independent of the slip length—the enhancement is lost—which is due to the importance of nonuniform surface conduction within the thin-diffuse layer, at large ζ and large, but finite, κa. Our asymptotic calculations for thick and thin-diffuse layers are corroborated and bridged by computation of the mobility from the numerical solution of the full electrokinetic equations (using the method of O'Brien and White [J. Chem. Soc., Faraday Trans. 2 74, 1607 (1978)]). In summary, then, we demonstrate that hydrodynamic slip can indeed produce an enhancement in the electrophoretic mobility; however, such enhancements will not be as dramatic as the previously studied κa →∞ limit would suggest. Importantly, this conclusion applies not only to electrophoresis but also to

  16. Control and Reversal of the Electrophoretic Force on DNA in a Charged Nanopore

    PubMed Central

    Luan, Binquan

    2011-01-01

    Electric field-driven transport of DNA through solid-state nanopores is the key process in nanopore-based DNA sequencing that promises dramatic reduction of genome sequencing costs. A major hurdle in the development of this sequencing method is that DNA transport through the nanopores occurs too quickly for the DNA sequence to be detected. By means of all-atom molecular dynamics simulations, we demonstrate in this communication that velocity of DNA transport through a nanopore can be controlled by the charge state of the nanopore surface. In particular, we show that the charge density of the nanopore surface controls the magnitude and/or direction of the electro-osmotic flow through the nanopore and thereby can significantly reduce or even reverse the effective electrophoretic force on DNA. Our work suggests a physical mechanism to control DNA transport in a nanopore by chemical, electrical or electrochemical modification of the nanopore surface. PMID:21339610

  17. Electrophoretic mobility as a tool to separate immune adjuvant saponins from Quillaja saponaria Molina.

    PubMed

    Gilabert-Oriol, Roger; Weng, Alexander; von Mallinckrodt, Benedicta; Stöshel, Anja; Nissi, Linda; Melzig, Matthias F; Fuchs, Hendrik; Thakur, Mayank

    2015-06-20

    Quillaja saponins are used as adjuvants in animal vaccines but their application in human vaccination is still under investigation. Isolation and characterization of adjuvant saponins is very tedious. Furthermore, standardization of Quillaja saponins is critical pertaining to its application in humans. In this study, a convenient method based on agarose gel electrophoresis was developed for the separation of Quillaja saponins. Six different commercial Quillaja saponins were segregated by size/charge into numerous fractions. Each of the fractions was characterized by ESI-TOF-MS spectroscopy and thin layer chromatography. Real-time impedance-based monitoring and red blood cell lysis assay were used to evaluate cytotoxicity and hemolytic activities respectively. Two specific regions in the agarose gel (delimited by specific relative electrophoretic mobility values) were identified and characterized by exclusive migration of acylated saponins known to possess immune adjuvant properties (0.18-0.58), and cytotoxic and hemolytic saponins (0.18-0.94). In vivo experiments in mice with the isolated fractions for evaluation of adjuvant activity also correlated with the relative electrophoretic mobility. In addition to the separation of specific Quillaja saponins with adjuvant effects as a pre-purification step to HPLC, agarose gel electrophoresis stands out as a new method for rapid screening, separation and quality control of saponins. PMID:25839418

  18. Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic Acid Interactions

    PubMed Central

    Hellman, Lance M.; Fried, Michael G.

    2009-01-01

    The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided. PMID:17703195

  19. DNA mobility modifier

    DOEpatents

    Barron, Annelise E.

    2004-04-20

    Polyamides comprising at least one hydrophilic C.sub.1 -C.sub.10 hydrocarbyl substituent on an amide nitrogen atom, and methods for producing and using the same is provided. In particular, polyamides of the formula: ##STR1## and methods for using the same for altering the ratio of charge/translational frictional drag of binding polymers to allow electrophoretic separation of polynucleotides or analogs thereof in a non-sieving liquid medium is provided, where a, q, L.sup.1, P.sup.1, Q.sup.1, R, R.sup.1, R.sup.10 and R.sup.11 are those described herein.

  20. DNA mobility modifier

    DOEpatents

    Barron, Annelise

    2002-01-01

    Polyamides comprising at least one hydrophilic C.sub.1 -C.sub.10 hydrocarbyl substituent on an amide nitrogen atom, and methods for producing and using the same is provided. In particular, polyamides of the formula: ##STR1## and methods for using the same for altering the ratio of charge/translational frictional drag of binding polymers to allow electrophoretic separation of polynucleotides or analogs thereof in a non-sieving liquid medium is provided, where a, q, L.sup.1, P.sup.1, Q.sup.1, R, R.sup.1, R.sup.10 and R.sup.11 are those described herein.

  1. Cell surface adhesiveness of mouse sarcoma lines evaluated by latex particle adherence assay: correlation with growth behavior and electrophoretic mobility.

    PubMed

    Bubeník, J; Jandlová, T; Suhajová, E; Malkovský, M

    1979-01-01

    Using the latex particle adherence assay and five mouse sarcoma cell lines of the identical origin, etiology and genotype but differing in malignancy we attempted to correlate the degree of cell surface adhesiveness with growth behavior and electrophoretic mobility of cells. Higher tumorigenicity of four of the cell lines (Mc11--Mc14) was associated with lower cell surface adhesiveness and, conversely, lower malignancy of the fifth line (Mc15) with higher cell surface adhesiveness. No simple correlation or causal relationship was found among the electrophoretic mobility of the lines and other cellular characteristics. PMID:522921

  2. Observation of separate cation and anion electrophoretic mobilities in pure ionic liquids

    NASA Astrophysics Data System (ADS)

    Zhang, Zhiyang; Madsen, Louis A.

    2014-02-01

    Ionic liquids (ILs) continue to show relevance in many fields, from battery electrolytes, to carbon capture, to advanced separations. These highly ion-dense fluids present unique challenges in understanding their electrochemical properties due to deviations in behavior from existing electrolyte theories. Here we present a novel characterization of ILs using electrophoretic NMR (ENMR) to determine separate cation and anion mobilities. This method uses an applied electric field coincident with a pulsed magnetic field gradient to encode the E-field driven flow into NMR signals for cations (1H) and anions (19F). We describe the detailed design of these experiments, including quantitative analysis of artifact mitigation and necessary control experiments. We then explore mobilities and diffusion coefficients for two representative ILs: 1-ethyl-3-methyl imidazolium tetrafluoroborate ([C2mim][BF4]) and 1-ethyl-3-methyl imidazolium trifluoromethanesulfonate ([C2mim][TfO]). We further use the individual ion mobilities to calculate the bulk net conductivity, which closely agrees with bulk conductivity measurements obtained using impedance spectroscopy. These observations represent the first reliable measurements of cation and anion mobilities in pure ILs, with errors of ±7%. We discuss this advanced experimental methodology in detail, as well as implications of these sensitive measurements for understanding conduction mechanisms in ion-dense electrolytes.

  3. Observation of separate cation and anion electrophoretic mobilities in pure ionic liquids.

    PubMed

    Zhang, Zhiyang; Madsen, Louis A

    2014-02-28

    Ionic liquids (ILs) continue to show relevance in many fields, from battery electrolytes, to carbon capture, to advanced separations. These highly ion-dense fluids present unique challenges in understanding their electrochemical properties due to deviations in behavior from existing electrolyte theories. Here we present a novel characterization of ILs using electrophoretic NMR (ENMR) to determine separate cation and anion mobilities. This method uses an applied electric field coincident with a pulsed magnetic field gradient to encode the E-field driven flow into NMR signals for cations ((1)H) and anions ((19)F). We describe the detailed design of these experiments, including quantitative analysis of artifact mitigation and necessary control experiments. We then explore mobilities and diffusion coefficients for two representative ILs: 1-ethyl-3-methyl imidazolium tetrafluoroborate ([C2mim][BF4]) and 1-ethyl-3-methyl imidazolium trifluoromethanesulfonate ([C2mim][TfO]). We further use the individual ion mobilities to calculate the bulk net conductivity, which closely agrees with bulk conductivity measurements obtained using impedance spectroscopy. These observations represent the first reliable measurements of cation and anion mobilities in pure ILs, with errors of ±7%. We discuss this advanced experimental methodology in detail, as well as implications of these sensitive measurements for understanding conduction mechanisms in ion-dense electrolytes. PMID:24588161

  4. Difference in microchip electrophoretic mobility between partially and fully PEGylated poly(amidoamine) dendrimers.

    PubMed

    Park, Eun Ji; Na, Dong Hee

    2015-11-01

    The objective of this study was to investigate the difference in electrophoretic mobility between partially and fully poly(ethylene glycol)-conjugated poly(amidoamine) dendrimers (part-PEG-PAMAM and full-PEG-PAMAM, respectively) using a microchip capillary gel electrophoresis (MCGE). While MCGE allowed size-based separation of PEG-PAMAMs prepared with monomethoxy PEG-nitrophenyl carbonate, full-PEG-PAMAMs migrated slower than part-PEG-PAMAMs that were similar in size or larger. When the measured molecular weights obtained from MCGE analysis and the calculated molecular weights were plotted, each part-PEG-PAMAM and full-PEG-PAMAM showed correlation coefficients greater than 0.98. This study indicates that MCGE would be useful for characterizing PEG-PAMAMs with different PEGylation degrees. PMID:26253023

  5. Electrophoretic mobility shift assays: analysis of tRNA binding to the T box riboswitch antiterminator RNA.

    PubMed

    Anupam, R; Zhou, S; Hines, J V

    2015-01-01

    Changes in electrophoretic mobility upon complex formation with RNA can be used to probe structure-function relationships that are critical for complex formation. Here, we describe the application of this technique to monitor tRNA binding to the T box riboswitch antiterminator RNA. PMID:25352142

  6. Effect of pH on the Electrophoretic Mobility of Spores of Bacillus anthracis and Its Surrogates in Aqueous Solutions

    EPA Science Inventory

    Electrophoretic mobility (EPM) of endospores of Bacillus anthracis and surrogates were measured in aqueous solution across a broad pH range and several ionic strengths. EPM values trended around phylogenetic clustering based on the 16S rRNA gene. Measurements reported here prov...

  7. Rapid agarose gel electrophoretic mobility shift assay for quantitating protein: RNA interactions.

    PubMed

    Ream, Jennifer A; Lewis, L Kevin; Lewis, Karen A

    2016-10-15

    Interactions between proteins and nucleic acids are frequently analyzed using electrophoretic mobility shift assays (EMSAs). This technique separates bound protein:nucleic acid complexes from free nucleic acids by electrophoresis, most commonly using polyacrylamide gels. The current study utilizes recent advances in agarose gel electrophoresis technology to develop a new EMSA protocol that is simpler and faster than traditional polyacrylamide methods. Agarose gels are normally run at low voltages (∼10 V/cm) to minimize heating and gel artifacts. In this study we demonstrate that EMSAs performed using agarose gels can be run at high voltages (≥20 V/cm) with 0.5 × TB (Tris-borate) buffer, allowing for short run times while simultaneously yielding high band resolution. Several parameters affecting band and image quality were optimized for the procedure, including gel thickness, agarose percentage, and applied voltage. Association of the siRNA-binding protein p19 with its target RNA was investigated using the new system. The agarose gel and conventional polyacrylamide gel methods generated similar apparent binding constants in side-by-side experiments. A particular advantage of the new approach described here is that the short run times (5-10 min) reduce opportunities for dissociation of bound complexes, an important concern in non-equilibrium nucleic acid binding experiments. PMID:27495142

  8. Easy measurement and analysis method of zeta potential and electrophoretic mobility of water-dispersed colloidal particles by using a self-mixing solid-state laser

    NASA Astrophysics Data System (ADS)

    Sudo, S.; Ohtomo, T.; Otsuka, K.

    2013-08-01

    We describe a highly sensitive method of measuring electrophoretic mobility and zeta potential of water-dispersed colloidal particles by using a self-mixing laser Doppler velocimeter with a laser-diode-pumped, thin-slice solid-state laser with extremely high optical sensitivity. The power spectra of laser output modulated by reinjected laser light scattered by the electrophoretic particles were observed. The power spectrum cannot be described by the well-known formula for translational motion or flowing Brownian motion, i.e., a combination of Doppler shift, diffusion, and translation. The power spectra shape is found to reflect the velocity distribution of electrophoretic particles in a capillary tube due to the electro-osmotic flow contribution. Not only evaluation of the electrophoretic mobility and zeta potential but also the particle diameter undergoing electrophoretic motion can be performed from the shape of the power spectrum.

  9. A computer program for determining the electrophoretic mobility of cells in a rectangular chamber during asymmetric electroosmotic flow

    NASA Technical Reports Server (NTRS)

    1985-01-01

    In the field of cell electrophoresis, computer programs have been used for the estimation of zeta potential and surface charge density of specific charged chemical species from electrophoretic mobility data. The union of computer and microscope cell electrophoresis in the laboratory has yielded several satisfying results. The method organizes and error checks these data and stores them on permanent file for future reference or reanalysis. This computer analysis, accomplished in two steps by two main programs and a major subroutine, provides a quick, useful and realistic presentation of mobility data in histogram form, and is appropriate for any microelectrophoretic work using rectangular chambers. Computer program 1 is the first step of two in the analysis of mobility data. Program 2 consists of the main program, POLY2, which fits a least squares parabola to the apparent mobility data generated by Program 1, and the subroutine EXTRA which determines actual cell mobilities using the regression curve equation.

  10. Electrophoretic concentration of DNA at nanoporous polymer membranes for separations and diagnostics.

    SciTech Connect

    Thaitrong, Numrin; Meagher, Robert J.; Singh, Anup K.

    2010-11-01

    We report on the use of thin ({approx}30 micron) photopatterned polymer membranes for on-line preconcentration of single- or double-stranded DNA samples prior to electrophoretic analysis. Shaped UV laser light is used to quickly ({approx}10 seconds) polymerize a highly crosslinked polyacrylamide plug. By applying an electric field across the membrane, DNA from a dilute sample can be concentrated into a narrow zone (<100 micron wide) at the outside edge of the membrane. The field at the membrane can then be reversed, allowing the narrow plug to be cleanly injected into a separation channel filled with a sieving polymer for analysis. Concentration factors >100 are possible, increasing the sensitivity of analysis for dilute samples. We have fabricated both neutral membranes (purely size-based exclusion) as well as anionic membranes (size and charge exclusion), and characterized the rate of preconcentration as well as the efficiency of injection from both types of membrane, for DNA, ranging from a 20 base ssDNA oligonucleotide to >14 kbp dsDNA. We have also investigated the effects of concentration polarization on device performance for the charged membrane. Advantages of the membrane preconcentration approach include the simplicity of device fabrication and operation, and the generic (non-sequence specific) nature of DNA capture, which is useful for complex or poorly characterized samples where a specific capture sequence is not present. The membrane preconcentration approach is well suited to simple single-level etch glass chips, with no need for patterned electrodes, integrated heaters, valves, or other elements requiring more complex chip fabrication. Additionally, the ability to concentrate multiple charged analytes into a narrow zone enables a variety of assay functionalities, including enzyme-based and hybridization-based analyses.

  11. An electrophoretic mobility shift assay identifies a mechanistically unique inhibitor of protein sumoylation.

    PubMed

    Kim, Yeong Sang; Nagy, Katelyn; Keyser, Samantha; Schneekloth, John S

    2013-04-18

    The dynamic, posttranslational modification of proteins with a small ubiquitin-like modifier (SUMO) tag has been recognized as an important cellular regulatory mechanism relevant to a number of cancers as well as normal embryonic development. As part of a program aimed toward the identification of inhibitors of SUMO-conjugating enzymes, we developed a microfluidic electrophoretic mobility shift assay to monitor sumoylation events in real time. We disclose herein the use of this assay to identify a cell-permeable compound capable of blocking the transfer of SUMO-1 from the E2 enzyme Ubc9 to the substrate. We screened a small collection of compounds and identified an oxygenated flavonoid derivative that inhibits sumoylation in vitro. Next, we carried out an in-depth mechanistic analysis that ruled out many common false-positive mechanisms such as aggregation or alkylation. Furthermore, we report that this flavonoid inhibits a single step in the sumoylation cascade: the transfer of SUMO from the E2 enzyme (Ubc9) thioester conjugate to the substrate. In addition to having a unique mechanism of action, this inhibitor has a discrete structure-activity relationship uncharacteristic of a promiscuous inhibitor. Cell-based studies showed that the flavonoid inhibits the sumoylation of topoisomerase-I in response to camptothecin treatment in two different breast cancer cell lines, while isomeric analogs are inactive. Importantly, this compound blocks sumoylation while not affecting ubiquitylation in cells. This work identifies a point of entry for pharmacologic inhibition of the sumoylation cascade and may serve as the basis for continued study of additional pharmacophores that modulate SUMO-conjugating enzymes such as Ubc9. PMID:23601649

  12. Electrophoretic Transport of Single DNA Nucleotides through Nanoslits: A Molecular Dynamics Simulation Study.

    PubMed

    Xia, Kai; Novak, Brian R; Weerakoon-Ratnayake, Kumuditha M; Soper, Steven A; Nikitopoulos, Dimitris E; Moldovan, Dorel

    2015-09-01

    There is potential for flight time based DNA sequencing involving disassembly into individual nucleotides which would pass through a nanochannel with two or more detectors. We performed molecular dynamics simulations of electrophoretic motion of single DNA nucleotides through 3 nm wide hydrophobic slits with both smooth and rough walls. The electric field (E) varied from 0.0 to 0.6 V/nm. The nucleotides adsorb and desorb from walls multiple times during their transit through the slit. The nucleotide-wall interactions differed due to nucleotide hydrophobicities and wall roughness which determined duration and frequency of nucleotide adsorptions and their velocities while adsorbed. Transient association of nucleotides with one, two, or three sodium ions occurred, but the mean association numbers (ANs) were weak functions of nucleotide type. Nucleotide-wall interactions contributed more to separation of nucleotide flight time distributions than ion association and thus indicate that nucleotide-wall interactions play a defining role in successfully discriminating between nucleotides on the basis of their flight times through nanochannels/slits. With smooth walls, smaller nucleotides moved faster, but with rough walls larger nucleotides moved faster due to fewer favorable wall adsorption sites. This indicates that roughness, or surface patterning, might be exploited to achieve better time-of-flight based discrimination between nucleotides. PMID:26237155

  13. Solubilization of Minerals by Bacteria: Electrophoretic Mobility of Thiobacillus ferrooxidans in the Presence of Iron, Pyrite, and Sulfur

    PubMed Central

    Blake, Robert C.; Shute, Elizabeth A.; Howard, Gary T.

    1994-01-01

    Thiobacillus ferroxidans is an obligate acidophile that respires aerobically on pyrite, elemental sulfur, or soluble ferrous ions. The electrophoretic mobility of the bacterium was determined by laser Doppler velocimetry under physiological conditions. When grown on pyrite or ferrous ions, washed cells were negatively charged at pH 2.0. The density of the negative charge depended on whether the conjugate base was sulfate, perchlorate, chloride, or nitrate. The addition of ferric ions shifted the net charge on the surface asymptotically to a positive value. When grown on elemental sulfur, washed cells were close to their isoelectric point at pH 2.0. Both pyrite and colloidal sulfur were negatively charged under the same conditions. The electrical double layer around the bacterial cells under physiological conditions exerted minimal electrostatic repulsion in possible interactions between the cell and either of its charged insoluble substrates. When Thiobacillus ferrooxidans was mixed with either pyrite or colloidal sulfur at pH 2.0, the mobility spectra of the free components disappeared with time to be replaced with a new colloidal particle whose electrophoretic properties were intermediate between those of the starting components. This new particle had the charge and size properties anticipated for a complex between the bacterium and its insoluble substrates. The utility of such measurements for the study of the interactions of chemolithotrophic bacteria with their insoluble substrates is discussed. Images PMID:16349387

  14. Electrophoretic mobility of concentrated carbon black dispersions in a low-permittivity solvent by optical coherence tomography.

    PubMed

    Patel, Mehul N; Smith, P Griffin; Kim, Jihoon; Milner, Thomas E; Johnston, Keith P

    2010-05-15

    Electrophoretic mobilities of concentrated dispersions of carbon black particles in a low-permittivity solvent were measured using differential-phase optical coherence tomography (DP-OCT). An electrode spacing of only 0.18 mm enables measurement of highly concentrated dispersions up to 1 wt.% of highly absorbing carbon black particles with high electric fields at low potentials. The capabilities of this DP-OCT method, including high sensitivity, high spatial resolution, and strong electric fields, enable enhanced measurement of low electrophoretic mobilities encountered in low-permittivity solvents. The zeta potential of carbon black particles ranged from -24 mV to -12 mV as the concentration of surfactant sodium bis(2-ethyl-1-hexyl)sulfosuccinate (AOT) was increased from 1 mM to 100 mM. A mechanism is presented to explain the electrostatic charging of carbon black particles in terms of the partitioning of the ions between the reverse micelles in the double layer and the surfactant adsorbed on the particle surface, as AOT concentration is varied. PMID:20176365

  15. Liquid phase separation of proteins based on electrophoretic effects in an electrospray setup during sample introduction into a gas-phase electrophoretic mobility molecular analyzer (CE–GEMMA/CE–ES–DMA)

    PubMed Central

    Weiss, Victor U.; Kerul, Lukas; Kallinger, Peter; Szymanski, Wladyslaw W.; Marchetti-Deschmann, Martina; Allmaier, Günter

    2014-01-01

    Nanoparticle characterization is gaining importance in food technology, biotechnology, medicine, and pharmaceutical industry. An instrument to determine particle electrophoretic mobility (EM) diameters in the single-digit to double-digit nanometer range receiving increased attention is the gas-phase electrophoretic mobility molecular analyzer (GEMMA) separating electrophoretically single charged analytes in the gas-phase at ambient pressure. A fused-silica capillary is used for analyte transfer to the gas-phase by means of a nano electrospray (ES) unit. The potential of this capillary to separate analytes electrophoretically in the liquid phase due to different mobilities is, at measurement conditions recommended by the manufacturer, eliminated due to elevated pressure applied for sample introduction. Measurements are carried out upon constant feeding of analytes to the system. Under these conditions, aggregate formation is observed for samples including high amounts of non-volatile components or complex samples. This makes the EM determination of individual species sometimes difficult, if not impossible. With the current study we demonstrate that liquid phase electrophoretic separation of proteins (as exemplary analytes) occurs in the capillary (capillary zone electrophoresis, CE) of the nano ES unit of the GEMMA. This finding was consecutively applied for on-line desalting allowing EM diameter determination of analytes despite a high salt concentration within samples. The present study is to our knowledge the first report on the use of the GEMMA to determine EM diameters of analytes solubilized in the ES incompatible electrolyte solutions by the intended use of electrophoresis (in the liquid phase) during sample delivery. Results demonstrate the proof of concept of such an approach and additionally illustrate the high potential of a future on-line coupling of a capillary electrophoresis to a GEMMA instrument. PMID:25109866

  16. Affinity Capillary Electrophoresis for Selective Control of Electrophoretic Mobility of Sialic Acid Using Lanthanide-Hexadentate Macrocyclic Polyazacarboxylate Complexes.

    PubMed

    Goto, Daiki; Ouchi, Kazuki; Shibukawa, Masami; Saito, Shingo

    2015-01-01

    It is difficult to control the electrophoretic mobility in order to obtain high resolution among saccharides in complex samples. We report herein on a new affinity capillary electrophoresis (ACE) method for an anionic monosaccharide, N-acetylneuraminic acid (Neu5Ac), which is important in terms of pathological diagnosis, using lanthanide-hexadentate macrocyclic polyazacarboxylate complexes (Ln-NOTA) as affinity reagents. It was shown that Ln-NOTA complexes increased the anionic mobility of Neu5Ac by approximately 40% through selective complexation with Neu5Ac. The extent of change in the mobility strongly depended on the type of central metal ion of Ln-NOTA. The stability constant (K) of Lu-NOTA with Neu5Ac was determined by ACE to be log Kb = 3.62 ± 0.04, which is the highest value among artificial receptors for Neu5Ac reported so far. Using this ACE, the Neu5Ac content in a glycoprotein sample, α1-acid glycoprotein (AGP), was determined after acid hydrolysis. Complete separation between Neu5Ac and hydrolysis products was successful by controlling the mobility to determine the concentration of Neu5Ac. PMID:26561258

  17. Patterns of Molecular Variation. II. Associations of Electrophoretic Mobility and Larval Substrate within Species of the DROSOPHILA MULLERI Complex

    PubMed Central

    Richardson, R. H.; Smouse, Peter E.; Richardson, Martha E.

    1977-01-01

    Electromorphic variation among populations of Drosophila mojavensis, D. arizonensis and D. longicornis was examined for seven genetic loci. The average electrophoretic mobility for a population was used as the metric. D. mojavensis and D. arizonensis use larval substrates in different parts of their geographic ranges, while D. longicornis is more narrowly restricted to different species of the cactus Opuntia in different localities. There is marked electromorphic variation among populations of either D. mojavensis or D. arizonensis, and the bulk of this variation is accounted for by differences in laval substrate. There is somewhat less variation among populations of D. longicornis, and only a moderate portion of this is accounted for by larval substrate differences. There appears to be an association between the taxonomic diversity of the larval substrates and the electromorphic diversity of the Drosophila populations utilizing those substrates. Evidence is reviewed that suggests physiological mechanisms for these possibly adaptive associations. PMID:838268

  18. Gel electrophoretic restriction fragment length polymorphism analysis of DNA derived from individual nematodes, using the PhastSystem.

    PubMed

    Triga, D; Pamjav, H; Vellai, T; Fodor, A; Buzás, Z

    1999-06-01

    The DNA sequences constituting the internal transcribed spacer region, located between 18S and 26S rDNA genes within the rRNA operon, derived from single nematodes of two genera (Steinernema and Heterorhabditis) were amplified by polymerase chain reaction (PCR) and subjected to digestion by four restriction enzymes. The digests were analyzed by restriction fragment length polymorphism (RFLP) gel electrophoresis on the PhastSystem, using 7.5%T, 5%C(Bis) polyacrylamide. The downscaling from conventional agarose to PhastSystem gels permitted the analysis to be done on individual nematodes, rather than on mixed samples with average properties. The analysis time was reduced so as to allow for the electrophoretic separation on 200 samples/workday. The resulting patterns of DNA fragments differed from those obtained by agarose gel electrophoresis under conventional conditions by an increased number of detected fragments. The PhastSystem gel analysis provides the basis for taxonomical revisions. PMID:10380768

  19. Gel mobilities of linking-number topoisomers and their dependence on DNA helical repeat and elasticity

    PubMed Central

    Vetcher, Alexandre A.; McEwen, Abbye E.; Abujarour, Ramzey; Hanke, Andreas; Levene, Stephen D.

    2010-01-01

    Agarose-gel electrophoresis has been used for more than thirty years to characterize the linking-number (Lk) distribution of closed-circular DNA molecules. Although the physical basis of this technique remains poorly understood, the gel-electrophoretic behavior of covalently closed DNAs has been used to determine the local unwinding of DNA by proteins and small-molecule ligands, characterize supercoiling-dependent conformational transitions in duplex DNA, and to measure helical-repeat changes due to shifts in temperature and ionic strength. Those results have been analyzed by assuming that the absolute mobility of a particular topoisomer is mainly a function of the integral number of superhelical turns, and thus a slowly varying function of plasmid molecular weight. In examining the mobilities of Lk topoisomers for a series of plasmids that differ incrementally in size over more than one helical turn, we found that the size-dependent agarose-gel mobility of individual topoisomers with identical values of Lk (but different values of the excess linking number, ΔLk) vary dramatically over a duplex turn. Our results suggest that a simple semi-empirical relationship holds between the electrophoretic mobility of linking-number topoisomers and their average writhe in solution. PMID:20346570

  20. Characterization of the cell surface properties of drinking water pathogens by microbial adhesion to hydrocarbon and electrophoretic mobility measurements.

    PubMed

    Popovici, Jonathan; White, Colin P; Hoelle, Jill; Kinkle, Brian K; Lytle, Darren A

    2014-06-01

    The surface characteristics of microbial cells directly influence their mobility and behavior within aqueous environments. The cell surface hydrophobicity (CSH) and electrophoretic mobility (EPM) of microbial cells impact a number of interactions and processes including aggregation, adhesion to surfaces, and stability of the cells within the aqueous environments. These cell characteristics are unique to the bacterial species and are a reflection of the large diversity of surface structures, proteins, and appendages of microorganisms. CSH and EPM of bacterial cells contribute substantially to the effectiveness of drinking water treatment to remove them, and therefore an investigation of these properties will be useful in predicting their removal through drinking water treatment processes and transport through drinking water distribution systems. EPM and CSH measurements of six microbiological pathogen or surrogate species suspended in phosphate-buffered water are reported in this work. Two strains of Vibrio cholerae were hydrophobic, while three strains of Escherichia coli were hydrophilic. Bacillus cereus was categorized as moderately hydrophobic. The strains of E. coli had the highest (most negative) EPM. Based on the measurements, E. coli species is predicted to be most difficult to remove from water while V. cholerae will be the easiest to remove. PMID:24815929

  1. A 502-Base Free-Solution Electrophoretic DNA Sequencing Method Using End-Attached Wormlike Micelles.

    PubMed

    Istivan, Stephen B; Bishop, Daniel K; Jones, Angela L; Grosser, Shane T; Schneider, James W

    2015-11-17

    We demonstrate that the use of wormlike nonionic micelles as drag-tags in end-labeled free-solution electrophoresis ("micelle-ELFSE") provides single-base resolution of Sanger sequencing products up to 502 bases in length, a nearly 2-fold improvement over reported ELFSE separations. "CiEj" running buffers containing 48 mM C12E5, 6 mM C10E5, and 3 M urea (32.5 °C) form wormlike micelles that provide a drag equivalent to an uncharged DNA fragment with a length (α) of 509 bases (effective Rh = 27 nm). Runtime in a 40 cm capillary (30 kV) was 35 min for elution of all products down to the 26-base primer. We also show that smaller Triton X-100 micelles give a read length of 103 bases in a 4 min run, so that a combined analysis of the Sanger products using the two buffers in separate capillaries could be completed in 14 min for the full range of lengths. A van Deemter analysis shows that resolution is limited by diffusion-based peak broadening and wall adsorption. Effects of drag-tag polydispersity are not observed, despite the inherent polydispersity of the wormlike micelles. We ascribe this to a stochastic size-sampling process that occurs as micelle size fluctuates rapidly during the runtime. A theoretical model of the process suggests that fluctuations occur with a time scale less than 10 ms, consistent with the monomer exchange process in nonionic micelles. The CiEj buffer has a low viscosity (2.7 cP) and appears to be semidilute in micelle concentration. The large drag-tag size of the CiEj buffers leads to steric segregation of the DNA and tag for short fragments and attendant mobility shifts. PMID:26455271

  2. Determination of electrophoretic mobilities and hydrodynamic radii of three humic substances as a function of pH and ionic strength.

    PubMed

    Hosse, M; Wilkinson, K J

    2001-11-01

    Capillary electrophoresis (CE) and fluorescence correlation spectroscopy (FCS) were employed to determine electrophoretic mobilities and hydrodynamic sizes of three humic substances (IHSS aquatic fulvic acid (FA), IHSS aquatic humic acid (HA), and IHSS peat humic acid (PHA)) as a function of pH and ionic strength. A slight aggregation corresponding to the formation of dimers and trimers was observed at low pH using fluorescence correlation spectroscopy (FCS). For example, for the peat humic acid, diffusion coefficients decreased from 2.1 x 10(-10) m2 s(-1) at pH 4 to 2.4 x 10(-10) m2 s(-1) at pH 11. For all three humic substances, electrophoretic mobilities were also shown to decrease significantly below pH 6. Calculated zeta potentials observed at high pH of -69 mV (FA), -62 mV (HA), and -63 mV (PHA) decreased to -39, -50, and -47 mV, respectively, under slightly acidic pH (4.5-4.8) conditions. No evidence of ionic strength induced aggregation was found using fluorescence correlation spectroscopy (FCS); diffusion coefficients increased slightly (<25%) with increasing ionic strength (up to 1 M). Negative electrophoretic mobilities decreased to a maximum measured ionic strength of 0.18 M. Above this ionic strength, no peaks were observed due to an increased HS adsorption to the capillary wall and an important decrease in electroosmotic flow. Interpretation of electrophoretic mobilities determined by CE is complicated by the fact that under certain conditions, HS appeared to be complexed by CE buffer systems, including MES, BES, and AMPSO. PMID:11718346

  3. Understanding the poor iontophoretic transport of lysozyme across the skin: when high charge and high electrophoretic mobility are not enough.

    PubMed

    Dubey, S; Kalia, Y N

    2014-06-10

    The original aim of the study was to investigate the transdermal iontophoretic delivery of lysozyme and to gain further insight into the factors controlling protein electrotransport. Initial experiments were done using porcine skin. Lysozyme transport was quantified by using an activity assay based on the lysis of Micrococcus lysodeikticus and was corrected for the release of endogenous enzyme from the skin during current application. Cumulative iontophoretic permeation of lysozyme during 8h at 0.5mA/cm(2) (0.7mM; pH6) was surprisingly low (5.37±3.46μg/cm(2) in 8h) as compared to electrotransport of cytochrome c (Cyt c) and ribonuclease A (RNase A) under similar conditions (923.0±496.1 and 170.71±92.13μg/cm(2), respectively) - despite its having a higher electrophoretic mobility. The focus of the study then became to understand and explain the causes of its poor iontophoretic transport. Lowering formulation pH to 5 increased histidine protonation in the protein and decreased the ionisation of fixed negative charges in the skin (pI ~4.5) and resulted in a small but statistically significant increase in permeation. Co-iontophoresis of acetaminophen revealed a significant inhibition of electroosmosis; inhibition factors of 12-16 were indicative of strong lysozyme binding to skin. Intriguingly, lidocaine electrotransport, which is due almost exclusively to electromigration, was also decreased (approximately 2.7-fold) following skin pre-treatment by lysozyme iontophoresis (cf. iontophoresis of buffer solution) - suggesting that lysozyme was also able to influence subsequent cation electromigration. In order to elucidate the site of skin binding, different porcine skin models were tested (dermatomed skin with thicknesses of 250 and 750μm, tape-stripped skin and heat-separated dermis). Although no difference was seen between permeation across 250 and 750μm dermatomed skin (13.57±12.20 and 5.37±3.46μg/cm(2), respectively), there was a statistically significant

  4. Alpha chain hemoglobins with electrophoretic mobility similar to that of hemoglobin S in a newborn screening program

    PubMed Central

    Silva, Marcilene Rezende; Sendin, Shimene Mascarenhas; Araujo, Isabela Couto de Oliveira; Pimentel, Fernanda Silva; Viana, Marcos Borato

    2013-01-01

    Objective To characterize alpha-chain variant hemoglobins with electric mobility similar to that of hemoglobin S in a newborn screening program. Methods βS allele and alpha-thalassemia deletions were investigated in 14 children who had undefined hemoglobin at birth and an electrophoretic profile similar to that of hemoglobin S when they were six months old. Gene sequencing and restriction enzymes (DdeI, BsaJI, NlaIV, Bsu36I and TaqI) were used to identify hemoglobins. Clinical and hematological data were obtained from children who attended scheduled medical visits. Results The following alpha chain variants were found: seven children with hemoglobin Hasharon [alpha2 47(CE5) Asp>His, HbA2:c.142G>C], all associated with alpha-thalassemia, five with hemoglobin Ottawa [alpha1 15(A13) Gly>Arg, HBA1:c.46G>C], one with hemoglobin St Luke's [alpha1 95(G2) Pro>Arg, HBA1:c.287C>G] and another one with hemoglobin Etobicoke [alpha212 84(F5) Ser>Arg, HBA212:c.255C>G]. Two associations with hemoglobin S were found: one with hemoglobin Ottawa and one with hemoglobin St Luke's. The mutation underlying hemoglobin Etobicoke was located in a hybrid α212 allele in one child. There was no evidence of clinically relevant hemoglobins detected in this study. Conclusion Apparently these are the first cases of hemoglobin Ottawa, St Luke's, Etobicoke and the α212 gene described in Brazil. The hemoglobins detected in this study may lead to false diagnosis of sickle cell trait or sickle cell disease when only isoelectric focusing is used in neonatal screening. Additional tests are necessary for the correct identification of hemoglobin variants. PMID:23741188

  5. Capillary electrophoretic separation of DNA restriction fragments using dilute polymer solutions

    SciTech Connect

    Braun, B.; Blanch, W.; Prausnitz, J.M.

    1997-02-01

    Because the mechanism of DNA separation in capillary electrophoresis is not well understood, selection of polymers is a {open_quotes}trial-and-error{close_quotes} procedure. We investigated dilute-solution DNA separations by capillary electrophoresis using solutions of four polymers that differ in size, shape and stiffness. Hydroxyethylcellulose of high molecular weight provides excellent separation of large DNA fragments (2027 bp - 23130 bp). Polyvinylpyrrolidone separates DNA from 72 bp to 23 kbp and star-(polyethylene oxide), like linear poly (ethylene oxide), provides separation of fragments up to 1353 bp.

  6. Simulation guided design of a microfluidic device for electrophoretic stretching of DNA.

    PubMed

    Hsieh, Chih-Chen; Lin, Tsung-Hsien; Huang, Chiou-De

    2012-01-01

    We have used Brownian dynamics-finite element method (BD-FEM) to guide the optimization of a microfluidic device designed to stretch DNA for gene mapping. The original design was proposed in our previous study [C. C. Hsieh and T. H. Lin, Biomicrofluidics 5(4), 044106 (2011)] for demonstrating a new pre-conditioning strategy to facilitate DNA stretching through a microcontraction using electrophoresis. In this study, we examine the efficiency of the original device for stretching DNA with different sizes ranging from 48.5 kbp (λ-DNA) to 166 kbp (T4-DNA). The efficiency of the device is found to deteriorate with increasing DNA molecular weight. The cause of the efficiency loss is determined by BD-FEM, and a modified design is proposed by drawing an analogy between an electric field and a potential flow. The modified device does not only regain the efficiency for stretching large DNA but also outperforms the original device for stretching small DNA. PMID:24155866

  7. DNA Electrophoretic Migration Patterns Change after Exposure of Jurkat Cells to a Single Intense Nanosecond Electric Pulse

    PubMed Central

    Romeo, Stefania; Zeni, Luigi; Sarti, Maurizio; Sannino, Anna; Scarfì, Maria Rosaria; Vernier, P. Thomas; Zeni, Olga

    2011-01-01

    Intense nanosecond pulsed electric fields (nsPEFs) interact with cellular membranes and intracellular structures. Investigating how cells respond to nanosecond pulses is essential for a) development of biomedical applications of nsPEFs, including cancer therapy, and b) better understanding of the mechanisms underlying such bioelectrical effects. In this work, we explored relatively mild exposure conditions to provide insight into weak, reversible effects, laying a foundation for a better understanding of the interaction mechanisms and kinetics underlying nsPEF bio-effects. In particular, we report changes in the nucleus of Jurkat cells (human lymphoblastoid T cells) exposed to single pulses of 60 ns duration and 1.0, 1.5 and 2.5 MV/m amplitudes, which do not affect cell growth and viability. A dose-dependent reduction in alkaline comet-assayed DNA migration is observed immediately after nsPEF exposure, accompanied by permeabilization of the plasma membrane (YO-PRO-1 uptake). Comet assay profiles return to normal within 60 minutes after pulse delivery at the highest pulse amplitude tested, indicating that our exposure protocol affects the nucleus, modifying DNA electrophoretic migration patterns. PMID:22164287

  8. Sequence Dependent Electrophoretic Separations of DNA in Pluronic F127 Gels

    NASA Astrophysics Data System (ADS)

    You, Seungyong; van Winkle, David H.

    2010-03-01

    Two-dimensional (2-D) electrophoresis has successfully been used to visualize the separation of DNA fragments of the same length. We electrophorese a double-stranded DNA ladder in an Agarose gel for the first dimension and in gels of Pluronic F127 for the second dimension at room temperature. The 1000 bp band that travels together as a single band in an Agarose gel is split into two bands in Pluronic gels. The slower band follows the exponential decay trend that the other ladder constituents do. After sequencing the DNA fragments, the faster band has an apparently random sequence, while the slower band and the others have two A-tracts in each 250 bp segment. The A-tracts consist of a series of at least five adenine bases pairing with thymine bases. This result leads to the conclusion that the migration of the DNA molecules bent with A-tracts is more retarded in Pluronic gels than the wild-type of DNA molecules.

  9. Electrophoretic detection and separation of mutant DNA using replaceable polymer matrices

    DOEpatents

    Karger, B.L.; Thilly, W.G.; Foret, F.; Khrapko, K.; Koehavong, P.; Cohen, A.S.; Giese, R.W.

    1997-05-27

    The disclosure relates to a method for resolving double-stranded DNA species differing by at least one base pair. Each of the species is characterized by an iso-melting domain with a unique melting temperature contiguous with a melting domain of higher thermal stability. 18 figs.

  10. Electrophoretic detection and separation of mutant DNA using replaceable polymer matrices

    DOEpatents

    Karger, Barry L.; Thilly, William G.; Foret, Frantisek; Khrapko, Konstaintin; Koehavong, Phouthone; Cohen, Aharon S.; Giese, Roger W.

    1997-01-01

    The disclosure relates to a method for resolving double-stranded DNA species differing by at least one base pair. Each of the species is characterized by an iso-melting domain with a unique melting temperature contiguous with a melting domain of higher thermal stability.

  11. Impact of chemical and structural anisotropy on the electrophoretic mobility of spherical soft multilayer particles: the case of bacteriophage MS2.

    PubMed

    Langlet, Jérémie; Gaboriaud, Fabien; Gantzer, Christophe; Duval, Jérôme F L

    2008-04-15

    We report a theoretical investigation of the electrohydrodynamic properties of spherical soft particles composed of permeable concentric layers that differ in thickness, soft material density, chemical composition, and flow penetration degree. Starting from a recent numerical scheme developed for the computation of the direct-current electrophoretic mobility (mu) of diffuse soft bioparticles, the dependence of mu on the electrolyte concentration and solution pH is evaluated taking the known three-layered structure of bacteriophage MS2 as a supporting model system (bulk RNA, RNA-protein bound layer, and coat protein). The electrokinetic results are discussed for various layer thicknesses, hydrodynamic flow penetration degrees, and chemical compositions, and are discussed on the basis of the equilibrium electrostatic potential and hydrodynamic flow field profiles that develop within and around the structured particle. This study allows for identifying the cases where the electrophoretic mobility is a function of the inner structural and chemical specificity of the particle and not only of its outer surface properties. Along these lines, we demonstrate the general inapplicability of the notions of zeta potential (zeta) and surface charge for quantitatively interpreting electrokinetic data collected for such systems. We further shed some light on the physical meaning of the isoelectric point. In particular, numerical and analytical simulations performed on structured soft layers in indifferent electrolytic solution demonstrate that the isoelectric point is a complex ionic strength-dependent signature of the flow permeation properties and of the chemical and structural details of the particle. Finally, the electrophoretic mobilities of the MS2 virus measured at various ionic strength levels and pH values are interpreted on the basis of the theoretical formalism aforementioned. It is shown that the electrokinetic features of MS2 are to a large extent determined not only

  12. Impact of Chemical and Structural Anisotropy on the Electrophoretic Mobility of Spherical Soft Multilayer Particles: The Case of Bacteriophage MS2

    PubMed Central

    Langlet, Jérémie; Gaboriaud, Fabien; Gantzer, Christophe; Duval, Jérôme F. L.

    2008-01-01

    We report a theoretical investigation of the electrohydrodynamic properties of spherical soft particles composed of permeable concentric layers that differ in thickness, soft material density, chemical composition, and flow penetration degree. Starting from a recent numerical scheme developed for the computation of the direct-current electrophoretic mobility (μ) of diffuse soft bioparticles, the dependence of μ on the electrolyte concentration and solution pH is evaluated taking the known three-layered structure of bacteriophage MS2 as a supporting model system (bulk RNA, RNA-protein bound layer, and coat protein). The electrokinetic results are discussed for various layer thicknesses, hydrodynamic flow penetration degrees, and chemical compositions, and are discussed on the basis of the equilibrium electrostatic potential and hydrodynamic flow field profiles that develop within and around the structured particle. This study allows for identifying the cases where the electrophoretic mobility is a function of the inner structural and chemical specificity of the particle and not only of its outer surface properties. Along these lines, we demonstrate the general inapplicability of the notions of zeta potential (ζ) and surface charge for quantitatively interpreting electrokinetic data collected for such systems. We further shed some light on the physical meaning of the isoelectric point. In particular, numerical and analytical simulations performed on structured soft layers in indifferent electrolytic solution demonstrate that the isoelectric point is a complex ionic strength-dependent signature of the flow permeation properties and of the chemical and structural details of the particle. Finally, the electrophoretic mobilities of the MS2 virus measured at various ionic strength levels and pH values are interpreted on the basis of the theoretical formalism aforementioned. It is shown that the electrokinetic features of MS2 are to a large extent determined not only by

  13. A simple method for assessment and minimization of errors in determination of electrophoretic or electroosmotic mobilities and velocities associated with the axial electric field distortion.

    PubMed

    Nowak, Paweł Mateusz; Woźniakiewicz, Michał; Kościelniak, Paweł

    2015-12-01

    It is commonly accepted that the modern CE instruments equipped with efficient cooling system enable accurate determination of electrophoretic or electroosmotic mobilities. It is also often assumed that velocity of migration in a given buffer is constant throughout the capillary length. It is simultaneously neglected that the noncooled parts of capillary produce extensive Joule heating leading to an axial electric field distortion, which contributes to a difference between the effective and nominal electric field potentials and between velocities in the cooled and noncooled parts of capillary. This simplification introduces systematic errors, which so far were however not investigated experimentally. There was also no method proposed for their elimination. We show a simple and fast method allowing for estimation and elimination of these errors that is based on combination of a long-end and short-end injections. We use it to study the effects caused by variation of temperature, electric field, capillary length, and pH. PMID:26383237

  14. Anti-epileptic drugs and bone loss: Phenytoin reduces pro-collagen I and alters the electrophoretic mobility of osteonectin in cultured bone cells.

    PubMed

    Wilson, Emma L; Garton, Mark; Fuller, Heidi R

    2016-05-01

    Phenytoin is an antiepileptic drug used in the management of partial and tonic-clonic seizures. In previous studies we have shown that valproate, another antiepileptic drug, reduced the amount of two key bone proteins, pro-collagen I and osteonectin (SPARC, BM-40), in both skin fibroblasts and cultured osteoblast-like cells. Here we show that phenytoin also reduces pro-collagen I production in osteoblast-like cells, but does not appear to cause a decrease in osteonectin message or protein production. Instead, a 24h exposure to a clinically relevant concentration of phenytoin resulted in a dose-dependent change in electrophoretic mobility of osteonectin, which was suggestive of a change in post-translational modification status. The perturbation of these important bone proteins could be one of the mechanisms to explain the bone loss that has been reported following long-term treatment with phenytoin. PMID:26999801

  15. Measurement of Electrophoretic Mobility of Human Promyelocytic Leukemia Cell Lines (HL60) During Neutrophil Differentiation Using On-Chip Cell Electrophoresis

    NASA Astrophysics Data System (ADS)

    Matsuhashi, Ryutaro; Akagi, Takanori; Ichiki, Takanori

    Electrophoretic mobility (EPM) of human promyelocytic leukemia cell lines (HL60) during neutrophil differentiation induced by all-trans retinoic acid (ATRA) or dimethyl sulfoxide (DMSO) was measured using microcapillary electrophoresis chips. Prior to EPM measurement of HL60 cells, neutrophil differentiation of the cells was confirmed by morphological classification. Subsequently, EPM of HL60 cells was measured using an on-chip cell electrophoresis system before and after neutrophil differentiation. The EPM changed gradually with the progress of the neutrophil differentiation. From the analysis of experimental data by principal component analysis, it was revealed that there is a strong correlation between morphologic classification and EPM during the neutrophilic differentiation. The present result suggests that on-chip EPM measurement system can be used as a monitoring tool for the cell differentiation.

  16. Comprehensive size-determination of whole virus vaccine particles using gas-phase electrophoretic mobility macromolecular analyzer, atomic force microscopy, and transmission electron microscopy.

    PubMed

    Havlik, Marlene; Marchetti-Deschmann, Martina; Friedbacher, Gernot; Winkler, Wolfgang; Messner, Paul; Perez-Burgos, Laura; Tauer, Christa; Allmaier, Günter

    2015-09-01

    Biophysical properties including particle size distribution, integrity, and shape of whole virus vaccine particles at different stages in tick-borne encephalitis (TBE) vaccines formulation were analyzed by a new set of methods. Size-exclusion chromatography (SEC) was used as a conservative sample preparation for vaccine particle fractionation and gas-phase electrophoretic mobility macromolecular analyzer (GEMMA) for analyzing electrophoretic mobility diameters of isolated TBE virions. The derived particle diameter was then correlated with molecular weight. The diameter of the TBE virions determined after SEC by GEMMA instrumentation was 46.8 ± 1.1 nm. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) were implemented for comparison purposes and to gain morphological information on the virion particle. Western blotting (Dot Blot) as an immunological method confirmed biological activity of the particles at various stages of the developed analytical strategy. AFM and TEM measurements revealed higher diameters with much higher SD for a limited number of virions, 60.4 ± 8.5 and 53.5 ± 5.3 nm, respectively. GEMMA instrumentation was also used for fractionation of virions with specifically selected diameters in the gas-phase, which were finally collected by means of an electrostatic sampler. At that point (i.e., after particle collection), AFM and TEM showed that the sampled virions were still intact, exhibiting a narrow size distribution (i.e., 59.8 ± 7.8 nm for AFM and 47.5 ± 5.2 nm for TEM images), and most importantly, dot blotting confirmed immunological activity of the collected samples. Furthermore dimers and virion artifacts were detected, too. PMID:26266988

  17. Comprehensive Size-Determination of Whole Virus Vaccine Particles Using Gas-Phase Electrophoretic Mobility Macromolecular Analyzer, Atomic Force Microscopy, and Transmission Electron Microscopy

    PubMed Central

    Havlik, Marlene; Marchetti-Deschmann, Martina; Friedbacher, Gernot; Winkler, Wolfgang; Messner, Paul; Perez-Burgos, Laura; Tauer, Christa; Allmaier, Günter

    2015-01-01

    Biophysical properties including particle size distribution, integrity, and shape of whole virus vaccine particles at different stages in tick-borne encephalitis (TBE) vaccines formulation were analyzed by a new set of methods. Size-exclusion chromatography (SEC) was used as a conservative sample preparation for vaccine particle fractionation and gas-phase electrophoretic mobility macromolecular analyzer (GEMMA) for analyzing electrophoretic mobility diameters of isolated TBE virions. The derived particle diameter was then correlated with molecular weight. The diameter of the TBE virions determined after SEC by GEMMA instrumentation was 46.8 ± 1.1 nm. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) were implemented for comparison purposes and to gain morphological information on the virion particle. Western blotting (Dot Blot) as an immunological method confirmed biological activity of the particles at various stages of the developed analytical strategy. AFM and TEM measurements revealed higher diameters with much higher SD for a limited number of virions, 60.4 ± 8.5 and 53.5 ± 5.3 nm, respectively. GEMMA instrumentation was also used for fractionation of virions with specifically selected diameters in the gas-phase, which were finally collected by means of an electrostatic sampler. At that point (i.e., after particle collection), AFM and TEM showed that the sampled virions were still intact, exhibiting a narrow size distribution (i.e., 59.8 ± 7.8 nm for AFM and 47.5 ± 5.2 nm for TEM images), and most importantly, dot blotting confirmed immunological activity of the collected samples. Furthermore dimers and virion artifacts were detected, too. PMID:26266988

  18. Analysis of a Common Cold Virus and Its Subviral Particles by Gas-Phase Electrophoretic Mobility Molecular Analysis and Native Mass Spectrometry

    PubMed Central

    2015-01-01

    Gas-phase electrophoretic mobility molecular analysis (GEMMA) separates nanometer-sized, single-charged particles according to their electrophoretic mobility (EM) diameter after transition to the gas-phase via a nano electrospray process. Electrospraying as a soft desorption/ionization technique preserves noncovalent biospecific interactions. GEMMA is therefore well suited for the analysis of intact viruses and subviral particles targeting questions related to particle size, bioaffinity, and purity of preparations. By correlating the EM diameter to the molecular mass (Mr) of standards, the Mr of analytes can be determined. Here, we demonstrate (i) the use of GEMMA in purity assessment of a preparation of a common cold virus (human rhinovirus serotype 2, HRV-A2) and (ii) the analysis of subviral HRV-A2 particles derived from such a preparation. (iii) Likewise, native mass spectrometry was employed to obtain spectra of intact HRV-A2 virions and empty viral capsids (B-particles). Charge state resolution for the latter allowed its Mr determination. (iv) Cumulatively, the data measured and published earlier were used to establish a correlation between the Mr and EM diameter for a range of globular proteins and the intact virions. Although a good correlation resulted from this analysis, we noticed a discrepancy especially for the empty and subviral particles. This demonstrates the influence of genome encapsulation (preventing analytes from shrinking upon transition into the gas-phase) on the measured analyte EM diameter. To conclude, GEMMA is useful for the determination of the Mr of intact viruses but needs to be employed with caution when subviral particles or even empty viral capsids are targeted. The latter could be analyzed by native MS. PMID:26221912

  19. Electrophoretic cell separation by means of microspheres

    NASA Technical Reports Server (NTRS)

    Smolka, A. J. K.; Nerren, B. H.; Margel, S.; Rembaum, A.

    1979-01-01

    The electrophoretic mobility of fixed human erythrocytes immunologically labeled with poly(vinylpyridine) or poly(glutaraldehyde) microspheres was reduced by approximately 40%. This observation was utilized in preparative scale electrophoretic separations of fixed human and turkey erythrocytes, the mobilities of which under normal physiological conditions do not differ sufficiently to allow their separation by continuous flow electrophoresis. We suggest that resolution in the electrophoretic separation of cell subpopulations, currently limited by finite and often overlapping mobility distributions, may be significantly enhanced by immunospecific labeling of target populations using microspheres.

  20. Electrophoretic transport of biomolecules across liquid-liquid interfaces.

    PubMed

    Hahn, Thomas; Münchow, Götz; Hardt, Steffen

    2011-05-11

    The mass transfer resistance of a liquid-liquid interface in an aqueous two-phase system composed of poly(ethylene glycol) and dextran is investigated. Different types of proteins and DNA stained with fluorescent dyes serve as probes to study the transport processes close to the interface. A microfluidic device is employed to enable the electrophoretic transport of biomolecules from one phase to another. The results obtained for proteins can be explained solely via the different electrophoretic mobilities and different affinities of the molecules to the two phases, without any indications of a significant mass transfer resistance of the liquid-liquid interface. By contrast, DNA molecules adsorb to the interface and only desorb under an increased electric field strength. The desorption process carries the signature of a thermally activated escape from a metastable state, as reflected in the exponential decay of the fluorescence intensity at the interface as a function of time. PMID:21508474

  1. Assessment of DNA-binding affinity of cholinesterase reactivators and electrophoretic determination of their effect on topoisomerase I and II activity.

    PubMed

    Janockova, J; Zilecka, E; Kasparkova, J; Brabec, V; Soukup, O; Kuca, K; Kozurkova, M

    2016-08-16

    In this paper, we describe the biochemical properties and biological activity of a series of cholinesterase reactivators (symmetrical bisquaternary xylene-linked compounds, K106-K114) with ctDNA. The interaction of the studied derivatives with ctDNA was investigated using UV-Vis, fluorescence, CD and LD spectrometry, and electrophoretic and viscometric methods. The binding constants K were estimated to be in the range 1.05 × 10(5)-5.14 × 10(6) M(-1) and the percentage of hypochromism was found to be 10.64-19.28% (from UV-Vis titration). The used methods indicate that the studied samples are groove binders. Electrophoretic methods proved that the studied compounds clearly influence calf thymus Topo I (at 5 μM concentration, except for compounds K107, K111 and K114 which were effective at higher concentrations) and human Topo II (K110 partially inhibited Topo II effects even at 5 μM concentration) activity. PMID:27412811

  2. Fluctuations of DNA mobility in nanofluidic entropic traps

    PubMed Central

    Wu, Lingling; Levy, Stephen

    2014-01-01

    We studied the mobility of DNA molecules driven by an electric field through a nanofluidic device containing a periodic array of deep and shallow regions termed entropic traps. The mobility of a group of DNA molecules was measured by fluorescent video microscopy. Since the depth of a shallow region is smaller than the DNA equilibrium size, DNA molecules are trapped for a characteristic time and must compress themselves to traverse the boundary between deep and shallow regions. Consistent with previous experimental results, we observed a nonlinear relationship between the mobility and electric field strength, and that longer DNA molecules have larger mobility. In repeated measurements under seemingly identical conditions, we measured fluctuations in the mobility significantly larger than expected from statistical variation. The variation was more pronounced for lower electric field strengths where the trapping time is considerable relative to the drift time. To determine the origin of these fluctuations, we investigated the dependence of the mobility on several variables: DNA concentration, ionic strength of the solvent, fluorescent dye staining ratio, electroosmotic flow, and electric field strength. The mobility fluctuations were moderately enhanced in conditions of reduced ionic strength and electroosmotic flow. PMID:25379088

  3. An integrated electrophoretic mobility control device with split design for signal improvement in liquid chromatography-electrospray ionization mass spectrometry analysis of aminoglycosides using a heptafluorobutyric acid containing mobile phase.

    PubMed

    Hung, Sih-Hua; Yu, Meng-Ju; Wang, Nan-Hsuan; Hsu, Ren-Yu; Wei, Guor-Jien; Her, Guor-Rong

    2016-08-24

    Electrophoretic mobility control (EMC) was used to alleviate the adverse effect of the ion-pairing agent heptafluorobutyric acid (HFBA) in the liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) analysis of aminoglycosides. Aminoglycosides separated by LC were directed to a connecting column before their detection via ESI. Applying an electric field across the connecting column caused the positively charged aminoglycosides to migrate toward the mass spectrometer whereas the HFBA anions remained in the junction reservoir, thus alleviating the ion suppression caused by HFBA. To accommodate the flow rate of a narrow-bore column, minimize the effect of electrophoretic mobility on separation, and facilitate the operation, an integrated EMC device with a split design was fabricated. With the proposed EMC device, the signals of aminoglycosides were enhanced by a factor of 5-85 without affecting the separation efficiency or elution order. For the analysis of aminoglycosides in bovine milk, the proposed approach demonstrates a sensitivity that is at least 10 times below the maximum residue limits set by most countries. PMID:27497008

  4. Mobile DNA can drive lineage extinction in prokaryotic populations.

    PubMed

    Rankin, D J; Bichsel, M; Wagner, A

    2010-11-01

    Natural selection ultimately acts on genes and other DNA sequences. Adaptations that are good for the gene can have adverse effects at higher levels of organization, including the individual or the population. Mobile genetic elements illustrate this principle well, because they can self-replicate within a genome at a cost to their host. As they are costly and can be transmitted horizontally, mobile elements can be seen as genomic parasites. It has been suggested that mobile elements may cause the extinction of their host populations. In organisms with very large populations, such as most bacteria, individual selection is highly effective in purging genomes of deleterious elements, suggesting that extinction is unlikely. Here we investigate the conditions under which mobile DNA can drive bacterial lineages to extinction. We use a range of epidemiological and ecological models to show that harmful mobile DNA can invade, and drive populations to extinction, provided their transmission rate is high and that mobile element-induced mortality is not too high. Population extinction becomes more likely when there are more elements in the population. Even if elements are costly, extinction can still occur because of the combined effect of horizontal gene transfer, a mortality induced by mobile elements. Our study highlights the potential of mobile DNA to be selected at the population level, as well as at the individual level. PMID:20860700

  5. The pH dependence of predictive models relating electrophoretic mobility to peptide chemico-physical properties in capillary zone electrophoresis.

    PubMed

    Castagnola, M; Rossetti, D V; Corda, M; Pellegrini, M; Misiti, F; Olianas, A; Giardina, B; Messana, I

    1998-10-01

    We applied best fitting procedures to capillary electrophoresis (CE) mobility values, measured at varying acidic pH, of a set of 21 peptides with a molecular mass ranging from about 350 to 1850 Da. This method allowed the contemporary measurements of C-terminus and carboxylic group of the side-chain of aspartic and glutamic acid dissociation constants and of peptide Stokes radius at different protonation stages. Stokes radius was related to peptide molecular mass M at the power of a fractional coefficient, and best correlation was found at pH 2.25, the fractional coefficient being equal to 0.68. This value is close to that proposed by R. E. Offord (Nature 1966, 211, 591-593), who suggested a proportionality between the polymer Stokes radius and M(2/3). The coefficient value decreases at higher pH, reaching a value of 0.58 at pH 4.25, corresponding to a mean peptide conformational transition towards more compact structures as a consequence of C-terminus dissociation. The measurement of the dissociation constants of each peptide allowed us to determine the percentage error on peptide charge predictions performed utilizing mean dissociation constants. Even for the charge, the best predictive performance is obtained at the most acidic edge of the range of the pH studied, mainly at pH 2.25. Conclusively, this study shows that the best performance of predictive models for peptide CE mobility is obtainable in the very acidic pH range (2.25-2.50) and in the absence of electroosmotic flow, and that a satisfactory predictive equation of peptide electrophoretic mobility (m2V(-1)s(-1) is given by mu = 85.4(Z/M(0.68))10(-8). PMID:9788308

  6. Relatedness Analyses of Histoplasma capsulatum Isolates from Mexican Patients with AIDS-Associated Histoplasmosis by Using Histoplasmin Electrophoretic Profiles and Randomly Amplified Polymorphic DNA Patterns

    PubMed Central

    Reyes-Montes, M. R.; Bobadilla-Del Valle, M.; Martínez-Rivera, M. A.; Rodríguez-Arellanes, G.; Maravilla, E.; Sifuentes-Osornio, J.; Taylor, M. L.

    1999-01-01

    The present paper analyzes the histoplasmin electrophoretic profiles and the randomly amplified polymorphic DNA (RAPD) patterns of the fungus Histoplasma capsulatum isolated from Mexican patients with AIDS-associated histoplasmosis. Clinical isolates from Guatemala, Colombia, and Panama, as well as H. capsulatum isolates from different sources in nature, were also processed. All histoplasmin samples shared four antigenic fractions of 200, 49, 10.5, and 8.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). According to their percentage of relatedness, based on SDS-PAGE histoplasmin electrophoretic image analysis, H. capsulatum isolates were divided in two groups: group A contained all AIDS-associated isolates studied and two human reference strains from Mexican histoplasmosis patients without AIDS; group B included bat guano, infected bat, and cock excreta isolates from the State of Guerrero, Mexico, plus three human histoplasmosis strains from Guatemala, Panama, and Colombia. Polymorphic DNA patterns evaluated by RAPD-PCR showed three major bands of 4.4, 3.2, and 2.3 kb in most H. capsulatum isolates studied. Four groups were related by DNA polymorphisms: group I was formed by most of the AIDS-associated H. capsulatum isolates studied, one human histoplasmosis strain from Colombia, two human reference strains from Mexican patients without AIDS, and one human histoplasmosis strain from Guatemala. Group II consisted of only a single strain from Panama. Group III included three strains: one from a Mexican patient with AIDS and two isolated from nature in Guerrero (cock excreta and bat guano). The last, group IV, consisted of only one strain isolated from an infected bat, captured in Guerrero. A tight relationship between phenotypic and genotypic characterization was observed, and both analyses could be useful tools for typing H. capsulatum from different sources and geographic origins. PMID:10203495

  7. Carrier mobility characterization of DNA-surfactant complexes

    NASA Astrophysics Data System (ADS)

    Lin, Ting-Yu; Hung, Yu-Chueh

    2012-02-01

    Deoxyribonucleic acid (DNA) biopolymer has been emerging as a promising material for photonic applications. As many optoelectronic devices rely on carrier transportation to achieve desired functionality, carrier mobility is important for the exploitation of these biopolymer-based materials for practical implementation. In this study, we present the mobility measurement by employing time-of-flight technique and characterize the current-voltage (I-V) properties based on DNA-surfactant complexes. An additional NPB layer was introduced in the fabricated structure to serve as a charge generation layer (CGL). The dependency of hole mobility with respect to the applied electric field was characterized and a linear correlation was exhibited. Hole transport was found to be dispersive, indicating a high degree energetic disorder in these DNA-surfactant complexes. The characterization results show promises for the employment of DNA complexes in the applications of organic light-emitting devices and organic field-effect transistors.

  8. Mobility of Electron in DNA Crystals by Laser Radiation

    NASA Technical Reports Server (NTRS)

    Zhang, Kaixi; Zhao, Qingxun; Cui, Zhiyun; Zhang, Ping; Dong, Lifang

    1996-01-01

    The mobility of electrons in laser radiated DNA is closed to the energy transfer and energy migration of a biological molecule. Arrhenius has studied the conductivity of the electrons in a biological molecule. But his result is far from the experimental result and meanwhile the relation between some parameters in his theory and the micro-quantities in DNA is not very clear. In this paper, we propose a new phonon model of electron mobility in DNA and use Lippman-Schwinger equation and S-matrix theory to study the mobility of electrons in DNA crystal. The result is relatively close to the experiment result and some parameters in Arrhenius theory are explained in our work.

  9. Electrophoretic Focusing

    NASA Technical Reports Server (NTRS)

    Snyder, Robert S.

    2001-01-01

    Electrophoretic focusing is a new method of continuous flow electrophoresis that introduces precision flow control to achieve high resolution separations. The electric field is applied perpendicular to an incoming sample lamina and buffer but also perpendicular to the broad faces of the thin rectangular chamber. A uniform fluid cross-flow then enters and exits the separation chamber through the same broad faces which are porous. A balance is achieved by adjusting either the electric field or the cross-flow so the desired sample fraction with its specific migration velocity encounters an opposing flow of the same velocity. Applying an electric field transverse to the incoming sample lamina and opposing this field with a carefully configured buffer flow, a sample constituent can be selected and focused into a narrow stream for subsequent analysis. Monotonically changing either electric field or buffer cross-flow will yield a scan of all constituents of the sample. Stopping the scan increases the collection time for minor constituents to improve their analysis. Using the high voltage gradients and/or cross-flow to rapidly deflect extraneous sample through the porous screens and into either of the side (purge) chambers, the selected sample is focused in the center plane of the separation chamber and collected without contact or interaction with the separation chamber walls. Results will be presented on the separation of a range of materials including dyes, proteins, and monodisperse polystyrene latexes. Sources of sample dispersion inherent in other electrokinetic techniques will be shown to be negligible for a variety of sample concentrations, buffer properties and operating conditions.

  10. Identification and secondary structure analysis of a region affecting electrophoretic mobility of the STR locus SE33.

    PubMed

    Wang, Dennis Y; Green, Robert L; Lagacé, Robert E; Oldroyd, Nicola J; Hennessy, Lori K; Mulero, Julio J

    2012-05-01

    SE33 is one of the most informative markers in forensic use due to its high power of discrimination. During the course of developing the AmpFℓSTR(®) NGM SElect™ PCR Amplification Kit several SE33 primer designs were screened with one primer pair yielding a high frequency of discordant alleles when compared to the AmpFℓSTR(®) SEfiler Plus™ PCR Amplification Kit. This discordance was mostly specific to samples of African descent with an estimated frequency of 5.1% and was a result of a mobility shift of approximately +0.84nt. The sequence analysis of the affected alleles revealed that the only difference from the wild type sequence was a single nucleotide polymorphism (SNP) outside of the SE33 repeat but within the amplicon of this particular set of experimental primers. In total, we identified three different SNPs all within 9nt of each other, each of which could cause the mobility shift individually. Further characterization of this region via site directed mutagenesis and thermostability measurements strongly suggests that this polymorphic region contains a secondary structure that, when disrupted due to the presence of a variant SNP, results in a mobility shift relative to the wild type sequence. To overcome this problem, the SE33 primers used in the final configuration of the NGM SElect™ Kit avoided the amplification of this polymorphic region yielding in turn results highly concordant with the SEfiler Plus™ Kit. PMID:21757416

  11. A68 proteins in Alzheimer's disease are composed of several tau isoforms in a phosphorylated state which affects their electrophoretic mobilities.

    PubMed Central

    Brion, J P; Hanger, D P; Couck, A M; Anderton, B H

    1991-01-01

    The tau-immunoreactive A68 polypeptides found in brains from patients with Alzheimer's disease have been studied by Western blotting using (1) antibodies to synthetic peptides corresponding to sequences that span the complete human tau molecule, and (2) antibodies specific for inserts 1 and 2 found towards the N-terminus of some tau isoforms. The three major A68 polypeptides were labelled by all of the antibodies to sequences common to all tau isoforms, but the faster-migrating A68 polypeptides was not labelled by either of the two antibodies specific for inserts 1 and 2. Treatment with alkaline phosphatase of non-solubilized A68 did not change its electrophoretic mobility on SDS/PAGE under the conditions described here. However, A68 that was solubilized before treating it with alkaline phosphatase was found to move faster on SDS/PAGE than untreated A68, to a position similar to that of normal tau. We also confirmed that A68 preparations contain numerous paired helical filaments (PHF). These PHF were labelled by all anti-tau antibodies, including insert-specific antibodies. Our results further support the notion that PHF contain abnormally phosphorylated tau in an aggregated state, and indicate that these abnormally phosphorylated tau forms are composed of several tau isoforms and that the full length of the tau molecule is present in these polypeptides. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:1953678

  12. Charge regulation phenomenon predicted from the modeling of polypeptide electrophoretic mobilities as a relevant mechanism of amyloid-beta peptide oligomerization.

    PubMed

    Deiber, Julio A; Peirotti, Marta B; Piaggio, Maria V

    2016-03-01

    Electrophoretic mobilities of amyloid-beta (1-40) and (1-42) peptides and their aggregates are modeled to study the amyloidogenic pathway associated with Alzheimer´s Disease. The near molecule pH generated by the intraparticle charge regulation phenomenon during the oligomerization of amyloid-beta (1-40) and (1-42) peptides is evaluated and discussed as a relevant mechanism supporting the "amyloid cascade hypothesis" proposed in the literature. A theoretical framework associated with the oligomerization of amyloid-beta peptides including simple scaling laws and the consideration of electrokinetic and hydrodynamic global properties of oligomers is presented. The central finding is the explanation of the near molecule pH change toward the pI when the oligomerization number increases. These results allow one to rationalize consecutive physical stages that validate the amyloid cascade hypothesis. Concluding remarks involving mainly the effects of pair and intraparticle charge regulation phenomena on the amyloidogenic pathway with some suggestions for future research are provided. PMID:26718015

  13. High Sensitivity Method to Estimate Distribution of Hyaluronan Molecular Sizes in Small Biological Samples Using Gas-Phase Electrophoretic Mobility Molecular Analysis

    PubMed Central

    Do, Lan; Dahl, Christen P.; Kerje, Susanne; Hansell, Peter; Mörner, Stellan; Lindqvist, Ulla; Engström-Laurent, Anna; Larsson, Göran; Hellman, Urban

    2015-01-01

    Hyaluronan is a negatively charged polydisperse polysaccharide where both its size and tissue concentration play an important role in many physiological and pathological processes. The various functions of hyaluronan depend on its molecular size. Up to now, it has been difficult to study the role of hyaluronan in diseases with pathological changes in the extracellular matrix where availability is low or tissue samples are small. Difficulty to obtain large enough biopsies from human diseased tissue or tissue from animal models has also restricted the study of hyaluronan. In this paper, we demonstrate that gas-phase electrophoretic molecular mobility analyzer (GEMMA) can be used to estimate the distribution of hyaluronan molecular sizes in biological samples with a limited amount of hyaluronan. The low detection level of the GEMMA method allows for estimation of hyaluronan molecular sizes from different parts of small organs. Hence, the GEMMA method opens opportunity to attain a profile over the distribution of hyaluronan molecular sizes and estimate changes caused by disease or experimental conditions that has not been possible to obtain before. PMID:26448761

  14. Estimation of electrokinetic and hydrodynamic global properties of relevant amyloid-beta peptides through the modeling of their effective electrophoretic mobilities and analysis of their propensities to aggregation.

    PubMed

    Deiber, Julio A; Piaggio, Maria V; Peirotti, Marta B

    2014-09-01

    Neuronal activity loss may be due to toxicity caused by amyloid-beta peptides forming soluble oligomers. Here amyloid-beta peptides (1-42, 1-40, 1-39, 1-38, and 1-37) are characterized through the modeling of their experimental effective electrophoretic mobilities determined by a capillary zone electrophoresis method as reported in the literature. The resulting electrokinetic and hydrodynamic global properties are used to evaluate amyloid-beta peptide propensities to aggregation through pair particles interaction potentials and Brownian aggregation kinetic theories. Two background electrolytes are considered at 25°C, one for pH 9 and ionic strength I = 40 mM (aggregation is inhibited through NH4OH) the other for pH 10 and I = 100 mM (without NH4OH). Physical explanations of peptide oligomerization mechanisms are provided. The effect of hydration, electrostatic, and dispersion forces in the amyloidogenic process of amyloid-beta peptides (1-40 and 1-42) are quantitatively presented. The interplay among effective charge number, hydration, and conformation of chains is described. It is shown that amyloid-beta peptides (1-40 and 1-42) at pH 10, I = 100 mM and 25°C, may form soluble oligomers, mainly of order 2 and 4, after an incubation of 48 h, which at higher times evolve and end up in complex structures (protofibrils and fibrils) found in plaques associated with Alzheimer's disease. PMID:24975363

  15. Mobile small RNAs regulate genome-wide DNA methylation

    PubMed Central

    Lewsey, Mathew G.; Hardcastle, Thomas J.; Melnyk, Charles W.; Molnar, Attila; Valli, Adrián; Urich, Mark A.; Nery, Joseph R.; Baulcombe, David C.; Ecker, Joseph R.

    2016-01-01

    RNA silencing at the transcriptional and posttranscriptional levels regulates endogenous gene expression, controls invading transposable elements (TEs), and protects the cell against viruses. Key components of the mechanism are small RNAs (sRNAs) of 21–24 nt that guide the silencing machinery to their nucleic acid targets in a nucleotide sequence-specific manner. Transcriptional gene silencing is associated with 24-nt sRNAs and RNA-directed DNA methylation (RdDM) at cytosine residues in three DNA sequence contexts (CG, CHG, and CHH). We previously demonstrated that 24-nt sRNAs are mobile from shoot to root in Arabidopsis thaliana and confirmed that they mediate DNA methylation at three sites in recipient cells. In this study, we extend this finding by demonstrating that RdDM of thousands of loci in root tissues is dependent upon mobile sRNAs from the shoot and that mobile sRNA-dependent DNA methylation occurs predominantly in non-CG contexts. Mobile sRNA-dependent non-CG methylation is largely dependent on the DOMAINS REARRANGED METHYLTRANSFERASES 1/2 (DRM1/DRM2) RdDM pathway but is independent of the CHROMOMETHYLASE (CMT)2/3 DNA methyltransferases. Specific superfamilies of TEs, including those typically found in gene-rich euchromatic regions, lose DNA methylation in a mutant lacking 22- to 24-nt sRNAs (dicer-like 2, 3, 4 triple mutant). Transcriptome analyses identified a small number of genes whose expression in roots is associated with mobile sRNAs and connected to DNA methylation directly or indirectly. Finally, we demonstrate that sRNAs from shoots of one accession move across a graft union and target DNA methylation de novo at normally unmethylated sites in the genomes of root cells from a different accession. PMID:26787884

  16. On electrophoretic NMR. Exploring high conductivity samples

    NASA Astrophysics Data System (ADS)

    Bielejewski, Michał; Giesecke, Marianne; Furó, István

    2014-06-01

    The performance of a new electrophoretic NMR (eNMR) method that uses a Carr-Purcell-Meiboom-Gill echo train with repeated electric field reversal is investigated. We show that this pulse sequence, with acronym CPMGER, yields strongly reduced artifacts from convective flow effects caused by the simultaneous presence of electroosmotic and thermal driving forces. We demonstrate the achieved improvements in various aqueous solutions. Ultimately, the method can be used for obtaining electrophoretic mobilities by eNMR without relying on uncharged reference molecules, otherwise a significant limitation for electrophoretic experiments performed with nuclei other than 1H.

  17. Quantifying the Heterogeneity of Chemical Structures in Complex Charged Polymers through the Dispersity of Their Distributions of Electrophoretic Mobilities or of Compositions.

    PubMed

    Thevarajah, Joel J; Sutton, Adam T; Maniego, Alison R; Whitty, Elizabeth G; Harrisson, Simon; Cottet, Hervé; Castignolles, Patrice; Gaborieau, Marianne

    2016-02-01

    The complexity of synthetic and natural polymers used in industrial and medical applications is expanding; thus, it becomes increasingly important to improve and develop methods for their molecular characterization. Free-solution capillary electrophoresis is a robust technique for the separation and characterization of both natural and synthetic complex charged polymers. In the case of polyelectrolytes, free-solution capillary electrophoresis is in the "critical conditions" (CE-CC): it allows their separation by factors other than molar mass for molar masses typically higher than 20000 g/mol. This method is thus complementary to size-exclusion chromatography (SEC). SEC is widely used to determine molar mass distributions and their dispersities. Utilizing CE-CC, an analogous calculation of dispersity based on the distributions of electrophoretic mobilities was derived and the heterogeneity of composition or branching in different polysaccharides or synthetic polymers was obtained in a number of experimental cases. Calculations are based on a ratio of moments and could therefore be compared to simulations of polymerization processes, in analogy to the work performed on molar mass distributions. Among four possible types of dispersity, the most precise values were obtained with the calculation analogous with the dispersity of molar mass distribution Mw/Mn. In addition, the dispersity value allows conclusions based on a single value: the closer the dispersity is to 1, the more homogeneous the polymer is in terms of composition or branching. This approach allows the analysis of dispersity of important molecular attributes of polymers other than molar mass and aims at improving the overall molecular characterization of both synthetic and natural polymers. The dispersity can also be monitored online while performing a chemical reaction within the CE instrument. PMID:26674535

  18. Instrument and method to determine the electrophoretic mobility of nanoparticles and proteins by combining electrical and flow field-flow fractionation.

    PubMed

    Johann, Christoph; Elsenberg, Stephan; Schuch, Horst; Rösch, Ulrich

    2015-04-21

    A new FFF method is presented which combines asymmetrical flow-FFF (AF4) and electrical FFF (ElFFF) in one channel to electrical asymmetrical flow-FFF (EAF4) to overcome the restrictions of pure ElFFF. It allows for measuring electrophoretic mobility (μ) as a function of size. The method provides an absolute value and does not require calibration. Results of μ for two particle standards are in good agreement with values determined by phase analysis light scattering (PALS). There is no requirement for low ionic strength carriers with EAF4. This overcomes one of the main limitations of ElFFF, making it feasible to measure proteins under physiological conditions. EAF4 has the capability to determine μ for individual populations which are resolved into separate peaks. This is demonstrated for a mixture of three polystyrene latex particles with different sizes as well as for the monomer and dimer of BSA and an antibody. The experimental setup consists of an AF4 channel with added electrodes; one is placed beneath the frit at the bottom wall and the other covers the inside of the upper channel plate. This design minimizes contamination from the electrolysis reactions by keeping the particles distant from the electrodes. In addition the applied voltage range is low (1.5-5 V), which reduces the quantity of gaseous electrolysis products below a threshold that interferes with the laminar flow profile or detector signals. Besides measuring μ, the method can be useful to improve the separation between sample components compared to pure flow-FFF. For two proteins (BSA and a monoclonal antibody), enhanced resolution of the monomer and dimer is achieved by applying an electric field. PMID:25789885

  19. Mobility of long-chain DNA in two-dimensional artificial gels

    NASA Astrophysics Data System (ADS)

    Turner, Stephen W. P.; Han, Jongyoon; Craighead, Harold G.

    2000-03-01

    In this study, a two-dimensional array of nanofabricated obstacles is used as an artificial gel to study the electrophoretic mobility dependence of DNA as a function of pore size, molecule length and electric field. Limitations in feature size have prevented previous studies from testing the crossover from the separating to the non-separating regime predicted by the biased reptation model of Lumpkin, Dejardin and Zimm[1] and the modified model of Duke, Semenov and Viovy.[2] That limitation is overcome in this work with the use of electron beam lithography to define features as small as 30 nm. Attainment of these feature sizes was made possible by the use of a sacrificial-layer-based technique for fluidics fabrication.[3] A novel band-launching strategy is used to provide band separation data for the first time in this system. Molecule lengths between 5 and 150 kilobases are studied for electric field strengths from 0.1 to 20 Volts per meter. [1] O. Lumpkin, P. Dejardin and B. Zimm, Biopolymers, Vol. 24, 1573-1593 (1985) [2] T. Duke, A. Semenov and J. Viovy, Phys. Rev. Lett. Vol. 69, No. 22, 3260-3263 (1992) [3] S. Turner, A. Perez, A. Lopez, and H. Craighead, J. Vac. Sci. Technol. B 16(6) 3835-3840 (1998)

  20. Stability of DNA-Tethered Lipid Membranes with Mobile Tethers

    PubMed Central

    Chung, Minsub; Boxer, Steven G.

    2011-01-01

    We recently introduced two approaches for tethering planar lipid bilayers as membrane patches to either a supported lipid bilayer or DNA-functionalized surface using DNA hybridization (Chung, M., Lowe, R. D., Chan, Y-H. M., Ganesan, P. V., Boxer, S. G. J. Struct. Biol. 2009, 168, 190–9). When mobile DNA tethers are used, the tethered bilayer patches become unstable, while they are stable if the tethers are fixed on the surface. Because the mobile tethers between a patch and a supported lipid bilayer offer a particularly interesting architecture for studying the dynamics of membrane-membrane interactions, we have investigated the sources of instability, focusing on membrane composition. The most stable patches were made with a mixture of saturated lipids and cholesterol, suggesting an important role for membrane stiffness. Other factors such as the effect of tether length, lateral mobility and patch membrane edge were also investigated. Based on these results, a model for the mechanism of patch destruction is developed. PMID:21452847

  1. Electrophoretic transport equations - Electrophoretic models based on migration only and their interrelationships

    NASA Technical Reports Server (NTRS)

    Thormann, Wolfgang; Mosher, Richard A.

    1985-01-01

    The general equations which describe the electrophoretic transport of components in solution are restated using Newman's general concept of mobilities. A concise derivation of the moving boundary equation and the regulating function from the continuity equation is presented. Various other regulating principles across moving and stationary boundaries are also discussed, which permits a review of the features and interrelationships of the electrophoretic models based on electromigration only. The effect of considering an interactive (dissociating) solvent on the mathematical treatment is discussed.

  2. Mobile DNA and evolution in the 21st century

    PubMed Central

    2010-01-01

    Scientific history has had a profound effect on the theories of evolution. At the beginning of the 21st century, molecular cell biology has revealed a dense structure of information-processing networks that use the genome as an interactive read-write (RW) memory system rather than an organism blueprint. Genome sequencing has documented the importance of mobile DNA activities and major genome restructuring events at key junctures in evolution: exon shuffling, changes in cis-regulatory sites, horizontal transfer, cell fusions and whole genome doublings (WGDs). The natural genetic engineering functions that mediate genome restructuring are activated by multiple stimuli, in particular by events similar to those found in the DNA record: microbial infection and interspecific hybridization leading to the formation of allotetraploids. These molecular genetic discoveries, plus a consideration of how mobile DNA rearrangements increase the efficiency of generating functional genomic novelties, make it possible to formulate a 21st century view of interactive evolutionary processes. This view integrates contemporary knowledge of the molecular basis of genetic change, major genome events in evolution, and stimuli that activate DNA restructuring with classical cytogenetic understanding about the role of hybridization in species diversification. PMID:20226073

  3. Global properties and propensity to dimerization of the amyloid-beta (12-28) peptide fragment through the modeling of its monomer and dimer diffusion coefficients and electrophoretic mobilities.

    PubMed

    Deiber, Julio A; Peirotti, Marta B; Piaggio, Maria V

    2015-03-01

    Neuronal activity loss may be due to toxicity caused mainly by amyloid-beta (1-40) and (1-42) peptides forming soluble oligomers. Here the amyloid-beta (12-28) peptide fragment (monomer) and its dimer are characterized at low pH through the modeling of their diffusion coefficients and effective electrophoretic mobilities. Translational diffusion coefficient experimental values of monomer and dimer analogs of this peptide fragment and monomer and dimer mixtures at thermodynamic equilibrium are used as reported in the literature for different monomer initial concentrations. The resulting electrokinetic and hydrodynamic global properties are employed to evaluate the amyloid-beta (12-28) peptide fragment propensity to dimerization through a thermodynamic theoretical framework. Therefore equilibrium constants are considered at pH 2.9 to elucidate one of the amyloidogenic mechanisms involving the central hydrophobic region LVFFA of the peptide spanning residues 17-21 associated with phenylalanine at positions 19 and 20 in the amino acid sequence of amyloid-beta peptides. An analysis demonstrating that peptide aggregation is a concentration-dependent process is provided, where both pair and intraparticle charge regulation phenomena become relevant. It is shown that the modeling of the effective electrophoretic mobility of the amyloid-beta (12-28) peptide fragment is crucial to understand the effect of hydrophobic region LVFFA in the amyloidogenic process. PMID:25403948

  4. Reversible Light Switch for Macrocycle Mobility in a DNA Rotaxane

    PubMed Central

    2012-01-01

    A recent trend in DNA nanotechnology consists of the assembly of architectures with dynamic properties that can be regulated by employing external stimuli. Reversible processes are important for implementing molecular motion into DNA architectures as they allow for the regeneration of the original state. Here we describe two different approaches for the reversible switching of a double-stranded DNA rotaxane architecture from a stationary pseudorotaxane mode into a state with movable components. Both states only marginally differ in their respective topologies but their mechanical properties are fundamentally different. In the two approaches, the switching operation is based on strand-displacement reactions. One of them employs toehold-extended oligodeoxynucleotides whereas in the other one the switching is achieved by light-irradiation. In both cases, multiple back and forth switching between the stationary and the mobile states was achieved in nearly quantitative fashion. The ability to reversibly operate mechanical motion in an interlocked DNA nanostructure opens exciting new avenues in DNA nanotechnology. PMID:22780815

  5. Surface modification of inorganic black particles for electrophoretic display

    NASA Astrophysics Data System (ADS)

    Kim, Sang Deuk; Ahn, Woo Jin; Choi, Hyoung Jin

    2014-11-01

    Inorganic black particles (Black 444) were modified with poly(methyl methacrylate) as a shell material by using dispersion polymerization to improve their dispersion stability in a medium oil for electrophoretic display applications. They were also positively charged with vinylimidazole to enhance their electrophoretic mobility. The morphology and the shape of the composite particles were characterized by using scanning electron microscopy. The thermal properties and the chemical structure of the samples were examined by using thermogravimetric analysis and Fourier-transform infrared spectroscopy, respectively. In addition, the electrophoretic mobility and the zeta-potential of the black444/PMMA/vinylimidazole particles in a dielectric fluid were measured by using optical microscopy and electrophoretic light scattering. With increasing positive charge, the black444/PMMA/vinylimidazole particles showed improved electrophoretic characteristics compared to pristine Black 444.

  6. Simultaneous measurements of mobility, dispersion, and orientation of DNA during steady-field gel electrophoresis coupling a fluorescence recovery after photobleaching apparatus with a fluorescence detected linear dichroism setup

    NASA Astrophysics Data System (ADS)

    Tinland, B.; Meistermann, L.; Weill, G.

    2000-06-01

    Orientation of molecules is responsible for the loss of separability during steady-field gel electrophoresis. In this work we develop a technique to measure simultaneously the relevant parameters involved in the separation mechanism: electrophoretic mobility, band broadening, and molecular orientation. To do that we have associated a fluorescence recovery after photobleaching (FRAP) apparatus with a fluorescence detected linear dichroism setup. This coupling allows one to follow the buildup of orientation during the FRAP experiment. Because orientation involves a change in the angular distribution of fluorescence, we have added a fluorescence polarization setup which can be used in parallel with the FRAP and gives an exact value of the steady-state orientation factor. We illustrate the possibilities of these combined experiments by analyzing the coupling of electrophoretic transport and orientation of λ DNA in 1% agarose gels.

  7. DNA electrophoresis in agarose gels: A new mobility vs. DNA length dependence

    NASA Astrophysics Data System (ADS)

    Beheshti, Afshin

    2002-04-01

    Separations were performed on double stranded DNA (dsDNA) using electrophoresis. Electrophoresis is the steady transport of particles under the influence of an external electric field. Double stranded DNA fragments ranging in length from 200 base pairs (bp) to 194,000 bp (0.34 nm = 1 bp) were electrophoresed at agarose gel concentrations T = 0.4%--1.5%. The electric field was varied from 0.62 V/cm to 6.21 V/cm. A wide range of electric fields and gel concentrations were used to study the usefulness of a new interpolation equation, 1mL =1mL-( 1mL-1 ms)e-L/g , where mL,ms , and g are independent free fitting parameters. The long length mobility limit is interpreted as mL , the short length mobility limit is ms , and g is the crossover between the long length limit and the short length limit. This exponential relation fit very well (chi2 ≥ 0.999) when there are two smooth transitions observed in the "reptation plots" (plotting 3mL/m∘ vs. L) (J. Rousseau, G. Drouin, and G. W. Slater, Phys Rev Lett. 1997, 79, 1945--1948). Fits deviate from the data when three different slopes were observed in the reptation plots. Reptation plots were used to determine a phase diagram for dsDNA migration regimes. The phase diagrams define different regions where mechanisms for molecular transport affect the migration of dsDNA in agarose gels during electrophoresis. The parameters from the equation have also been interpreted to provide a physical description of the structure of the agarose gel by calculating the pore sizes. The relations between the values for the pore sizes and the phase diagrams are interpreted to better understand the migration of the DNA through agarose gels.

  8. Studies on the effect of mobile phone radiation on DNA using laser induced fluorescence technique

    NASA Astrophysics Data System (ADS)

    Vishnu, K.; Nithyaja, B.; Pradeep, C.; Sujith, R.; Mohanan, P.; Nampoori, V. P. N.

    2011-11-01

    In the present study we have investigated the effect of mobile phone radiation on deoxyribonucleic acid by using fluorescence technique. Absorption spectra shows increase in absorption of DNA after exposure to radiation from mobile phone with different SAR values and microwave frequency which give information about unwinding of the DNA double strand. Fluorescence intensity of dye doped DNA solution is getting reduced suggesting that the absorbed energy is used for unwinding of double strand of DNA after irradiating with microwave radiation. Unwinding of the DNA is very sensitive to power of the microwave radiation.

  9. Multistage Electrophoretic Separators

    NASA Technical Reports Server (NTRS)

    Thomas, Nathan; Doyle, John F.; Kurk, Andy; Vellinger, John C.; Todd, Paul

    2006-01-01

    A multistage electrophoresis apparatus has been invented for use in the separation of cells, protein molecules, and other particles and solutes in concentrated aqueous solutions and suspensions. The design exploits free electrophoresis but overcomes the deficiencies of prior free-electrophoretic separators by incorporating a combination of published advances in mathematical modeling of convection, sedimentation, electro-osmotic flow, and the sedimentation and aggregation of droplets. In comparison with other electrophoretic separators, these apparatuses are easier to use and are better suited to separation in relatively large quantities characterized in the art as preparative (in contradistinction to smaller quantities characterized in the art as analytical). In a multistage electrophoretic separator according to the invention, an applied vertical steady electric field draws the electrically charged particles of interest from within a cuvette to within a collection cavity that has been moved into position of the cuvette. There are multiple collection cavities arranged in a circle; each is aligned with the cuvette for a prescribed short time. The multistage, short-migration-path character of the invention solves, possibly for the first time, the fluid-instability problems associated with free electrophoresis. The figure shows a prototype multistage electrophoretic separator that includes four sample stations and five collection stages per sample. At each sample station, an aqueous solution or suspension containing charged species to be separated is loaded into a cuvette, which is machined into a top plate. The apparatus includes a lower plate, into which 20 collection cavities have been milled. Each cavity is filled with an electrophoresis buffer solution. For the collection of an electrophoretic fraction, the lower plate is rotated to move a designated collection cavity into alignment with the opening of the cuvette. An electric field is then applied between a non

  10. Pulsed-Field Electrophoresis: Application of a Computer Model to the Separation of Large DNA Molecules

    NASA Astrophysics Data System (ADS)

    Lalande, Marc; Noolandi, Jaan; Turmel, Chantal; Rousseau, Jean; Slater, Gary W.

    1987-11-01

    The biased reptation theory has been applied to the pulsed-field electrophoresis of DNA in agarose gels. A computer simulation of the theoretical model that calculates the mobility of large DNA molecules as a function of agarose pore size, DNA chain properties, and electric field conditions has been used to generate mobility curves for DNA molecules in the size range of the larger yeast chromosomes. Pulsed-field electrophoresis experiments resulting in the establishment of an electrophoretic karyotype for yeast, where the mobility of the DNA fragments is a monotonic function of molecular size for the entire size range that is resolved (200-2200 kilobase pairs), has been compared to the theoretical mobility curves generated by the computer model. The various physical mechanisms and experimental conditions responsible for band inversion and improved electrophoretic separation are identified and discussed in the framework of the model.

  11. Ion Mobility Spectrometry Reveals Duplex DNA Dissociation Intermediates

    NASA Astrophysics Data System (ADS)

    Burmistrova, Anastasia; Gabelica, Valérie; Duwez, Anne-Sophie; De Pauw, Edwin

    2013-11-01

    Electrospray ionization (ESI) soft desolvation is widely used to investigate fragile species such as nucleic acids. Tandem mass spectrometry (MS/MS) gives access to the gas phase energetics of the intermolecular interactions in the absence of solvent, by following the dissociation of mass-selected ions. Ion mobility mass spectrometry (IMS) provides indications on the tridimensional oligonucleotide structure by attributing a collision cross section (CCS) to the studied ion. Electrosprayed duplexes longer than eight bases pairs retain their helical structure in a solvent-free environment. However, the question of conformational changes under activation in MS/MS studies remains open. The objective of this study is to probe binding energetics and characterize the unfolding steps occurring prior to oligonucleotide duplex dissociation. Comparing the evolution of CCS with collision energy and breakdown curves, we characterize dissociation pathways involved in CID-activated DNA duplex separation into single strands, and we demonstrate here the existence of stable dissociation intermediates. At fixed duplex length, dissociation pathways were found to depend on the percentage of GC base pairs and on their position in the duplex. Our results show that pure GC sequences undergo a gradual compaction until reaching the dissociation intermediate: A-helix. Mixed AT-GC sequences were found to present at least two conformers: a classic B-helix and an extended structure where the GC tract is a B-helix and the AT tract(s) fray. The dissociation in single strands takes place from both conformers when the AT base pairs are enclosed between two GC tracts or only from the extended conformer when the AT tract is situated at the end(s) of the sequence.

  12. Effects of Crowder Structure and Salt on DNA Mobility and Conformation in Crowded Environments

    NASA Astrophysics Data System (ADS)

    Gorczyca, Stephanie M.; Robertson-Anderson, Rae M.

    Biological cells are crowded environments in which DNA must move through to perform specific functions. We study how the properties of crowded cell-like environments impact DNA dynamics by tracking individual 115 kbp ring and linear DNA in different crowded environments using single-molecule fluorescence microscopy. We determine the role of crowder structure and salt on DNA diffusion and conformation by measuring the mean-squared center-of-mass displacements, as well as the conformational shape, size, and fluctuations of each molecule. Previously, we used 10 and 500 kDa dextran as crowders and showed that mobility of both ring and linear DNA decreased exponentially with increased crowding, but rings compact while linear DNA elongate. These effects were dependent solely on the reduction in available volume for DNA rather than size or number of crowders. Here we use crowders of similar molecular weight, but different structure to dextran (10 kDa PEG and 400 kDa Ficoll). We find that DNA mobility reduction is independent of crowder structure and that ring and linear DNA undergo more significant compaction. Finally, we characterize the role of salt on DNA mobility and conformation to determine the relative roles of enthalpic versus entropic effects on crowding-induced DNA dynamics. This research was funded by the AFOSR Young Investigator Program, Grant No. FA95550-12-1-0315 and the Arnold and Mabel Beckman Scholarship Foundation.

  13. Electrophoretic Transport of Biomolecules through Carbon Nanotube Membranes

    PubMed Central

    Sun, Xinghua; Su, Xin; Wu, Ji; Hinds, Bruce J.

    2013-01-01

    Electrophoretic transport of proteins across electrochemically oxidized multi-walled carbon nanotube (MWCNT) membranes has been investigated. Small charged protein, lysozyme, was successfully pumped across MWCNT membranes by electric field while rejecting larger bovine serum albumin (BSA). Transport of the lysozome was reduced by a factor of about 30 in comparison to bulk mobility and consistent with prediction for hindered transport. Mobilities between 0.33-1.4×10-9 m2/V-s were observed and are approximately 10 fold faster than comparable ordered nanoporous membranes and are consistent with continuum models. For mixtures of BSA and lysozyme, complete rejection of BSA is seen with electrophoretic separations PMID:21338104

  14. Construction and evaluation of a capillary electrophoresis DNA sequencer

    SciTech Connect

    Drossman, H.

    1992-01-01

    This dissertation describes the construction and evaluation of an automated DNA sequencer using capillary gel electrophoresis (CGE) for separating single-strand DNA fragments and a fluorescence detector for analyzing labeled fragments. Theories governing the electrophoretic separation of DNA, dispersion processes in CGE and high sensitivity fluorescence detection are reviewed. The CGE DNA sequencer is compared with current DNA sequencing instruments and with projections of future DNA sequencing instruments. Parameters affecting the limits of detection, DNA sample loading, sample mobility and resolution are evaluated. Predictions for the future of capillary electrophoresis for large-scale sequencing projects are presented.

  15. Convection Compensated Electrophoretic NMR

    NASA Astrophysics Data System (ADS)

    He, Qiuhong; Wei, Zhaohui

    2001-06-01

    A novel method of convection compensated ENMR (CC-ENMR) has been developed to detect electrophoretic motion of ionic species in the presence of bulk solution convection. This was accomplished using a gradient moment nulling technique to remove spectral artifacts from heat-induced convection and using the polarity switch of the applied electric field to retain spin phase modulations due to electrophoretic flow. Experiments were carried out with a mixture of 100 mM L-aspartic acid and 100 mM 4,9-dioxa-1,12-dodecanediamine to demonstrate this new method of ENMR. CC-ENMR enhances our previously developed capillary array ENMR (CA-ENMR) in solving the convection problem. The combined CA- and CC-ENMR approach strengthens the potential of multidimensional ENMR in simultaneous structural determination of coexisting proteins and protein conformations in biological buffer solutions of high ionic strength. Structural mapping of interacting proteins during biochemical reactions becomes possible in the future using ENMR techniques, which may have a profound impact on the understanding of biological events, including protein folding, genetic control, and signal transduction in general.

  16. Two high-mobility group box domains act together to underwind and kink DNA

    SciTech Connect

    Sánchez-Giraldo, R.; Acosta-Reyes, F. J.; Malarkey, C. S.; Saperas, N.; Churchill, M. E. A.; Campos, J. L.

    2015-06-30

    The crystal structure of HMGB1 box A bound to an unmodified AT-rich DNA fragment is reported at a resolution of 2 Å. A new mode of DNA recognition for HMG box proteins is found in which two box A domains bind in an unusual configuration generating a highly kinked DNA structure. High-mobility group protein 1 (HMGB1) is an essential and ubiquitous DNA architectural factor that influences a myriad of cellular processes. HMGB1 contains two DNA-binding domains, box A and box B, which have little sequence specificity but have remarkable abilities to underwind and bend DNA. Although HMGB1 box A is thought to be responsible for the majority of HMGB1–DNA interactions with pre-bent or kinked DNA, little is known about how it recognizes unmodified DNA. Here, the crystal structure of HMGB1 box A bound to an AT-rich DNA fragment is reported at a resolution of 2 Å. Two box A domains of HMGB1 collaborate in an unusual configuration in which the Phe37 residues of both domains stack together and intercalate the same CG base pair, generating highly kinked DNA. This represents a novel mode of DNA recognition for HMGB proteins and reveals a mechanism by which structure-specific HMG boxes kink linear DNA.

  17. Combinative exposure effect of radio frequency signals from CDMA mobile phones and aphidicolin on DNA integrity.

    PubMed

    Tiwari, R; Lakshmi, N K; Surender, V; Rajesh, A D V; Bhargava, S C; Ahuja, Y R

    2008-01-01

    The aim of present study is to assess DNA integrity on the effect of exposure to a radio frequency (RF) signal from Code Division Multiple Access (CDMA) mobile phones. Whole blood samples from six healthy male individuals were exposed for RF signals from a CDMA mobile phone for 1 h. Alkaline comet assay was performed to assess the DNA damage. The combinative exposure effect of the RF signals and APC at two concentrations on DNA integrity was studied. DNA repair efficiency of the samples was also studied after 2 h of exposure. The RF signals and APC (0.2 microg/ml) alone or in synergism did not have any significant DNA damage as compared to sham exposed. However, univariate analysis showed that DNA damage was significantly different among combinative exposure of RF signals and APC at 0.2 microg/ml (p < 0.05) and at 2 microg/ml (p < 0.02). APC at 2 microg/ml concentration also showed significant damage levels (p < 0.05) when compared to sham exposed. DNA repair efficiency also varied in a significant way in combinative exposure sets (p < 0.05). From these results, it appears that the repair inhibitor APC enhances DNA breaks at 2 microg/ml concentration and that the damage is possibly repairable. Thus, it can be inferred that the in vitro exposure to RF signals induces reversible DNA damage in synergism with APC. PMID:19037791

  18. Epigenetic control of mobile DNA as an interface between experience and genome change

    PubMed Central

    Shapiro, James A.

    2014-01-01

    Mobile DNA in the genome is subject to RNA-targeted epigenetic control. This control regulates the activity of transposons, retrotransposons and genomic proviruses. Many different life history experiences alter the activities of mobile DNA and the expression of genetic loci regulated by nearby insertions. The same experiences induce alterations in epigenetic formatting and lead to trans-generational modifications of genome expression and stability. These observations lead to the hypothesis that epigenetic formatting directed by non-coding RNA provides a molecular interface between life history events and genome alteration. PMID:24795749

  19. INFLUENCE OF BORATE BUFFERS ON THE ELECTROPHORETIC BEHAVIOR OF HUMIC SUBSTANCES IN CAPILLARY ZONE ELECTROPHORESIS

    EPA Science Inventory

    The influence of tetrahydroxyborate ions on the electrophoretic mobility of humic acids was evaluated by capillary electrophoresis (CE). Depending on the molarity of borate ions in the separation buffer, the humic acids exhibit electropherograms with sharp peaks consistently exte...

  20. Using Measurements of Mobility, Diffusion, and Dispersion to Predict Separation Resolution in DNA Electrophoresis

    NASA Astrophysics Data System (ADS)

    Lo, Roger

    2005-03-01

    Electrophoresis of DNA continues to be a key component in a wide variety of genomic analysis assays. In order to customize and optimize these assay systems, much effort has been directed to improve and predict separation resolution using various sieving matrices and experimental platforms. Predicting separation resolution requires a much more detailed understanding of mobility, diffusion, and dispersion phenomena of DNA fragments migrating in the sieving matrix than is currently available in literature. In this study, we address this issue by obtaining a series of systematic measurements of mobility, diffusion, and dispersion using an automated DNA sequencer. Using this data, we are able to isolate key factors governing separation performance, and make comparisons with biased reptation theory to extract information on gel structure and predict achievable resolution under each set of operating conditions. We are also able to predict the separation resolution under specific run conditions, thereby giving researchers and engineers the ability to easily tailor DNA separation systems for required separation performance.

  1. Single DNA imaging and length quantification through a mobile phone microscope

    NASA Astrophysics Data System (ADS)

    Wei, Qingshan; Luo, Wei; Chiang, Samuel; Kappel, Tara; Mejia, Crystal; Tseng, Derek; Chan, Raymond Yan L.; Yan, Eddie; Qi, Hangfei; Shabbir, Faizan; Ozkan, Haydar; Feng, Steve; Ozcan, Aydogan

    2016-03-01

    The development of sensitive optical microscopy methods for the detection of single DNA molecules has become an active research area which cultivates various promising applications including point-of-care (POC) genetic testing and diagnostics. Direct visualization of individual DNA molecules usually relies on sophisticated optical microscopes that are mostly available in well-equipped laboratories. For POC DNA testing/detection, there is an increasing need for the development of new single DNA imaging and sensing methods that are field-portable, cost-effective, and accessible for diagnostic applications in resource-limited or field-settings. For this aim, we developed a mobile-phone integrated fluorescence microscopy platform that allows imaging and sizing of single DNA molecules that are stretched on a chip. This handheld device contains an opto-mechanical attachment integrated onto a smartphone camera module, which creates a high signal-to-noise ratio dark-field imaging condition by using an oblique illumination/excitation configuration. Using this device, we demonstrated imaging of individual linearly stretched λ DNA molecules (48 kilobase-pair, kbp) over 2 mm2 field-of-view. We further developed a robust computational algorithm and a smartphone app that allowed the users to quickly quantify the length of each DNA fragment imaged using this mobile interface. The cellphone based device was tested by five different DNA samples (5, 10, 20, 40, and 48 kbp), and a sizing accuracy of <1 kbp was demonstrated for DNA strands longer than 10 kbp. This mobile DNA imaging and sizing platform can be very useful for various diagnostic applications including the detection of disease-specific genes and quantification of copy-number-variations at POC settings.

  2. Skeleton versus fine earth: what information is stored in the mobile extracellular soil DNA fraction?

    NASA Astrophysics Data System (ADS)

    Ascher, Judith; Ceccherini, Maria Teresa; Agnelli, Alberto; Corti, Guiseppe; Pietramellara, Giacomo

    2010-05-01

    The soil genome consists of an intracellular and an extracellular fraction. Recently, soil extracellular DNA (eDNA) has been shown to be quantitatively relevant, with a high survival capacity and mobility, playing a crucial role in the gene transfer by transformation, in the formation of bacterial biofilm and as a source of nutrients for soil microorganisms. The eDNA fraction can be discriminated and classified by its interaction with clay minerals, humic acids and Al/Fe oxihydroxides, resulting in differently mobile components. The eDNA extractable in water, classified as DNA free in the extracellular soil environment or adsorbed on soil colloids (eDNAfree/adsorbed), is hypothesized to be the most mobile DNA in soil. Challenging to assess the information stored in this DNA fraction, eDNAfree/adsorbed was recovered from fine earth (< 4 mm) and highly altered rock fragments or skeleton (4-10 mm) of six consecutive horizons (A1-BCb2) of a forest soil profile by washing the two soil fractions with H2O. Quantitative analysis have been conducted in terms of DNA yields (fluorimeter and spectrophotometer), molecular weight and fragment length distribution (gel electrophoresis), and qualitative analysis in terms of the composition and distribution of fungal and bacterial communities (Denaturing Gradient Gel Electrophoresis- fingerprinting). The mobile soil eDNA, extracted from each horizon, was characterised by low molecular weight (< 2 kb) and amounts ranging from 3.96 (±0.179) to 0.17 (±0.023) µg g-1 for the fine earth and from 1.42 (±0.111) to 0.11 (±0.007) µg g-1 for the skeleton. Genetic fingerprinting of eDNA recovered from fine earth and skeleton revealed characteristic fungal and bacterial communities of each horizon, but also similarities among the microbial communities of both soil fractions and horizons. This could be interpreted also as a result of the movement of eDNA along the soil profile and from fine earth to skeleton. The molecular characterization

  3. Electrophoretic deposition of biomaterials

    PubMed Central

    Boccaccini, A. R.; Keim, S.; Ma, R.; Li, Y.; Zhitomirsky, I.

    2010-01-01

    Electrophoretic deposition (EPD) is attracting increasing attention as an effective technique for the processing of biomaterials, specifically bioactive coatings and biomedical nanostructures. The well-known advantages of EPD for the production of a wide range of microstructures and nanostructures as well as unique and complex material combinations are being exploited, starting from well-dispersed suspensions of biomaterials in particulate form (microsized and nanoscale particles, nanotubes, nanoplatelets). EPD of biological entities such as enzymes, bacteria and cells is also being investigated. The review presents a comprehensive summary and discussion of relevant recent work on EPD describing the specific application of the technique in the processing of several biomaterials, focusing on (i) conventional bioactive (inorganic) coatings, e.g. hydroxyapatite or bioactive glass coatings on orthopaedic implants, and (ii) biomedical nanostructures, including biopolymer–ceramic nanocomposites, carbon nanotube coatings, tissue engineering scaffolds, deposition of proteins and other biological entities for sensors and advanced functional coatings. It is the intention to inform the reader on how EPD has become an important tool in advanced biomaterials processing, as a convenient alternative to conventional methods, and to present the potential of the technique to manipulate and control the deposition of a range of nanomaterials of interest in the biomedical and biotechnology fields. PMID:20504802

  4. Electrophoretic separation of kidney and pituitary cells on STS-8

    NASA Astrophysics Data System (ADS)

    Morrison, D. R.; Nachtwey, D. S.; Barlow, G. H.; Cleveland, C.; Lanham, J. W.; Farrington, M. A.; Hatfield, J. M.; Hymer, W. C.; Todd, P.; Wilfinger, W.; Grindeland, R.; Lewis, M. L.

    A Continuous Flow Electrophoresis System (CFES) was used on Space Shuttle flight STS-8 to separate specific secretory cells from suspensions of cultured primary human embryonic kidney cells and rat pituitary cells. The objectives were to isolate the subfractions of kidney cells that produce the largest amounts of urokinase (plasminogen activator), and to isolate the subfractions of rat pituitary cells that secrete growth hormone, prolactin, and other hormones. Kidney cells were separated into more than 32 fractions in each of two electrophoretic runs. Electrophoretic mobility distributions in flight experiments were spread more than the ground controls. Multiple assay methods confirmed that all cultured kidney cell fractions produced some urokinase, and five to six fractions produced significantly more urokinase than the other fractions. Several fractions also produced tissue plasminogen activator. The pituitary cells were separated into 48 fractions in each of the two electrophoretic runs, and the amounts of growth hormone (GH) and prolactin (PRL) released into the medium for each cell fraction were determined. Cell fractions were grouped into eight mobility classes and immunocytochemically assayed for the presence of GH, PRL, ACTH, LH, TSH, and FSH. The patterns of hormone distribution indicate that the specialized cells producing GH and PRL are isolatable due to the differences in electrophoretic mobilities.

  5. A DNA-Inspired Encryption Methodology for Secure, Mobile Ad Hoc Networks

    NASA Technical Reports Server (NTRS)

    Shaw, Harry

    2012-01-01

    Users are pushing for greater physical mobility with their network and Internet access. Mobile ad hoc networks (MANET) can provide an efficient mobile network architecture, but security is a key concern. A figure summarizes differences in the state of network security for MANET and fixed networks. MANETs require the ability to distinguish trusted peers, and tolerate the ingress/egress of nodes on an unscheduled basis. Because the networks by their very nature are mobile and self-organizing, use of a Public Key Infra structure (PKI), X.509 certificates, RSA, and nonce ex changes becomes problematic if the ideal of MANET is to be achieved. Molecular biology models such as DNA evolution can provide a basis for a proprietary security architecture that achieves high degrees of diffusion and confusion, and resistance to cryptanalysis. A proprietary encryption mechanism was developed that uses the principles of DNA replication and steganography (hidden word cryptography) for confidentiality and authentication. The foundation of the approach includes organization of coded words and messages using base pairs organized into genes, an expandable genome consisting of DNA-based chromosome keys, and a DNA-based message encoding, replication, and evolution and fitness. In evolutionary computing, a fitness algorithm determines whether candidate solutions, in this case encrypted messages, are sufficiently encrypted to be transmitted. The technology provides a mechanism for confidential electronic traffic over a MANET without a PKI for authenticating users.

  6. Electrophoretic approach to the biochemical systematics of gammarids

    NASA Astrophysics Data System (ADS)

    Bulnheim, H.-P.; Scholl, A.

    1981-12-01

    By utilizing the techniques for electrophoretic separation of proteins by vertical starch gels, the biochemical systematics of 10 Gammaridae species obtained from marine, brackish and freshwater habitats was studied. They included Chaetogammarus marinus, Gammarus zaddachi, G. salinus, G. oceanicus, G. tigrinus, G. chevreuxi, G. locusta, G. duebeni duebeni, G. d. celticus, G. pulex pulex, and G. fossarum. For comparison of electrophoretic mobilities selected enzymes (phosphoglucose isomerase, glutamate oxalacetate transaminase, arginine phosphokinase, hexokinase, leucine amino peptidase, mannose 6-phosphate isomerase) were assayed. They were used as diagnostic characters in terms of electrophoretic identities or diversities of most frequent alleles at polymorphic gene loci. These criteria could be applied to estimate intrageneric enzymic variation and degrees of genetic relatedness between the crustacean amphipod species under consideration, thereby complementing traditional morphological classification.

  7. Bioinorganic Chemistry Special Feature: Gapped DNA is anisotropically bent

    NASA Astrophysics Data System (ADS)

    Guo, Hong; Tullius, Thomas D.

    2003-04-01

    Ionizing radiation damages DNA in several ways, including through formation of a single-nucleoside gap in one DNA strand. We have developed a two-dimensional gel electrophoresis method to investigate the effect of a strand gap on DNA structure. We generate a library of gapped DNA molecules by treating a DNA restriction fragment with the hydroxyl radical, generated by the reaction of Fe(II) EDTA with hydrogen peroxide. The DNA molecule studied contains a fixed bend produced by a set of phased adenine tracts. The A-tract bend serves as a reference bend for investigating the conformational nature of a strand gap. In the first electrophoretic dimension, a bent DNA molecule that has been treated with the hydroxyl radical is electrophoresed on a native gel. Smearing of the band on the native gel indicates that the library of gapped DNA molecules contains a variety of DNA conformations. In the second electrophoretic dimension, gapped DNA molecules having different native gel mobilities are electrophoresed on separate lanes of a denaturing gel to reveal how each strand gap affects the native gel mobility (and thus shape) of the DNA. Our results demonstrate that a single-nucleoside gap in a DNA duplex leads to an anisotropic, directional bend in the DNA helix axis. The implications of our findings for recognition of this lesion by DNA repair proteins are discussed.

  8. AN ELECTROPHORETIC STUDY OF A STREPTOCOCCAL PROTEINASE AND ITS PRECURSOR

    PubMed Central

    Shedlovsky, Theodore; Elliott, S. D.

    1951-01-01

    An electrophoretic study of crystalline preparations of a streptococcal proteinase and its precursor established their isoelectric points at pH values of 8.42 and 7.35 respectively (ionic strength 0.10). Preparations of the proteinase appeared to be electrophoretically homogeneous over a pH range of 5 to 8.5. Precursor preparations contained a relatively low concentration of the active enzyme visible as a separate peak in electrophoretic patterns of sufficiently concentrated solutions. Autocatalytic conversion of precursor to active enzyme was complete and resulted in a corresponding change in the electrophoretic pattern. Treatment of precursor preparations with trypsin produced incomplete conversion to the active enzyme and resulted in the formation of a modified precursor protein. This differed from the parent substance in electrophoretic mobility and in susceptibility to trypsin, but resembled it in immunological specificity and, as previously shown, in susceptibility to conversion to active enzyme by autocatalysis. Serological reactions of precursor and active enzyme components withdrawn from the cell after electrophoresis are described. It appears that the precursor protein may have two antigenic groups, one specific, the other shared by the active enzyme which behaves as a single antigen. PMID:14888818

  9. A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals.

    PubMed

    Wooten, Dennis C; Starr, Clarise R; Lyon, Wanda J

    2016-01-01

    Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and

  10. Nanoscale histone localization in live cells reveals reduced chromatin mobility in response to DNA damage

    PubMed Central

    Liu, Jing; Vidi, Pierre-Alexandre; Lelièvre, Sophie A.; Irudayaraj, Joseph M. K.

    2015-01-01

    ABSTRACT Nuclear functions including gene expression, DNA replication and genome maintenance intimately rely on dynamic changes in chromatin organization. The movements of chromatin fibers might play important roles in the regulation of these fundamental processes, yet the mechanisms controlling chromatin mobility are poorly understood owing to methodological limitations for the assessment of chromatin movements. Here, we present a facile and quantitative technique that relies on photoactivation of GFP-tagged histones and paired-particle tracking to measure chromatin mobility in live cells. We validate the method by comparing live cells to ATP-depleted cells and show that chromatin movements in mammalian cells are predominantly energy dependent. We also find that chromatin diffusion decreases in response to DNA breaks induced by a genotoxic drug or by the ISceI meganuclease. Timecourse analysis after cell exposure to ionizing radiation indicates that the decrease in chromatin mobility is transient and precedes subsequent increased mobility. Future applications of the method in the DNA repair field and beyond are discussed. PMID:25501817

  11. Electrophoretic Deposition for Fabricating Microbatteries

    NASA Technical Reports Server (NTRS)

    West, William; Whitacre, Jay; Bugga, Ratnakumar

    2003-01-01

    An improved method of fabrication of cathodes of microbatteries is based on electrophoretic deposition. Heretofore, sputtering (for deposition) and the use of photoresist and liftoff (for patterning) have been the primary methods of fabricating components of microbatteries. The volume of active electrode material that can be deposited by sputtering is limited, and the discharge capacities of prior microbatteries have been limited accordingly. In addition, sputter deposition is slow. In contrast, electrophoretic deposition is much faster and has shown promise for increasing discharge capacities by a factor of 10, relative to those of microbatteries fabricated by prior methods.

  12. Electrophoretic Process For Purifying Wastewater

    NASA Technical Reports Server (NTRS)

    Sammons, David W.; Twitty, Garland E.; Sharnez, Rizwan; Egen, Ned B.

    1992-01-01

    Microbes, poisonous substances, and colloidal particles removed by combination of electric fields. Electrophoretic process removes pathogenicorganisms, toxins, toxic metals, and cooloidal soil particles from wastewater. Used to render domestic, industrial, and agricultural wastewater streams potable. Process also useful in bioregenerative and other closed systems like in space stations and submarines, where water must be recycled.

  13. Diffusion of DNA during gel electrophoresis; a predictive function spanning the relevant regimes

    NASA Astrophysics Data System (ADS)

    McCormick, Laurette; Slater, Gary

    2004-03-01

    Gel electrophoresis is used extensively to separate DNA. Diffusion of the DNA bands during electrophoresis is an important phenomenon which reduces the resolution obtained. As with DNA mobility, the diffusion of DNA can be split into several different regimes, each described by relevant theory. Unfortunately, until recently there was no single formula for DNA mobility or diffusion that could be used in more than one regime. However, Van Winkle and co workers [Van Winkle DH, Beheshti A, Rill RL, ELECTROPHORESIS 23 (1): 15-19 JAN 2002] have successfully developed an analytical function to analyze DNA mobility data, throughout the relevant regimes. We present the development of a complementary function for the analysis of DNA diffusion. This function should be very useful both in analyzing DNA electrophoretic data, and as a predictive tool.

  14. An electrophoretic study of urinary protein in the rat.

    PubMed

    SELLERS, A L; ROBERTS, S; RASK, I; SMITH, S; MARMORSTON, J; GOODMAN, H C

    1952-05-01

    The nature of the proteins present in the urine of the normal rat has been investigated by electrophoretic analysis and by fractional precipitation of these proteins by ammonium sulfate. Components similar to serum alpha- and beta-globulin constitute the major portion of the urinary protein in both male and female rats. Following the intraperitoneal injection of renin, a massive proteinuria occurs. The proteins excreted are similar in proportion and electric mobility to those of normal rat serum. PMID:14927799

  15. AN ELECTROPHORETIC STUDY OF URINARY PROTEIN IN THE RAT

    PubMed Central

    Sellers, Alvin L.; Roberts, Sidney; Rask, Irene; Smith, Stephen; Marmorston, Jessie; Goodman, Howard C.

    1952-01-01

    The nature of the proteins present in the urine of the normal rat has been investigated by electrophoretic analysis and by fractional precipitation of these proteins by ammonium sulfate. Components similar to serum α- and β-globulin constitute the major portion of the urinary protein in both male and female rats. Following the intraperitoneal injection of renin, a massive proteinuria occurs. The proteins excreted are similar in proportion and electric mobility to those of normal rat serum. PMID:14927799

  16. Interaction of cis-diamminedichloroplatinum(II) with PM-2 DNA.

    PubMed

    Mong, S; Huang, C H; Prestayko, A W; Crooke, S T

    1980-09-01

    The interaction of cis-diamminedichloroplatinum(II) (CDDP) with PM-2 DNA was studied using two techniques: (a) agarose gel electrophoresis of PM-2 DNA conformation isomers after CDDP binding; and (b) viscometric measurement of different forms of CDDP-bound PM-2 DNA. In both systems, the results indicated that the DNA isomers interacted differently with CDDP. CDDP induced a decrease of viscosity upon interacting with single-strand broken relaxed circular (Form II) and double-strand broken linear (Form III) PM-2 DNA's. These observations are consistent with a "DNA shortening effect" proposed by Cohen et al. [Science (Wash. D. C.), 203: 1014-1016, 1979] and Macquet et al. [Biochimie (Paris), 60: 901-914, 1978] When covalently closed circular (Form I) PM-2 DNA was used, increasing concentrations of CDDP induced an initial slight increase and then decrease of electrophoretic mobility to the degree that it comigrated with CDDP-bound Form II DNA. Further addition of CDDP restored the electrophoretic mobility of Form I DNA. Corresponding changes in the viscosity of CDDP-bound Form I DNA showed an initial decrease, then an increase, and a final prolonged decrease of viscosity. These effects are similar but not identical to those induced by either DNA intercalators (e.g., ethidium bromide) or certain DNA denaturating agents (e.g., formaldehyde, ultraviolet light, alkali trichloroacetate, methylmercuric hydroxide, and carbodiimide). Thus, CDP may induce a DNA superhelix-unwinding process followed either by rewinding or a denaturation process or both. Quantitative analysis of the agarose gel electrophoretic pattern plus sucrose density gradient centrifugation studies also indicated that there was little DNA strand breakage induced by CDDP treatment. PMID:7191776

  17. Identification and quantitation of morphological cell types in electrophoretically separated human embryonic kidney cell cultures

    NASA Technical Reports Server (NTRS)

    Williams, K. B.; Kunze, M. E.; Todd, P. W.

    1985-01-01

    Four major cell types were identified by phase microscopy in early passage human embryonic kidney cell cultures. They are small and large epithelioid, domed, and fenestrated cells. Fibroblasts are also present in some explants. The percent of each cell type changes with passage number as any given culture grows. As a general rule, the fraction of small epithelioid cells increases, while the fraction of fenestrated cells, always small, decreases further. When fibroblasts are present, they always increase in percentage of the total cell population. Electrophoretic separation of early passage cells showed that the domed cells have the highest electrophoretic mobility, fibroblasts have an intermediate high mobility, small epithelioid cells have a low mobility, broadly distributed, and fenestrated cells have the lowest mobility. All cell types were broadly distributed among electrophoretic subfractions, which were never pure but only enriched with respect to a given cell type.

  18. Universal scaling of crowding-induced DNA mobility is coupled with topology-dependent molecular compaction and elongation.

    PubMed

    Gorczyca, Stephanie M; Chapman, Cole D; Robertson-Anderson, Rae M

    2015-10-21

    Using single-molecule fluorescence microscopy and particle-tracking techniques, we elucidate the role DNA topology plays in the diffusion and conformational dynamics of crowded DNA molecules. We focus on large (115 kbp), double-stranded ring and linear DNA crowded by varying concentrations (0-40%) of dextran (10, 500 kDa) that mimic cellular conditions. By tracking the center-of-mass and measuring the lengths of the major and minor axes of single DNA molecules, we characterize both DNA mobility reduction as well as crowding-induced conformational changes (from random spherical coils). We reveal novel topology-dependent conformations, with single ring molecules undergoing compaction to ordered spherical configurations ∼20% smaller than dilute random coils, while linear DNA elongates by ∼2-fold. Surprisingly, these highly different conformations result in nearly identical exponential mobility reduction dependent solely on crowder volume fraction Φ, revealing a universal critical crowding concentration of Φc≅ 2.3. Beyond Φc DNA exhibits topology-independent conformational relaxation dynamics despite highly distinct topology-driven conformations. Our collective results reveal that topology-dependent conformational changes, unique to crowded environments, enable DNA to overcome the classically expected mobility reduction that high-viscosity crowded environments impose. Such coupled universal dynamics suggest a mechanism for DNA to maintain sufficient mobility required for wide-ranging biological processes despite severe cellular crowding. PMID:26303877

  19. High mobility of flap endonuclease 1 and DNA polymerase eta associated with replication foci in mammalian S-phase nucleus.

    PubMed

    Solovjeva, Lioudmila; Svetlova, Maria; Sasina, Lioudmila; Tanaka, Kyoji; Saijo, Masafumi; Nazarov, Igor; Bradbury, Morton; Tomilin, Nikolai

    2005-05-01

    Originally detected in fixed cells, DNA replication foci (RFi) were later visualized in living cells by using green fluorescent protein (GFP)-tagged proliferating cell nuclear antigen (PCNA) and DNA ligase I. It was shown using fluorescence redistribution after photobleaching (FRAP) assay that focal GFP-PCNA slowly exchanged, suggesting the existence of a stable replication holocomplex. Here, we used the FRAP assay to study the dynamics of the GFP-tagged PCNA-binding proteins: Flap endonuclease 1 (Fen1) and DNA polymerase eta (Pol eta). We also used the GFP-Cockayne syndrome group A (CSA) protein, which does associate with transcription foci after DNA damage. In normal cells, GFP-Pol eta and GFP-Fen1 are mobile with residence times at RFi (t(m)) approximately 2 and approximately 0.8 s, respectively. GFP-CSA is also mobile but does not concentrate at discrete foci. After methyl methanesulfonate (MMS) damage, the mobile fraction of focal GFP-Fen1 decreased and t(m) increased, but it then recovered. The mobilities of focal GFP-Pol eta and GFP-PCNA did not change after MMS. The mobility of GFP-CSA did not change after UV-irradiation. These data indicate that the normal replication complex contains at least two mobile subunits. The decrease of the mobile fraction of focal GFP-Fen1 after DNA damage suggests that Fen1 exchange depends on the rate of movement of replication forks. PMID:15758026

  20. Detection, purification and characterization of a protein that binds the (6-4) photoproduct-containing DNA in HeLa cells.

    PubMed

    Fujiwara, Y; Masutani, C; Hanaoka, F; Iwai, S

    1997-01-01

    HeLa cell proteins that bind DNA containing the pyrimidine(6-4)pyrimidone photoproduct were detected by the electrophoretic mobility shift assay using synthetic oligonucleotide duplexes as probes. The major species was purified to near homogeneity, and the amino acid sequences of the proteolytic peptides revealed that it was the human damage-specific DNA-binding protein, which was reported previously. The substrate specificity of this protein was determined using damaged or modified DNA duplexes. PMID:9586107

  1. Sensitive and robust electrophoretic NMR: Instrumentation and experiments

    NASA Astrophysics Data System (ADS)

    Hallberg, Fredrik; Furó, István; Yushmanov, Pavel V.; Stilbs, Peter

    2008-05-01

    Although simple as a concept, electrophoretic NMR (eNMR) has so far failed to find wider application. Problems encountered are mainly due to disturbing and partly irreproducible convection-like bulk flow effects from both electro-osmosis and thermal convection. Additionally, bubble formation at the electrodes and rf noise pickup has constrained the typical sample geometry to U-tube-like arrangements with a small filling factor and a low resulting NMR sensitivity. Furthermore, the sign of the electrophoretic mobility cancels out in U-tube geometries. We present here a new electrophoretic sample cell based on a vertically placed conventional NMR sample tube with bubble-suppressing palladium metal as electrode material. A suitable radiofrequency filter design prevents noise pickup by the NMR sample coil from the high-voltage leads which extend into the sensitive sample volume. Hence, the obtained signal-to-noise ratio of this cell is one order of magnitude higher than that of our previous U-tube cells. Permitted by the retention of the sign of the displacement-related signal phase in the new cell design, an experimental approach is described where bulk flow effects by electro-osmosis and/or thermal convection are compensated through parallel monitoring of a reference signal from a non-charged species in the sample. This approach, together with a CPMG-like pulse train scheme provides a superior first-order cancellation of non-electrophoretic bulk flow effects.

  2. Design Modification of Electrophoretic Equipment

    NASA Technical Reports Server (NTRS)

    Reddick, J. M.; Hirsch, I.

    1973-01-01

    The improved design of a zone electrophoretic sampler is reported that can be used in mass screening for hemoglobin S, the cause of sickle cell anemia. Considered is a high voltage multicell cellulose acetate device that requires 5 to 6 minutes electrophoresis periods; cells may be activitated individually or simultaneously. A multisample hemoglobin applicator standardizes the amount of sample applied and transfers the homolysate to the electrical wires.

  3. Hole mobilities of periodic models of DNA double helices in the nucleosomes at different temperatures

    NASA Astrophysics Data System (ADS)

    Bende, Attila; Bogár, Ferenc; Ladik, János

    2013-04-01

    Using the Hartree-Fock crystal orbital method band structures of poly(G˜-C˜) and poly(A˜-T˜) were calculated (G˜, etc. means a nucleotide) including water molecules and Na+ ions. Due to the close packing of DNA in the ribosomes the motion of the double helix and the water molecules around it are strongly restricted, therefore the band picture can be used. The mobilities were calculated from the highest filled bands. The hole mobilities increase with decreasing temperatures. They are of the same order of magnitude as those of poly(A˜) and poly(T˜). For poly(G˜) the result is ˜5 times larger than in the poly(G˜-C˜) case.

  4. Electrophoresis of DNA on a disordered two-dimensional substrate

    NASA Astrophysics Data System (ADS)

    Olson Reichhardt, Cynthia J.; Reichhardt, Charles

    2006-03-01

    We propose a new method for electrophoretic separation of DNA in which adsorbed polymers are driven over a disordreed two-dimensional substrate which contains attractive sites for the polymers. Using simulations of a model for long polymer chains, we show that the mobility increases with polymer length, in contrast to gel electrophoresis techniques, and that separation can be achieved for a range of length scales. We demonstrate that the separation mechanism relies on excluded volume interactions between polymer segments.

  5. Time-of-flight studies of hole mobilities in DNA-CTMA films fabricated and passivated in a dry environment

    NASA Astrophysics Data System (ADS)

    Yaney, Perry P.; Gorman, Timothy T.; Ouchen, Fahima; Grote, James G.

    2012-01-01

    Salmon DNA-based films, including as-received DNA (molecular weight, MW>2000 kDa) and sonicated DNA of MW ˜200 kDa, both complexed with hexacetyltrimethyl-ammonium chloride (CTMA) surfactant, were studied. The DNA solutions were spin-coated on indium tin oxide (ITO)-coated quartz slides with vacuum deposited gold charge-collecting electrodes. The films were fabricated entirely at ˜0% humidity (0 to 86 ppmv of water) in a nitrogen-purged glove box and coated with 500 to 600 nm passivating layers of conformal urethane before exposure to room air. A quadrupled, 20 ns pulsed Nd:YAG laser with output at 266 nm was used for charge injection. The room temperature photoconductive transients were dispersive with hole mobilities in DNA films ranging between 7E-3 to 5E-5 cm2/Vs for fields ranging from 10 to 380 kV/cm. No electron response was observed in these films. The mobilities were determined from the transient curves at the intersections of initial and final tangent lines that defined the shoulders in the log-log plots. The results of these hole mobility studies, which appear to support predictions of high hole mobility in DNA, are the first on DNA-based films that were fabricated in a dry environment and passivated for measurements in room air.

  6. FLP-mediated DNA mobilization to specific target sites in Drosophila chromosomes.

    PubMed Central

    Golic, M M; Rong, Y S; Petersen, R B; Lindquist, S L; Golic, K G

    1997-01-01

    The ability to place a series of gene constructs at a specific site in the genome opens new possibilities for the experimental examination of gene expression and chromosomal position effects. We report that the FLP- FRT site-specific recombination system of the yeast 2mu plasmid can be used to integrate DNA at a chromosomal FRT target site in Drosophila. The technique we used was to first integrate an FRT- flanked gene by standard P element-mediated transformation. FLP was then used to excise the FRT- flanked donor DNA and screen for FLP-mediated re-integration at an FRT target at a different chromosome location. Such events were recovered from up to 5% of the crosses used to screen for mobilization and are easily detectable by altered linkage of a white reporter gene or by the generation of a white + gene upon integration. PMID:9278488

  7. High mobility group protein-mediated transcription requires DNA damage marker γ-H2AX

    PubMed Central

    Singh, Indrabahadur; Ozturk, Nihan; Cordero, Julio; Mehta, Aditi; Hasan, Diya; Cosentino, Claudia; Sebastian, Carlos; Krüger, Marcus; Looso, Mario; Carraro, Gianni; Bellusci, Saverio; Seeger, Werner; Braun, Thomas; Mostoslavsky, Raul; Barreto, Guillermo

    2015-01-01

    The eukaryotic genome is organized into chromatins, the physiological template for DNA-dependent processes including replication, recombination, repair, and transcription. Chromatin-mediated transcription regulation involves DNA methylation, chromatin remodeling, and histone modifications. However, chromatin also contains non-histone chromatin-associated proteins, of which the high-mobility group (HMG) proteins are the most abundant. Although it is known that HMG proteins induce structural changes of chromatin, the processes underlying transcription regulation by HMG proteins are poorly understood. Here we decipher the molecular mechanism of transcription regulation mediated by the HMG AT-hook 2 protein (HMGA2). We combined proteomic, ChIP-seq, and transcriptome data to show that HMGA2-induced transcription requires phosphorylation of the histone variant H2AX at S139 (H2AXS139ph; γ-H2AX) mediated by the protein kinase ataxia telangiectasia mutated (ATM). Furthermore, we demonstrate the biological relevance of this mechanism within the context of TGFβ1 signaling. The interplay between HMGA2, ATM, and H2AX is a novel mechanism of transcription initiation. Our results link H2AXS139ph to transcription, assigning a new function for this DNA damage marker. Controlled chromatin opening during transcription may involve intermediates with DNA breaks that may require mechanisms that ensure the integrity of the genome. PMID:26045162

  8. Diffusion, Dispersion, and Mobility of Single-stranded DNA in Polyacrylamide Gel Electrophoresis

    NASA Astrophysics Data System (ADS)

    Lo, Roger; Ugaz, Victor

    2004-03-01

    The ability to perform DNA electrophoresis in miniaturized microfluidic systems has the potential to provide a new generation of low-cost high-throughput genomic analysis technology. Further progress toward improving separation performance under these conditions, however, requires a more detailed understanding of diffusion and dispersion phenomena in the gel matrix. Unfortunately, it has thus far proven difficult to obtain extensive measurements of these quantities due in large part to the lack of a convenient experimental platform. In this paper, we demonstrate the use of microfabricated gel electrophoresis devices to measure diffusion, dispersion, and mobility of single-stranded DNA fragments in crosslinked and uncrosslinked polyacrylamide gels. The microdevice format allows a complete set of diffusion and dispersion data to be collected in approximately one hour, as opposed to experiment times lasting several days using conventional sequencing equipment. By comparing runs using identical DNA samples, gel formulations, and operating conditions in both microfabricated electrophoresis devices and an ALF Express automated DNA sequencer, we are able to isolate the key factors governing separation performance in each system. The results of these experiments are then compared with biased reptation theory to extract information about the gel structure and predict achievable resolution. The effects of gel composition and polymerization chemistry are also explored.

  9. ATM Alters the Otherwise Robust Chromatin Mobility at Sites of DNA Double-Strand Breaks (DSBs) in Human Cells

    PubMed Central

    Becker, Annabelle; Durante, Marco; Taucher-Scholz, Gisela; Jakob, Burkhard

    2014-01-01

    Ionizing radiation induces DNA double strand breaks (DSBs) which can lead to the formation of chromosome rearrangements through error prone repair. In mammalian cells the positional stability of chromatin contributes to the maintenance of genome integrity. DSBs exhibit only a small, submicron scale diffusive mobility, but a slight increase in the mobility of chromatin domains by the induction of DSBs might influence repair fidelity and the formation of translocations. The radiation-induced local DNA decondensation in the vicinity of DSBs is one factor potentially enhancing the mobility of DSB-containing chromatin domains. Therefore in this study we focus on the influence of different chromatin modifying proteins, known to be activated by the DNA damage response, on the mobility of DSBs. IRIF (ionizing radiation induced foci) in U2OS cells stably expressing 53BP1-GFP were used as a surrogate marker of DSBs. Low angle charged particle irradiation, known to trigger a pronounced DNA decondensation, was used for the defined induction of linear tracks of IRIF. Our results show that movement of IRIF is independent of the investigated chromatin modifying proteins like ACF1 or PARP1 and PARG. Also depletion of proteins that tether DNA strands like MRE11 and cohesin did not alter IRIF dynamics significantly. Inhibition of ATM, a key component of DNA damage response signaling, resulted in a pronounced confinement of DSB mobility, which might be attributed to a diminished radiation induced decondensation. This confinement following ATM inhibition was confirmed using X-rays, proving that this effect is not restricted to densely ionizing radiation. In conclusion, repair sites of DSBs exhibit a limited mobility on a small spatial scale that is mainly unaffected by depletion of single remodeling or DNA tethering proteins. However, it relies on functional ATM kinase which is considered to influence the chromatin structure after irradiation. PMID:24651490

  10. Electrophoretic separator for purifying biologicals

    NASA Technical Reports Server (NTRS)

    1975-01-01

    Mathematical expressions were developed to describe the interrelationships between operating requirements (capabilities), cell parameters, and system constraints in terms of design criteria definition. The mathematical model was programmed for computer solution. The model was exercised to identify performance-limiting characteristics, and analyses were conducted to predict operation in space of an experiment involving separation of four components. An engineering model of a flowing electrophoretic separator was constructed. The design is directed toward verifying improvements in resolution and throughput of a thicker cell than can be used on earth.

  11. Nonlinear electrophoretic response yields a unique parameter for separation of biomolecules

    PubMed Central

    Pel, Joel; Broemeling, David; Mai, Laura; Poon, Hau-Ling; Tropini, Giorgia; Warren, René L.; Holt, Robert A.; Marziali, Andre

    2009-01-01

    We demonstrate a unique parameter for biomolecule separation that results from the nonlinear response of long, charged polymers to electrophoretic fields and apply it to extraction and concentration of nucleic acids from samples that perform poorly under conventional methods. Our method is based on superposition of synchronous, time-varying electrophoretic fields, which can generate net drift of charged molecules even when the time-averaged molecule displacement generated by each field individually is zero. Such drift can only occur for molecules, such as DNA, whose motive response to electrophoretic fields is nonlinear. Consequently, we are able to concentrate DNA while rejecting high concentrations of contaminants. We demonstrate one application of this method by extracting DNA from challenging samples originating in the Athabasca oil sands. PMID:19706437

  12. Objective data on DNA success rates can aid the selection process of crime samples for analysis by rapid mobile DNA technologies.

    PubMed

    Mapes, A A; Kloosterman, A D; Poot, C J de; van Marion, V

    2016-07-01

    Mobile Rapid-DNA devices have recently become available on the market. These devices can perform DNA analyses within 90min with an easy 'sample in-answer out' system, with the option of performing comparisons with a DNA database or reference profile. However, these fast mobile systems cannot yet compete with the sensitivity of the standard laboratory analysis. For the future this implies that Scene of Crime Officers (SoCOs) need to decide on whether to analyse a crime sample with a Rapid-DNA device and to get results within 2h or to secure and analyse the sample at the laboratory with a much longer throughput time but with higher sensitivity. This study provides SoCOs with evidence-based information on DNA success rates, which can improve their decisions at the crime scene on whether or not to use a Rapid-DNA device. Crime samples with a high success rate in the laboratory will also have the highest potential for Rapid-DNA analysis. These include samples from e.g. headwear, cigarette ends, articles of clothing, bloodstains, and drinking items. PMID:27015156

  13. Electrophoretic separator for purifying biologicals, part 1

    NASA Technical Reports Server (NTRS)

    Mccreight, L. R.

    1978-01-01

    A program to develop an engineering model of an electrophoretic separator for purifying biologicals is summarized. An extensive mathematical modeling study and numerous ground based tests were included. Focus was placed on developing an actual electrophoretic separator of the continuous flow type, configured and suitable for flight testing as a space processing applications rocket payload.

  14. Urokinase production by electrophoretically separated cultured human embryonic kidney cells

    NASA Technical Reports Server (NTRS)

    Kunze, M. E.; Plank, L. D.; Giranda, V.; Sedor, K.; Todd, P. W.

    1985-01-01

    Urokinase is a plasminogen activator found in urine. Relatively pure preparations have been tested in Europe, Japan and the United States for the treatment of deep vein thrombosis and other dangerous blood clots. Human embryonic kidney cell cultures have been found to produce urokinase at much higher concentrations, but less than 5% of the cells in typical cultures are producers. Since human diploid cells become senescent in culture the selection of clones derived from single cells will not provide enough material to be useful, so a bulk purification method is needed for the isolation of urokinase producing cell populations. Preparative cell electrophoresis was chosen as the method, since evidence exists that human embryonic cell cultures are richly heterogeneous with respect to electrophoretic mobility, and preliminary electrophoretic separations on the Apollo-Soyuz space flight produced cell populations that were rich in urokinase production. Similarly, erythropoietin is useful in the treatment of certain anemias and is a kidney cell duct, and electrophoretically enriched cell populations producing this product have been reported. Thus, there is a clear need for diploid human cells that produce these products, and there is evidence that such cells should be separable by free-flow cell electrophoresis.

  15. Strain-specific mobilization and amplification of a transgenic defective-interfering DNA of the geminivirus beet curly top virus.

    PubMed

    Stenger, D C

    1994-09-01

    Transgenic Nicotiana benthamiana plants have been constructed which bear integrated, tandemly repeated copies of a beet curly top virus (BCTV) defective-interfering (DI) DNA derived from the Logan strain. Transgenic DI-DNA plant lines challenge-inoculated with BCTV-Logan exhibited delayed and attenuated symptoms compared to nontransgenic plants. Infection of transgenic plants with the Logan strain resulted in the mobilization of the integrated DI-DNA sequence, which was subsequently amplified as an episome. The accumulation of Logan helper virus DNA forms was reduced in transgenic plants, relative to nontransgenic plants. In contrast, no delay or attenuation of symptoms was observed for transgenic plants challenge-inoculated with the BCTV strains CFH and Worland. Infection by the CFH and Worland strains did not result in mobilization or amplification of the integrated Logan DI-DNA sequence, and no consistent differences in the accumulation of CFH or Worland genomic viral DNA forms were observed among transgenic and nontransgenic plants. These results, and a comparison of putative DNA replication origin sequences, suggest that BCTV strains display specificity with respect to recognition of heterologous DNA replication origin cis-elements. PMID:8053165

  16. Electrophoretic separation of kidney and pituitary cells on STS-8

    NASA Technical Reports Server (NTRS)

    Morrison, D. R.; Nachtwey, D. S.; Barlow, G. H.; Cleveland, C.; Lanham, J. W.; Farrington, M. A.; Hatfield, J. M.; Hymer, W. C.; Grindeland, R.; Lewis, M. L.

    1984-01-01

    Specific secretory cells were separated from suspensions of cultured primary human embryonic cells and rat pituitary cells in microgravity conditions, with an objective of isolating the subfractions of kidney cells that produce the largest amount of urakinase, and the subfractions of rat pituitary cells that secrete growth hormones (GH), prolactin (PRL), and other hormones. It is inferred from the experimental observations that the surface charge distributions of the GH-containing cells differ from those of the PRL-containing cells, which is explained by the presence of secretory products on the surface of pituitary cells. For kidney cells, the electrophoretic mobility distributions in flight experiments were spread more than the ground controls.

  17. Human immunodeficiency virus type 1 evolution in vivo tracked by DNA heteroduplex mobility assays.

    PubMed Central

    Delwart, E L; Sheppard, H W; Walker, B D; Goudsmit, J; Mullins, J I

    1994-01-01

    High mutation rates and strong selective pressures imposed on human immunodeficiency viruses in vivo result in the formation of pools of genetic variants known as quasispecies. DNA heteroduplex mobility and tracking analyses were used to monitor the generation of HIV sequence diversity, to estimate quasispecies complexity, and to assess the turnover of genetic variants to approach an understanding of the relationship between viral quasispecies evolution in vivo and disease progression. Proviral DNA pools were nearly homogeneous soon after sexual transmission. The emergence and clearance of individual variants then occurred at different rates in different individuals. High quasispecies complexity was found in long-term-infected, asymptomatic individuals, while rapid CD4+ cell decline and AIDS were often, but not always, associated with lower quasispecies complexity. Proviral genetic variation was often low following in vitro culture, because of the outgrowth of one or a few variants that often became more abundant only later as proviruses in peripheral blood mononuclear cells. These studies provide insight into the dynamics of human immunodeficiency virus sequence changes in vivo and illustrate the utility of heteroduplex analysis for the study of phenomena associated with rapid genetic changes. Images PMID:8084001

  18. Analyses of gonococcal H8 antigen. Surface location, inter- and intrastrain electrophoretic heterogeneity, and unusual two-dimensional electrophoretic characteristics.

    PubMed

    Hitchcock, P J; Hayes, S F; Mayer, L W; Shafer, W M; Tessier, S L

    1985-12-01

    The H8 protein is a surface-exposed antigen that is found, among members of the Neisseria genus, primarily on pathogenic species. In this study, the surface exposure of H8 was reassessed by four techniques. Results of slide agglutination, indirect fluorescent antibody binding, absorption of sera with whole gonococci, and immune electron microscopy all confirmed the presence of H8 in the outer membrane. The degree to which protein A-gold-labeled monoclonal antibodies bound to H8 was marked, and suggested that this antigen was present in abundant amounts in the outer membrane. Also in this study, the electrophoretic heterogeneity of this common surface antigen was examined. Because H8 stains poorly, electrophoretic mobility was assessed using polyclonal antibodies and a monoclonal antibody that recognizes a common H8 epitope. H8 was analyzed with respect to protein I, lipopolysaccharide (LPS), and pilus and opacity phenotypic variation; results confirmed that heterogeneity of Mr was the rule among strains (21 were examined), however, the variability in Mr was independent of protein I or LPS Mr. In one strain (FA1090), the heterogeneity of H8 was examined among 10 piliation/opacity variants; the H8 (and LPS) Mr was identical in all variants; similar data were generated in strains JS3 and JS1. The electrophoretic mobility of H8 was altered in serum-resistant and neutrophil enzyme-resistant gonococci compared to the sensitive gonococci. Some of the unusual electrophoretic migration characteristics of the antigen were also examined. H8 formed a unique mushroom-shaped band in one-dimensional gels; in a two-dimensional electrophoresis system, the antigen migrated aberrantly, very similarly to LPS. Also seen in the two-dimensional electrophoresis profile were multimers of the H8 antigen; in strain JS3 (Mr 23,500), these migrated at 43,600, 86,000, and greater than 150,000. In other strains, the Mr of the multimers differed depending upon the Mr of the monomer. The two

  19. Improved single-strand DNA sizing accuracy in capillary electrophoresis.

    PubMed Central

    Rosenblum, B B; Oaks, F; Menchen, S; Johnson, B

    1997-01-01

    Interpolation algorithms can be developed to size unknown single-stranded (ss) DNA fragments based on their electrophoretic mobilities, when they are compared with the mobilities of standard fragments of known sizes; however, sequence-specific anomalous electrophoretic migration can affect the accuracy and precision of the called sizes of the fragments. We used the anomalous migration of ssDNA fragments to optimize denaturation conditions for capillary electrophoresis. The capillary electrophoretic system uses a refillable polymer that both coats the capillary wall to suppress electro-osmotic flow and acts as the sieving matrix. The addition of 8 M urea to the polymer solution, as in slab gel electrophoresis, is insufficient to fully denature some anomalously migrating ssDNA fragments in this capillary electrophoresis system. The sizing accuracy of these fragments is significantly improved by the addition of 2-pyrrolidinone, or increased capillary temperature (60 degrees C). the effect of these two denaturing strategies is additive, and the best accuracy and precision in sizing results are obtained with a combination of chemical and thermal denaturation. PMID:9380518

  20. BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells

    PubMed Central

    Reuter, Marcel; Zelensky, Alex; Smal, Ihor; Meijering, Erik; van Cappellen, Wiggert A.; de Gruiter, H. Martijn; van Belle, Gijsbert J.; van Royen, Martin E.; Houtsmuller, Adriaan B.; Essers, Jeroen; Kanaar, Roland

    2014-01-01

    Genome maintenance by homologous recombination depends on coordinating many proteins in time and space to assemble at DNA break sites. To understand this process, we followed the mobility of BRCA2, a critical recombination mediator, in live cells at the single-molecule level using both single-particle tracking and fluorescence correlation spectroscopy. BRCA2-GFP and -YFP were compared to distinguish diffusion from fluorophore behavior. Diffusive behavior of fluorescent RAD51 and RAD54 was determined for comparison. All fluorescent proteins were expressed from endogenous loci. We found that nuclear BRCA2 existed in oligomeric clusters, and exhibited heterogeneous mobility. DNA damage increased BRCA2 transient binding, presumably including binding to damaged sites. Despite its very different size, RAD51 displayed mobility similar to BRCA2, which indicates physical interaction between these proteins both before and after induction of DNA damage. We propose that BRCA2-mediated sequestration of nuclear RAD51 serves to prevent inappropriate DNA interactions and that all RAD51 is delivered to DNA damage sites in association with BRCA2. PMID:25488918

  1. Characterization of DNA recognition by the human UV-damaged DNA-binding protein.

    PubMed

    Fujiwara, Y; Masutani, C; Mizukoshi, T; Kondo, J; Hanaoka, F; Iwai, S

    1999-07-01

    The UV-damaged DNA-binding (UV-DDB) protein is the major factor that binds DNA containing damage caused by UV radiation in mammalian cells. We have investigated the DNA recognition by this protein in vitro, using synthetic oligonucleotide duplexes and the protein purified from a HeLa cell extract. When a 32P-labeled 30-mer duplex containing the (6-4) photoproduct at a single site was used as a probe, only a single complex was detected in an electrophoretic mobility shift assay. It was demonstrated by Western blotting that both of the subunits (p48 and p127) were present in this complex. Electrophoretic mobility shift assays using various duplexes showed that the UV-DDB protein formed a specific, high affinity complex with the duplex containing an abasic site analog, in addition to the (6-4) photoproduct. By circular permutation analyses, these DNA duplexes were found to be bent at angles of 54 degrees and 57 degrees in the complexes with this protein. From the previously reported NMR studies and the fluorescence resonance energy transfer experiments in the present study, it can be concluded that the UV-DDB protein binds DNA that can be bent easily at the above angle. PMID:10391953

  2. Electrophoretic karyotypes of clinical isolates of Coccidioides immitis.

    PubMed Central

    Pan, S; Cole, G T

    1992-01-01

    Chromosomes of the fungal respiratory pathogen, Coccidioides immitis, were separated by contour-clamped homogeneous electric field gel electrophoresis. Twelve isolates were examined, the majority of which showed four chromosomes with a range of molecular size from 11.5 to 3.2 Mb. Three isolates (C634, C735, and L) revealed three chromosomal bands under the conditions employed for electrophoretic separation. However, in two of these isolates (C634 and C735), four chromosomes were visible on membrane transfers of pulsed-field gels after Southern hybridization between the chromosomal DNA and selected DNA probes. The probes included a conserved ribosomal gene and three previously described cDNAs isolated from C. immitis expression libraries. The L isolate was determined to have the same genome size as a typical four-chromosome isolate on the basis of microspectrophotometric comparison of fluorescence intensity of the ethidium bromide-stained nuclear DNA. The genome size of C. immitis determined by microspectrophotometry was approximately 28.2 +/- 2.6 Mb. The calculated genome size based on addition of the average molecular weights of chromosomal bands separated by contour-clamped homogeneous electric field gel electrophoresis was approximately equal to the estimate derived from the spectrophotometric analyses. This is the first report of the electrophoretic karyotype of C. immitis. Images PMID:1398998

  3. Direct Probing of Solvent Accessibility and Mobility at the Binding Interface of Polymerase (Dpo4)-DNA Complex

    NASA Astrophysics Data System (ADS)

    Qin, Yangzhong; Zhong, Dongping

    2014-03-01

    Water plays essential structural and dynamical roles in protein-DNA recognition through contributing to enthalpic or entropic stabilization of binding complex and by mediating intermolecular interactions and fluctuations for biological function. These interfacial water molecules are confined in nanospace but mostly highly mobile. Here, we report our studies of interfacial water dynamics in the binary and ternary complexes of a polymerase (Dpo4) with DNA and an incoming nucleotide using a site-specific tryptophan probe with femtosecond resolution. By systematic comparison of the interfacial water motions and local sidechain fluctuations in the apo, binary and ternary states of Dpo4, we observed that the DNA binding interface and active site is dynamically solvent accessible and the interfacial water dynamics are slightly slow but similar to the surface hydration water fluctuations on picosecond time scales. Our MD simulations also show the binding interface full of water molecules and nonspecific weak interactions with protein and DNA. Such a fluid binding interface facilitates the polymerase sliding on DNA for fast translocation while the spacious and mobile hydrated active site contributes to the low fidelity of the lesion-bypass Y-family DNA polymerase.

  4. Cohesin phosphorylation and mobility of SMC1 at ionizing radiation-induced DNA double-strand breaks in human cells

    SciTech Connect

    Bauerschmidt, Christina; Helleday, Thomas

    2011-02-01

    Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunits contributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage.

  5. Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks.

    PubMed

    Kruhlak, Michael J; Celeste, Arkady; Dellaire, Graham; Fernandez-Capetillo, Oscar; Müller, Waltraud G; McNally, James G; Bazett-Jones, David P; Nussenzweig, André

    2006-03-13

    The repair of DNA double-strand breaks (DSBs) is facilitated by the phosphorylation of H2AX, which organizes DNA damage signaling and chromatin remodeling complexes in the vicinity of the lesion. The disruption of DNA integrity induces an alteration of chromatin architecture that has been proposed to activate the DNA damage transducing kinase ataxia telangiectasia mutated. However, little is known about the physical properties of damaged chromatin. In this study, we use a photoactivatable version of GFP-tagged histone H2B to examine the mobility and structure of chromatin containing DSBs in living cells. We find that chromatin containing DSBs exhibits limited mobility but undergoes an energy-dependent local expansion immediately after DNA damage. The localized expansion observed in real time corresponds to a 30-40% reduction in the density of chromatin fibers in the vicinity of DSBs, as measured by energy-filtering transmission electron microscopy. The observed opening of chromatin occurs independently of H2AX and ATM. We propose that localized adenosine triphosphate-dependent decondensation of chromatin at DSBs establishes an accessible subnuclear environment that facilitates DNA damage signaling and repair. PMID:16520385

  6. 21 CFR 864.7440 - Electrophoretic hemoglobin analysis system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Electrophoretic hemoglobin analysis system. 864....7440 Electrophoretic hemoglobin analysis system. (a) Identification. An electrophoretic hemoglobin analysis system is a device that electrophoretically separates and identifies normal and...

  7. Properties of electrophoretic fractions of human embryonic kidney cells separated on space shuttle flight STS-8

    NASA Astrophysics Data System (ADS)

    Morrison, Dennis R.; Lewis, Marian L.; Barlow, Grant H.; Todd, Paul; Kunze, M. Elaine; Sarnoff, Burton E.; Li, Zhankui

    Suspensions of cultured primary human embryonic kidney cells were subjected to continuous flow electrophoresis on Space Shuttle flight STS-8. The objectives of the experiments were to obtain electrophoretically separated fractions of the original cell populations and to test these fractions for the amount and kind of urokinase (a kidney plasminogen activator that is used medically for digesting blood clots), the morphologies of cells in the individual fractions, and their cellular electrophoretic mobilities after separation and subsequent proliferation. Individual fractions were successfully cultured after return from orbit, and they were found to differ substantially from one another and from the starting sample with respect to all of these properties.

  8. Properties of electrophoretic fractions of human embryonic kidney cells separated on space shuttle flight STS-8

    NASA Technical Reports Server (NTRS)

    Morrison, D. R.; Lewis, M. L.; Barlow, G. H.; Todd, P. W.; Kunze, M. E.; Sarnoff, B. E.; Li, Z. K.

    1985-01-01

    Suspensions of cultured primary human embryonic kidney cells were subjected to continuous flow electrophoresis on Space Shuttle flight STS-8. The objectives of the experiments were to obtain electrophoretically separated fractions of the original cell populations and to test these fractions for the amount and kind of urokinase (a kidney plasminogen activator that is used medically for digesting blood clots), the morphologies of cells in the individual fractions, and their cellular electrophoretic mobilities after separation and subsequent proliferation. Individual fractions were successfully cultured after return from orbit, and they were found to differ substantially from one another and from the starting sample with respect to all of these properties.

  9. CAPILLARY ELECTROPHORETIC BEHAVIOR OF SEVEN SULFONYLUREAS

    EPA Science Inventory

    The electrophoretic behavior of bensulfuron Me, sulfometuron Me, nicosulfuron (Accent), chlorimuron Et, thifensulfuron Me (Harmony), metsulfuron Me, and chlorsulfuron was studied under capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) conditio...

  10. Automated Parallel Capillary Electrophoretic System

    DOEpatents

    Li, Qingbo; Kane, Thomas E.; Liu, Changsheng; Sonnenschein, Bernard; Sharer, Michael V.; Kernan, John R.

    2000-02-22

    An automated electrophoretic system is disclosed. The system employs a capillary cartridge having a plurality of capillary tubes. The cartridge has a first array of capillary ends projecting from one side of a plate. The first array of capillary ends are spaced apart in substantially the same manner as the wells of a microtitre tray of standard size. This allows one to simultaneously perform capillary electrophoresis on samples present in each of the wells of the tray. The system includes a stacked, dual carousel arrangement to eliminate cross-contamination resulting from reuse of the same buffer tray on consecutive executions from electrophoresis. The system also has a gel delivery module containing a gel syringe/a stepper motor or a high pressure chamber with a pump to quickly and uniformly deliver gel through the capillary tubes. The system further includes a multi-wavelength beam generator to generate a laser beam which produces a beam with a wide range of wavelengths. An off-line capillary reconditioner thoroughly cleans a capillary cartridge to enable simultaneous execution of electrophoresis with another capillary cartridge. The streamlined nature of the off-line capillary reconditioner offers the advantage of increased system throughput with a minimal increase in system cost.

  11. Reduced graphene oxide-functionalized high electron mobility transistors for novel recognition pattern label-free DNA sensors.

    PubMed

    Zhang, Xiaohui; Zhang, Yue; Liao, Qingliang; Song, Yu; Ma, Siwei

    2013-12-01

    We designed and constructed reduced graphene oxide (rGO) functionalized high electron mobility transistor (HEMT) for rapid and ultra-sensitive detection of label-free DNA in real time. The micrometer sized rGO sheets with structural defects helped absorb DNA molecules providing a facile and robust approach to functionalization. DNA was immobilized onto the surface of HEMT gate through rGO functionalization, and changed the conductivity of HEMT. The real time monitor and detection of DNA hybridization by rGO functionalized HEMT presented interesting current responses: a "two steps" signal enhancement in the presence of target DNA; and a "one step" signaling with random DNA. These two different recognition patterns made the HEMT capable of specifically detecting target DNA sequence. The working principle of the rGO functionalized HEMT can be demonstrated as the variation of the ambience charge distribution. Furthermore, the as constructed DNA sensors showed excellent sensitivity of detect limit at 0.07 fM with linear detect range from 0.1 fM to 0.1 pM. The results indicated that the HEMT functionalized with rGO paves a new avenue to design novel electronic devices for high sensitive and specific genetic material assays in biomedical applications. PMID:23828864

  12. EMSA Analysis of DNA Binding By Rgg Proteins

    PubMed Central

    LaSarre, Breah; Federle, Michael J.

    2016-01-01

    In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activities that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function (e.g. interruption of DNA-binding in some cases).

  13. Preferential binding of high mobility group 1 protein to UV-damaged DNA. Role of the COOH-terminal domain.

    PubMed

    Pasheva, E A; Pashev, I G; Favre, A

    1998-09-18

    Binding of chromosomal high mobility group 1 protein (HMG1) to UV-damaged DNA has been studied with oligonucleotides containing a single dipyrimidine site for formation of UV photolesions. Irradiation of an oligonucleotide with unique TT dinucleotide resulted in generation of cyclobutane pyrimidine dimer with no evidence for induction of (6-4) photoproducts, whereas the analysis of irradiated TC-containing oligonucleotide detected (6-4) photoproducts but not cyclobutane pyrimidine dimers. Mobility shift assays have revealed that HMG1 protein binds preferentially to irradiated TT and TC oligonucleotides. Photoreversal of cyclobutane pyrimidine dimers with DNA photolyase and hydrolysis of the (6-4) photoproducts with hot alkali substantially reduced but did not eliminate binding of HMG1. The protein, therefore, appears to bind the two main types of UV damages in DNA, but some other photolesion(s) contributes to the preferential binding of HMG1 to irradiated DNA. By quantifying gel shift assays and considering the efficiencies of lesion formation, we determined dissociation constants of 1.2 +/- 0.5 and 4.0 +/- 1.5 microM for irradiated TT and TC oligonucleotides, respectively, and 70 +/- 20 microM for the control non-irradiated probes. Tryptic removal of the acidic COOH-terminal domain of HMG1 significantly affected binding of the protein to both irradiated and intact oligonucleotides. The potential role of HMG1 in recognition of the UV lesions in DNA is discussed. PMID:9733773

  14. Direct Probing of Solvent Accessibility and Mobility at the Binding Interface of Polymerase (Dpo4)-DNA Complex

    PubMed Central

    Qin, Yangzhong; Yang, Yi; Zhang, Luyuan; Fowler, Jason D.; Qiu, Weihong; Wang, Lijuan; Suo, Zucai; Zhong, Dongping

    2014-01-01

    Water plays essential structural and dynamical roles in protein-DNA recognition through contributing to enthalpic or entropic stabilization of binding complex and by mediating intermolecular interactions and fluctuations for biological function. These interfacial water molecules are confined by the binding partners in nanospace but in many cases they are highly mobile and exchange with outside bulk solution. Here, we report our studies of the interfacial water dynamics in the binary and ternary complexes of a polymerase (Dpo4) with DNA and an incoming nucleotide using a site-specific tryptophan probe with femtosecond resolution. By systematic comparison of the interfacial water motions and local sidechain fluctuations in the apo, binary and ternary states of Dpo4, we observed that the DNA binding interface and active site is dynamically solvent accessible and the interfacial water dynamics are similar to the surface hydration water fluctuations on picosecond time scales. Our molecular dynamics simulations also show the binding interface full of water molecules and nonspecific weak interactions. Such a fluid binding interface facilitates the polymerase sliding on DNA for fast translocation while the spacious and mobile hydrated active site contributes to the low fidelity of the lesion-bypass Y-family DNA polymerase. PMID:24308461

  15. Methylated DNA-binding protein is present in various mammalian cell types

    SciTech Connect

    Supakar, P.C.; Weist, D.; Zhang, D.; Inamdar, N.; Zhang, Xianyang; Khan, R.; Ehrlich, M. ); Ehrlich, K.C. )

    1988-08-25

    A DNA-binding protein from human placenta, methylated DNA-binding protein (MDBP), binds to certain DNA sequences only when they contain 5-methylcytosine (m{sup 5}C) residues at specific positions. The authors found a very similar DNA-binding activity in nuclear extracts of rat tissues, calf thymus, human embryonal carcinoma cells, HeLa cells, and mouse LTK cells. Like human placental MDBP, the analogous DNA-binding proteins from the above mammalian cell lines formed a number of different low-electrophoretic-mobility complexes with a 14-bp MDBP-specific oligonucleotide duplex. All of these complexes exhibited the same DNA methylation specificity and DNA sequence specificity. Although MDBP activity was found in various mammalian cell types, it was not detected in extracts of cultured mosquito cells and so may be associated only with cells with vertebrate-type DNA methylation.

  16. Study of the Electrophoretic Behavior of Cephalosporins by Capillary Zone Electrophoresis

    PubMed Central

    Hancu, Gabriel; Sasebeşi, Adina; Rusu, Aura; Kelemen, Hajnal; Ciurba, Adriana

    2015-01-01

    Purpose: The aim of the study was the characterization of the electrophoretic behavior of cephalosporins from different generation having different structural characteristics in order to develop a rapid, simple and efficient capillary electrophoretic method for their identification and simultaneous separation from complex mixtures. Methods: Ten cephalosporin derivatives (cefaclor, cefadroxil, cefalexin, cefazolin, cefoxitin, cefuroxime, cefoperazone, cefotaxime, ceftazidime, ceftriaxone) were analyzed by capillary zone electrophoresis using different background electrolyte solutions at different pH values. Electrophoretic mobilities of the analytes were calculated, the influence of the electrophoretic parameteres on the separation was established and the analytical conditions were optimized. Results: Taking into consideration their structural and chemical properties cephalosporins can be detected over a pH range between 6 and 10. The best results were obtained using a buffer solution containing 25 mM disodium hydrogenophosphate - 25 mM sodium dihydrogenophosphate, at a pH – 7.00, + 25 kV voltage at a temperature of 25 °C, UV detection at 210 nm. Using the optimized analytical conditions we achieved the simultaneous baseline separation for seven cephalosporins in less then 10 minutes. Conclusion: Using the described optimized electrophoretic procedures, capillary electrophoresis can be used for the identification and determination of cephalosporins in formulated pharmaceutical products and for their separation from complex mixtures. PMID:26236661

  17. Yeast high mobility group protein HMO1 stabilizes chromatin and is evicted during repair of DNA double strand breaks

    PubMed Central

    Panday, Arvind; Xiao, LiJuan; Grove, Anne

    2015-01-01

    DNA is packaged into condensed chromatin fibers by association with histones and architectural proteins such as high mobility group (HMGB) proteins. However, this DNA packaging reduces accessibility of enzymes that act on DNA, such as proteins that process DNA after double strand breaks (DSBs). Chromatin remodeling overcomes this barrier. We show here that the Saccharomyces cerevisiae HMGB protein HMO1 stabilizes chromatin as evidenced by faster chromatin remodeling in its absence. HMO1 was evicted along with core histones during repair of DSBs, and chromatin remodeling events such as histone H2A phosphorylation and H3 eviction were faster in absence of HMO1. The facilitated chromatin remodeling in turn correlated with more efficient DNA resection and recruitment of repair proteins; for example, inward translocation of the DNA-end-binding protein Ku was faster in absence of HMO1. This chromatin stabilization requires the lysine-rich C-terminal extension of HMO1 as truncation of the HMO1 C-terminal tail phenocopies hmo1 deletion. Since this is reminiscent of the need for the basic C-terminal domain of mammalian histone H1 in chromatin compaction, we speculate that HMO1 promotes chromatin stability by DNA bending and compaction imposed by its lysine-rich domain and that it must be evicted along with core histones for efficient DSB repair. PMID:25979266

  18. Cell electrophoretic characterization of peripheral blood lymphocyte subpopulations enriched by rosette formation, from normal individuals and CLL patients.

    PubMed

    Rychly, J; Babusíková, O; Koníková, E; Anders, O

    1984-01-01

    Peripheral blood lymphocytes from healthy subjects and patients with chronic lymphatic leukemia (CLL) were isolated and their subpopulations enriched through formation of spontaneous rosettes with sheep or mouse red blood cells, respectively. Electrophoretic measurements were performed in unseparated as well as in fractionated cell populations. Normal blood lymphocytes showed two clearly distinguishable populations of different electrophoretic mobilities. After separation by SRBC rosette formation the rosette-forming cells could be identified as high mobility cells. CLL lymphocytes showed in most cases an unimodally distributed cytopherogram, the mean electrophoretic mobility being intermediate between the low and high mobility cells of control persons. After separation through mouse erythrocytes rosette formation these cells contained two cell fractions differing in their electrophoretic mobility: a fraction of slower mouse rosette-forming cells and a fraction of the non-MRFC which contained mainly cells of higher mobility that could be identified as enriched T cells. These both fractions showed unimodal distributions. This study shows that CLL lymphocyte subpopulations can be further characterized by surface charge density. PMID:6700796

  19. Tunable electrophoretic separations using a scalable, fabric-based platform.

    PubMed

    Narahari, Tanya; Dendukuri, Dhananjaya; Murthy, Shashi K

    2015-02-17

    There is a rising need for low-cost and scalable platforms for sensitive medical diagnostic testing. Fabric weaving is a mature, scalable manufacturing technology and can be used as a platform to manufacture microfluidic diagnostic tests with controlled, tunable flow. Given its scalability, low manufacturing cost (<$0.25 per device), and potential for patterning multiplexed channel geometries, fabric is a viable platform for the development of analytical devices. In this paper, we describe a fabric-based electrophoretic platform for protein separation. Appropriate yarns were selected for each region of the device and weaved into straight channel electrophoretic chips in a single step. A wide dynamic range of analyte molecules ranging from small molecule dyes (<1 kDa) to macromolecule proteins (67-150 kDa) were separated in the device. Individual yarns behave as a chromatographic medium for electrophoresis. We therefore explored the effect of yarn and fabric parameters on separation resolution. Separation speed and resolution were enhanced by increasing the number of yarns per unit area of fabric and decreasing yarn hydrophilicity. However, for protein analytes that often require hydrophilic, passivated surfaces, these effects need to be properly tuned to achieve well-resolved separations. A fabric device tuned for protein separations was built and demonstrated. As an analytical output parameter for this device, the electrophoretic mobility of a sedimentation marker, Naphthol Blue Black bovine albumin in glycine-NaOH buffer, pH 8.58 was estimated and found to be -2.7 × 10(-8) m(2) V(-1) s(-1). The ability to tune separation may be used to predefine regions in the fabric for successive preconcentrations and separations. The device may then be applied for the multiplexed detection of low abundance proteins from complex biological samples such as serum and cell lysate. PMID:25582166

  20. The high-mobility group protein T160 binds to both linear and cruciform DNA and mediates DNA bending as determined by ring closure.

    PubMed

    Gariglio, M; Ying, G G; Hertel, L; Gaboli, M; Clerc, R G; Landolfo, S

    1997-11-01

    The high-mobility group protein T160 was isolated by screening a phage library from a murine pre-B-cell line L1210. South-Western experiments have previously shown that this protein binds to V-(D)-J recombination signal sequences, suggesting that it may be a sequence-specific DNA-binding protein. However, neither gel-shift nor footprinting analyses have been successfully employed with the T160 protein, despite an extensive effort. In this study, the T160 protein or truncated forms made soluble through denaturing and renaturing cycles in urea were successfully used in gel-shift experiments showing that T160 binds to cruci-form or linear duplex DNA with no apparent sequence specificity. Furthermore, fragments longer than 100 bp efficiently formed covalently closed circular monomers in the presence of T160 and T4 DNA ligase, indicating that the protein is capable of introducing bends into the duplex. Last, tissue distribution by Western blotting analysis showed that the T160 protein is expressed in various murine tissues in addition to those of lymphoid origin. Considering its broad evolutionary conservation (from plants to mammals) also, these results suggest that the functional role of the T160 protein is not limited to V-(D)-J recombination, but might be involved in basic processes such as DNA replication and repairing, where irregular DNA structures are generated and very likely recognized by HMG domain proteins. PMID:9367632

  1. Numerical investigation of molecular nano-array in potential-energy profile for a single dsDNA.

    PubMed

    Alishahi, Marzieh; Kamali, Reza; Abouali, Omid

    2016-04-01

    A Rigorous numerical investigation on dsDNA translocation in quasi-2-dimensional nano-array filter is performed using Molecular Dynamics (MD) method. Various dsDNA molecules with different sizes are chosen in order to model Ogston sieving in a nano-array filter. The radius of gyration of dsDNA molecule is less than the characteristic length of the shallow region in nano-array. The dsDNA molecule is assumed to be in the 0.05M NaCl electrolyte. MD shows acceptable results for potential-energy profile for nano-array filter. According to the MD outcomes, the dsDNA electrophoretic mobility decreases almost linearly with dsDNA size and show the same trend as Ogston sieving for gel electrophoresis. In addition, different shapes for nano-array filter are studied for a unique dsDNA molecule. It is concluded that steeping the nano-array wall can cause the retardation of dsDNA translocation and decreases dsDNA electrophoretic mobility. PMID:27125679

  2. Electrophoretic characterization of insulin growth factor (IGF-1) functionalized magnetic nanoparticles.

    PubMed

    Viota, Julián L; Rudzka, Katarzyna; Trueba, Ángel; Torres-Aleman, Ignacio; Delgado, Ángel V

    2011-05-17

    The synthesis of composite nanoparticles consisting of a magnetite core coated with a layer of the hormone insulin growth factor 1 (IGF-1) is described. The adsorption of the hormone in the different formulations is first studied by electrophoretic mobility measurements as a function of pH, ionic strength, and time. Because of the permeable character expected for both citrate and IGF-1 coatings surrounding the magnetite cores, an appropriate analysis of their electrophoretic mobility must be addressed. Recent developments of electrokinetic theories for particles covered by soft surface layers have rendered possible the evaluation of the softness degree from raw electrophoretic mobility data. In the present contribution, the data are quantitatively analyzed based on the theoretical model of the electrokinetics of soft particles. As a result, information is obtained on both the thickness and the charge density of the surrounding layer. It is shown that IGF-1 adsorbs onto the surface of citrate-coated magnetite nanoparticles, and adsorption is confirmed by dot-blot analysis. In addition, it is also demonstrated that the external layer of IGF-1 exerts a shielding effect on the surface charge of citrate-magnetite particles, as suggested by the mobility reduction upon contacting the particles with the hormone. Aging effects are demonstrated, providing an electrokinetic fingerprint of changes in adsorbed protein configuration with time. PMID:21506536

  3. Connections between RNA splicing and DNA intron mobility in yeast mitochondria: RNA maturase and DNA endonuclease switching experiments.

    PubMed Central

    Goguel, V; Delahodde, A; Jacq, C

    1992-01-01

    The intron-encoded proteins bI4 RNA maturase and aI4 DNA endonuclease can be faithfully expressed in yeast cytoplasm from engineered forms of their mitochondrial coding sequences. In this work we studied the relationships between these two activities associated with two homologous intron-encoded proteins: the bI4 RNA maturase encoded in the fourth intron of the cytochrome b gene and the aI4 DNA endonuclease (I-SceII) encoded in the fourth intron of the gene coding for the subunit I of cytochrome oxidase. Taking advantage of both the high recombinogenic properties of yeast and the similarities between the two genes, we constructed in vivo a family of hybrid genes carrying parts of both RNA maturase and DNA endonuclease coding sequences. The presence of a sequence coding for a mitochondrial targeting peptide upstream from these hybrid genes allowed us to study the properties of their translation products within the mitochondria in vivo. We thus could analyze the ability of the recombinant proteins to complement RNA maturase deficiencies in different strains. Many combinations of the two parental intronic sequences were found in the recombinants. Their structural and functional analysis revealed the following features. (i) The N-terminal half of the bI4 RNA maturase could be replaced in total by its equivalent from the aI4 DNA endonuclease without affecting the RNA maturase activity. In contrast, replacing the C-terminal half of the bI4 RNA maturase with its equivalent from the aI4 DNA endonuclease led to a very weak RNA maturase activity, indicating that this region is more differentiated and linked to the maturase activity. (ii) None of the hybrid proteins carrying an RNA maturase activity kept the DNA endonuclease activity, suggesting that the latter requires the integrity of the aI4 protein. These observations are interesting because the aI4 DNA endonuclease is known to promote the propagation, at the DNA level, of the aI4 intron, whereas the bI4 RNA maturase

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

    NASA Astrophysics Data System (ADS)

    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

  5. Combined electrophoretic-separation and electrospray method and system

    DOEpatents

    Smith, Richard D.; Olivares, Jose A.

    1989-01-01

    A system and method for analyzing molecular constituents of a composition sample includes: forming a solution of the sample, separating the solution by capillary zone electrophoresis into an eluent of constituents longitudinally separated according to their relative electrophoretic mobilities, electrospraying the eluent to form a charged spray in which the molecular constituents have a temporal distribution; and detecting or collecting the separated constituents in accordance with the temporal distribution in the spray. A first high-voltage (e.g., 5-100 KVDC) is applied to the solution. The spray is charged by applying a second high voltage (e.g., .+-.2-8 KVDC) between the eluent at the capillary exit and a cathode spaced in front of the exit. A complete electrical circuit is formed by a conductor which directly contacts the eluent at the capillary exit.

  6. Preparation of guinea pig macrophage for electrophoretic experiments in space

    NASA Technical Reports Server (NTRS)

    1979-01-01

    Methods of storage and cultivation of macrophage cells in preparation for space experiments were investigated. Results show that freezing and thawing immediately after extraction did not cause any change in viability or electrophoretic mobility of the cells. A prolonged storage at -80 C did cause cell damage as indicated by a 95% reduction in variable cells. Cell damage was decreased when Glycerol or Dimethyl Sulfoxide (DMSO) was added as a cryogenic protective agent. A 100% viability was observed in cultivation experiments after two weeks due to the additional serum. Results from gamma-glutamyl transpeptidase study showed a zero activity rate. It is suggested that a flat stationary field be used for the collection and use of macrophage. It was found that a 24-hour delay in obtaining macrophage cells helps to maintain a pure culture.

  7. Combined electrophoretic-separation and electrospray method and system

    DOEpatents

    Smith, R.D.; Olivares, J.A.

    1989-06-27

    A system and method for analyzing molecular constituents of a composition sample includes: forming a solution of the sample, separating the solution by capillary zone electrophoresis into an eluent of constituents longitudinally separated according to their relative electrophoretic mobilities, electrospraying the eluent to form a charged spray in which the molecular constituents have a temporal distribution; and detecting or collecting the separated constituents in accordance with the temporal distribution in the spray. A first high-voltage (e.g., 5--100 kVDC) is applied to the solution. The spray is charged by applying a second high voltage (e.g., [+-]2--8 kVDC) between the eluent at the capillary exit and a cathode spaced in front of the exit. A complete electrical circuit is formed by a conductor which directly contacts the eluent at the capillary exit. 10 figs.

  8. Mobilization of copper ions in human peripheral lymphocytes by catechins leading to oxidative DNA breakage: A structure activity study.

    PubMed

    Farhan, Mohd; Zafar, Atif; Chibber, Sandesh; Khan, Husain Yar; Arif, Hussain; Hadi, S M

    2015-08-15

    Epidemiological studies suggest that dietary consumption of plant polyphenols is related to a lower incidence of various cancers. Among these compounds catechins (present in green tea and other beverages) are considered to be potent inducers of apoptosis and cytotoxicity to cancer cells. Thus these compounds can be used as leads to synthesize novel anticancer drugs with greater bioavailability. In view of this in this paper we have examined the chemical basis of cytotoxicity of catechins by studying the structure-activity relationship between catechin (C), epicatechin (EC), epigallocatechin (EGC) and epigallocatechin-3-gallate (EGCG). Using single cell alkaline gel electrophoresis (comet assay) we have established the relative efficiency of cellular DNA breakage as EGCG>EGC>EC>C. We also show that cellular DNA breakage is the result of mobilization of copper ions bound to chromatin and the generation of reactive oxygen species. Further the relative DNA binding affinity order was confirmed using molecular docking and thermodynamic studies by studying the interaction of catechins with calf thymus DNA. The results suggest that the synthesis of any novel anti cancer molecule based on the structure of catechins should have as many galloyl moieties as possible resulting in an increased number of hydroxyl groups that may facilitate the binding of the molecule to cellular DNA. PMID:26142371

  9. The 2100MHz radiofrequency radiation of a 3G-mobile phone and the DNA oxidative damage in brain.

    PubMed

    Sahin, Duygu; Ozgur, Elcin; Guler, Goknur; Tomruk, Arın; Unlu, Ilhan; Sepici-Dinçel, Aylin; Seyhan, Nesrin

    2016-09-01

    We aimed to evaluate the effect of 2100MHz radiofrequency radiation emitted by a generator, simulating a 3G-mobile phone on the brain of rats during 10 and 40 days of exposure. The female rats were randomly divided into four groups. Group I; exposed to 3G modulated 2100MHz RFR signal for 6h/day, 5 consecutive days/wk for 2 weeks, group II; control 10 days, were kept in an inactive exposure set-up for 6h/day, 5 consecutive days/wk for 2 weeks, group III; exposed to 3G modulated 2100MHz RFR signal for 6h/day, 5 consecutive days/wk for 8 weeks and group IV; control 40 days, were kept in an inactive exposure set-up for 6h/day, 5 consecutive days/wk for 8 weeks. After the genomic DNA content of brain was extracted, oxidative DNA damage (8-hydroxy-2'deoxyguanosine, pg/mL) and malondialdehyde (MDA, nmoL/g tissue) levels were determined. Our main finding was the increased oxidative DNA damage to brain after 10 days of exposure with the decreased oxidative DNA damage following 40 days of exposure compared to their control groups. Besides decreased lipid peroxidation end product, MDA, was observed after 40 days of exposure. The measured decreased quantities of damage during the 40 days of exposure could be the means of adapted and increased DNA repair mechanisms. PMID:26775761

  10. Stretching and Controlled Motion of Single-Stranded DNA in Locally-Heated Solid-State Nanopores

    PubMed Central

    Belkin, Maxim; Maffeo, Christopher; Wells, David B.

    2013-01-01

    Practical applications of solid-state nanopores for DNA detection and sequencing require the electrophoretic motion of DNA through the nanopores to be precisely controlled. Controlling the motion of single-stranded DNA presents a particular challenge, in part because of the multitude of conformations that a DNA strand can adopt in a nanopore. Through continuum, coarse-grained and atomistic modeling, we demonstrate that local heating of the nanopore volume can be used to alter the electrophoretic mobility and conformation of single-stranded DNA. In the nanopore systems considered, the temperature near the nanopore is modulated via a nanometer-size heater element that can be radiatively switched on and off. The local enhancement of temperature produces considerable stretching of the DNA fragment confined within the nanopore. Such stretching is reversible, so that the conformation of DNA can be toggled between compact (local heating is off) and extended (local heating is on) states. The effective thermophoretic force acting on single-stranded DNA in the vicinity of the nanopore is found to be sufficiently large (4–8 pN) to affect such changes in the DNA conformation. The local heating of the nanopore volume is observed to promote single-file translocation of DNA strands at transmembrane biases as low as 10 mV, which opens new avenues for using solid-state nanopores for detection and sequencing of DNA. PMID:23876013

  11. Electrophoretic purification of cells in space - Evaluation of results from STS-3

    NASA Technical Reports Server (NTRS)

    Sarnoff, B. E.; Kunze, M. E.; Todd, P.

    1983-01-01

    The procedure and results of Electrophoresis Equipment Verification Test, designed to examine electrophoretic behavior of animal cells is suspension more concentrated than possible on earth and flown on the Shuttle flight STS-3, were discussed. Ground-based laboratory values of electrophoretic mobilities of a mixture of human and rabbit aldehyde-fixed red blood cells (RBC) were compared with those recorded at 11 minute intervals on the Shuttle STS-3. RBC migration and separation observed through photographic records were not as expected. However, cell mobilities and migrating band profiles were consistent with the results of laboratory simulation experiments. It was concluded that zero G electrophoresis of very high concentrations (1 x 10 to the 9th) is possible and similar to electrophoresis of normal cell concentrations on earth.

  12. Gel Electrophoresis of Gold-DNA Nanoconjugates

    DOE PAGESBeta

    Pellegrino, T.; Sperling, R. A.; Alivisatos, A. P.; Parak, W. J.

    2007-01-01

    Gold-DNA conjugates were investigated in detail by a comprehensive gel electrophoresis study based on 1200 gels. A controlled number of single-stranded DNA of different length was attached specifically via thiol-Au bonds to phosphine-stabilized colloidal gold nanoparticles. Alternatively, the surface of the gold particles was saturated with single stranded DNA of different length either specifically via thiol-Au bonds or by nonspecific adsorption. From the experimentally determined electrophoretic mobilities, estimates for the effective diameters of the gold-DNA conjugates were derived by applying two different data treatment approaches. The first method is based on making a calibration curve for the relation between effectivemore » diameters and mobilities with gold nanoparticles of known diameter. The second method is based on Ferguson analysis which uses gold nanoparticles of known diameter as reference database. Our study shows that effective diameters derived from gel electrophoresis measurements are affected with a high error bar as the determined values strongly depend on the method of evaluation, though relative changes in size upon binding of molecules can be detected with high precision. Furthermore, in this study, the specific attachment of DNA via gold-thiol bonds to Au nanoparticles is compared to nonspecific adsorption of DNA. Also, the maximum number of DNA molecules that can be bound per particle was determined.« less

  13. The effect of pH on charge inversion and condensation of DNA.

    PubMed

    Guo, Zilong; Wang, Yanwei; Yang, Anthony; Yang, Guangcan

    2016-08-21

    Charge inversion and condensation of DNA in solutions of trivalent and quadrivalent counterions are significantly influenced by the pH value of the solution. We systematically investigated the condensation and charge compensation of DNA by spermidine, hexammine cobalt(iii) (cohex, [Co(NH3)6](3+)) and spermine in solutions of a wide range of pH values from 3 to 9.3 by dynamic light scattering, magnetic tweezers, and atomic force microscopy. In trivalent counterion solution, we found that there is a critical concentration (0.75 mM for cohex and 0.5 mM for spermidine), under which the electrophoresis mobility of DNA initially increases, reaches a maximum, and finally decreases when the pH value is decreased. In contrast, above the critical concentration, the electrophoretic mobility of DNA increases monotonously with decreasing pH value of the solution. The corresponding condensing force has the same dependence on the pH value. However, for the case of quadrivalent counterions, the electrophoretic mobility of DNA is monotonously promoted by lowering the pH value of the solution at any concentration of counterions in which charge inversion of DNA may occur. In atomic force microscopy images and force spectroscopy of magnetic tweezers, we found that maximal charge neutralization and condensation force correspond to the most compact DNA condensation. We propose a mechanism of promoting DNA charge neutralization: small and highly mobile hydrogen ions tend to attach to the DNA-counterion complex to further neutralize its remaining charge, which is related to the surface area of the complex. Therefore, this further neutralization is prominent when the complex is toroidal which corresponds to the case of mild ion concentration while it is less prominent for more compact globules or rod complexes at high counterion concentration. PMID:27427090

  14. Yield, Purity and Mobility of a Silver-DNA Fluorophore in Solution

    NASA Astrophysics Data System (ADS)

    O'Neill, Patrick; Velazquez, Lourdes; Goodwin, Peter; Driehorst, Til; Pennathur, Sumita; Fygenson, Deborah

    2010-03-01

    Chemical reduction of DNA oligonucleotide:Ag+ mixtures leads to the formation of fluorescent few-atom Ag clusters stabilized by the DNA. This reaction typically produces many species, some of which are fluorescent, with emission wavelengths and stabilities that vary widely with DNA sequence. While most DNA sequences studied produce many different Ag:DNA products, we identify a specific DNA sequence that strongly favors the formation of a green 11Ag cluster, stable for months under ambient conditions. We generate pure solutions of this emitter by synthesizing in the presence of excess silver and then removing free silver from solution. We report on results enabled by the purity of these samples, including determination of the extinction coefficient (using FCS), diffusion coefficient (using microfluidics) and bulk chemical yield of this fluorophore, and comment on the challenges that remain on the path to production of sufficient quantity and purity for high-resolution structure determination.

  15. Experimental strategies for cloning or identifying genes encoding DNA-binding proteins.

    PubMed

    Carey, Michael F; Peterson, Craig L; Smale, Stephen T

    2012-02-01

    This article describes experimental strategies for cloning or identifying genes encoding DNA-binding proteins. DNA-binding proteins are most commonly identified by electrophoretic mobility-shift assay (EMSA) or DNase I footprinting. To identify the gene encoding a protein detected by EMSA or DNase footprinting, the protein often needs to be purified and its sequence analyzed, as described here. Other methods are also available which do not resort to protein purification, including the one-hybrid screen, in vitro expression library screen, and mammalian expression cloning. These methods are outlined, and their advantages and disadvantages are discussed. PMID:22301659

  16. Method for in-situ calibration of electrophoretic analysis systems

    DOEpatents

    Liu, Changsheng; Zhao, Hequan

    2005-05-08

    An electrophoretic system having a plurality of separation lanes is provided with an automatic calibration feature in which each lane is separately calibrated. For each lane, the calibration coefficients map a spectrum of received channel intensities onto values reflective of the relative likelihood of each of a plurality of dyes being present. Individual peaks, reflective of the influence of a single dye, are isolated from among the various sets of detected light intensity spectra, and these can be used to both detect the number of dye components present, and also to establish exemplary vectors for the calibration coefficients which may then be clustered and further processed to arrive at a calibration matrix for the system. The system of the present invention thus permits one to use different dye sets to tag DNA nucleotides in samples which migrate in separate lanes, and also allows for in-situ calibration with new, previously unused dye sets.

  17. Mobilization of Copper ions by Flavonoids in Human Peripheral Lymphocytes Leads to Oxidative DNA Breakage: A Structure Activity Study.

    PubMed

    Arif, Hussain; Rehmani, Nida; Farhan, Mohd; Ahmad, Aamir; Hadi, Sheikh Mumtaz

    2015-01-01

    Epidemiological studies have linked dietary consumption of plant polyphenols with lower incidence of various cancers. In particular, flavonoids (present in onion, tomato and other plant sources) induce apoptosis and cytotoxicity in cancer cells. These can therefore be used as lead compounds for the synthesis of novel anticancer drugs with greater bioavailability. In the present study, we examined the chemical basis of cytotoxicity of flavonoids by studying the structure-activity relationship of myricetin (MN), fisetin (FN), quercetin (QN), kaempferol (KL) and galangin (GN). Using single cell alkaline gel electrophoresis (comet assay), we established the relative efficiency of cellular DNA breakage as MN > FN > QN > KL > GN. Also, we determined that the cellular DNA breakage was the result of mobilization of chromatin-bound copper ions and the generation of reactive oxygen species. The relative DNA binding affinity order was further confirmed using molecular docking and thermodynamic studies through the interaction of flavonoids with calf thymus DNA. Our results suggest that novel anti-cancer molecules should have ortho-dihydroxy groups in B-ring and hydroxyl groups at positions 3 and 5 in the A-ring system. Additional hydroxyl groups at other positions further enhance the cellular cytotoxicity of the flavonoids. PMID:26569217

  18. Mobilization of Copper ions by Flavonoids in Human Peripheral Lymphocytes Leads to Oxidative DNA Breakage: A Structure Activity Study

    PubMed Central

    Arif, Hussain; Rehmani, Nida; Farhan, Mohd; Ahmad, Aamir; Hadi, Sheikh Mumtaz

    2015-01-01

    Epidemiological studies have linked dietary consumption of plant polyphenols with lower incidence of various cancers. In particular, flavonoids (present in onion, tomato and other plant sources) induce apoptosis and cytotoxicity in cancer cells. These can therefore be used as lead compounds for the synthesis of novel anticancer drugs with greater bioavailability. In the present study, we examined the chemical basis of cytotoxicity of flavonoids by studying the structure–activity relationship of myricetin (MN), fisetin (FN), quercetin (QN), kaempferol (KL) and galangin (GN). Using single cell alkaline gel electrophoresis (comet assay), we established the relative efficiency of cellular DNA breakage as MN > FN > QN > KL > GN. Also, we determined that the cellular DNA breakage was the result of mobilization of chromatin-bound copper ions and the generation of reactive oxygen species. The relative DNA binding affinity order was further confirmed using molecular docking and thermodynamic studies through the interaction of flavonoids with calf thymus DNA. Our results suggest that novel anti-cancer molecules should have ortho-dihydroxy groups in B-ring and hydroxyl groups at positions 3 and 5 in the A-ring system. Additional hydroxyl groups at other positions further enhance the cellular cytotoxicity of the flavonoids. PMID:26569217

  19. B-DNA to Z-DNA structural transitions in the SV40 enhancer: stabilization of Z-DNA in negatively supercoiled DNA minicircles

    NASA Technical Reports Server (NTRS)

    Gruskin, E. A.; Rich, A.

    1993-01-01

    During replication and transcription, the SV40 control region is subjected to significant levels of DNA unwinding. There are three, alternating purine-pyrimidine tracts within this region that can adopt the Z-DNA conformation in response to negative superhelix density: a single copy of ACACACAT and two copies of ATGCATGC. Since the control region is essential for both efficient transcription and replication, B-DNA to Z-DNA transitions in these vital sequence tracts may have significant biological consequences. We have synthesized DNA minicircles to detect B-DNA to Z-DNA transitions in the SV40 enhancer, and to determine the negative superhelix density required to stabilize the Z-DNA. A variety of DNA sequences, including the entire SV40 enhancer and the two segments of the enhancer with alternating purine-pyrimidine tracts, were incorporated into topologically relaxed minicircles. Negative supercoils were generated, and the resulting topoisomers were resolved by electrophoresis. Using an anti-Z-DNA Fab and an electrophoretic mobility shift assay, Z-DNA was detected in the enhancer-containing minicircles at a superhelix density of -0.05. Fab saturation binding experiments demonstrated that three, independent Z-DNA tracts were stabilized in the supercoiled minicircles. Two other minicircles, each with one of the two alternating purine-pyrimidine tracts, also contained single Z-DNA sites. These results confirm the identities of the Z-DNA-forming sequences within the control region. Moreover, the B-DNA to Z-DNA transitions were detected at superhelix densities observed during normal replication and transcription processes in the SV40 life cycle.

  20. Electrophoretic mobility of magnetite particles in high temperature water

    SciTech Connect

    Vidojkovic, Sonja; Rodriguez-Santiago, V; Fedkin, Mark V.; Wesolowski, David J; Lvov, Serguei N.

    2011-01-01

    Magnetite(Fe3O4) isoneofthemostcommonoxidesformingdepositsandparticulatephasesin industrialhightemperaturewatercircuits.Itscolloidalcharacteristicsplayaprincipalroleinthe mechanismofdepositformationandcanbeusedascontrollingfactorstopreventorminimizedeposit formationanddamageofindustrialpipelinesduetounder-depositcorrosion.Inthisstudy,ahigh temperatureparticleelectrophoresistechniquewasemployedtomeasurethezetapotentialatthe magnetite/waterinterface the parameterthatcontrolscolloidalstabilityofparticles,theiraggrega- tion, anddeposition.Themeasurementsweremadeattemperaturesupto200 1C overawiderangeofpH. The isoelectricpointsofmagnetite,atwhichthedepositionofparticlesisincreased,weredeterminedatpH 6.35, 6.00,5.25,and5.05fortemperatures25,100,150,and200 1C, respectively.Theobserved temperaturedependenceofzetapotentialandtheisoelectricpHpointofmagnetitecanhelptoexplain the extentofinteractionsbetweenthecolloidalparticlesandthesteelwallsurfacesunderhydro- thermalconditions,andindicatemethodsforcontrollingandmitigatingoxidedepositioninhigh temperaturewatercycles.

  1. 8-Oxo-7, 8-dihydro-2'-deoxyguanosine as a biomarker of DNA damage by mobile phone radiation.

    PubMed

    Khalil, Ahmad M; Gagaa, M H; Alshamali, A M

    2012-07-01

    We examined the effect of exposure to mobile phone 1800 MHz radio frequency radiation (RFR) upon the urinary excretion of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG), one major form of oxidative DNA damage, in adult male Sprague-Dawley rats. Twenty-four rats were used in three independent experiments (RFR exposed and control, 12 rats, each). The animals were exposed to RFR for 2 h from Global System for Mobile Communications (GSM) signal generator with whole-body-specific absorption rate of 1.0 W/kg. Urine samples were collected from the rat while housed in a metabolic cage during the exposure period over a 4-h period at 0.5, 1.0, 2.0 and 4.0 h from the beginning of exposure. In the control group, the signal generator was left in the turn-off position. The creatinine-standardized concentrations of 8-oxodG were measured. With the exception of the urine collected in the last half an hour of exposure, significant elevations were noticed in the levels of 8-oxodG in urine samples from rats exposed to RFR when compared to control animals. Significant differences were seen overall across time points of urine collection with a maximum at 1 h after exposure, suggesting repair of the DNA lesions leading to 8-oxodG formation. PMID:22249391

  2. Comparison of detection platforms and post-polymerase chain reaction DNA purification methods for use in conjunction with Cleavase fragment length polymorphism analysis.

    PubMed

    Sander, T; Olson, S; Hall, J; Siebert, M; Grooms, K; Heisler, L; de Arruda, M; Neri, B

    1999-06-01

    The removal of impurities and contaminants from PCR-amplified fragments is important for mutation detection methods which identify mutations based on shifts in electrophoretic mobility. This is particularly critical for assays and detection methods which use target DNA that is labeled prior to analysis and electrophoretic detection. We examined several procedures for purifying DNA amplified by the polymerase chain reaction (PCR) and their use in conjunction with a novel DNA scanning method, the Cleavase fragment length polymorphism (CFLP)* assay. In this study, a 480 bp DNA fragment, fluorescently labeled on the 5'-end of one strand, was amplified and subjected to various widely used purification procedures, including several commercially available clean-up kits. We demonstrate that visualization of the fluorescent label, as opposed to simple ethidium bromide staining, reveals the presence of considerable levels of labeled, truncated, amplification products. The various procedures were evaluated on the basis of their ability to remove these unwanted DNA fragments as well as on the degree to which they inhibited or promoted the CFLP reaction. Several procedures are recommended for use with CFLP analysis, including isopropanol precipitation, gel excision, and several commercially available spin columns. Concurrently, we evaluated (compared) a number of commonly used visualization platforms, including fluorescence imaging, chemiluminescence, and post-electrophoretic staining, for the ability to detect CFLP pattern changes. The advantages and disadvantages of different methods are discussed and amounts of DNA to be used for CFLP analysis on different detection platforms are recommended. PMID:10380752

  3. DNA electrophoresis in agarose gels: Effects of electric field and gel concentration on the exponential dependence of reciprocal mobility on DNA length

    NASA Astrophysics Data System (ADS)

    Beheshti, Afshin; van Winkle, David; Randolph, Rill

    2002-03-01

    Electrophoresis was performed on double stranded DNA fragments ranging in length from 200 bp to 48502 bp at agarose gel concentrations T = 0.5% - 1.5% and electric fields E = 0.71 V/cm to 5 V/cm. A wide range of electric fields and gel concentrations were used to find what range of conditions work with the new interpolation equation, 1/μ(L) = 1/μl - (1/μl - 1/μ_s)e^-L/γ. The equation fit extremely well (\\chi^2 >= 0.999) to data with E = 2.5 V/cm to 5 V/cm and for lower fields (E < 2.5 V/cm) at low gel concentrations (T = 0.5% and 0.7%). This exponential relation seemed to hold when there is a smooth transition from the Ogston sieving regime to the reptation regime when looking at the “reptation plots” (plotting 3μL/μo vs. L) (Rousseau, J., Drouin, G., and Slater, G. W., Phys Rev Lett. 1997, 79, 1945-1948). For separations of single-stranded DNA in polyacrylamide, similar reptation plots have a region with a negative slope between the Ogston sieving regime and the reptation regime which has been interpreted as the signature of entropic trapping. When separating double-stranded DNA in agarose it was observed that fits deviate from the data when three different slopes are observed in the reptation plots. Failure of the simple exponential relationship between reciprocal mobility and DNA length appears to be the consequence of entropic trapping.

  4. New reflective-type electrophoretic display

    NASA Astrophysics Data System (ADS)

    Orsaev, A. M.; Orsaev, T. M.; Gaev, D. S.

    2001-06-01

    Advantages and problems of the electrophoretic display design are considered. To increase its lifetime a new EPD version is proposed, and operation process is described. Main distinguish feature is the present of an agile film as the third electrode. Such a display can be manufactured on the base of standard technological processes and promises to be X-Y multiplex addressed, simple and cheap for manufacturing.

  5. A unified mathematical theory of electrophoretic processes

    NASA Technical Reports Server (NTRS)

    Bier, M.; Palusinski, O. A.; Mosher, R. A.; Graham, A.; Saville, D. A.

    1983-01-01

    A mathematical theory is presented which shows that each of the four classical electrophoretic modes (zone electrophoresis, moving boundary electrophoresis, isotachophoresis, and isoelectric focusing) is based on the same general principles and can collectively be described in terms of a single set of equations. This model can predict the evolution of the four electrophoretic modes as a function of time. The model system is one-dimensional, neglecting the effects of electroosmosis, temperature gradients, and any bulk flows of liquid. The model is based on equations which express the components' dissociation equilibria, the mass transport due to electromigration and diffusion, electroneutrality, and the conservation of mass and charge. The model consists of a system of coupled partial differential and nonlinear algebraic equations which can be solved numerically by use of a computer. The versatility of this model was verified using an example of a three-component system containing cacodylate, tris hydroxylmethylaminomethane, and histidine. Results show that this model not only correctly predicts the characteristic features of each electrophoretic mode, but also gives details of the concentration, pH, and conductivity profiles not easily amenable to direct experimental measurement.

  6. A novel DNA-binding protein from Campylobacter jejuni bacteriophage NCTC12673.

    PubMed

    Arutyunov, Denis; Szymanski, Christine M

    2015-11-01

    We previously suggested that the double-stranded genomic DNA of Campylobacter jejuni bacteriophage NCTC12673 was complexed with proteins. Mass spectrometry of peptides obtained from tryptic digests of purified phage DNA indicated that phage protein Gp001 co-purified with the DNA. Gp001 is an acidic protein that lacks any obvious homology or conserved domains found in known DNA-binding proteins. The DNA-binding ability of recombinant Gp001 was examined using an electrophoretic mobility shift assay. Slow DNA-Gp001 complex formation was observed at pH 5.5, but not at neutral or basic pH. This nucleoprotein complex had difficulty entering agarose gels used in the assay while proteinase K pretreatment released the DNA from the complex. No mobility shift was observed when the DNA was immediately subjected to electrophoresis after mixing with Gp001, even if both components were separately pre-incubated at pH 5.5. The complexed DNA was unable to transform chemically competent Escherichia coli cells and was less susceptible to degradation by nucleases. The formation of Gp001-DNA complexes at low pH may provide a mechanism for maintaining DNA integrity while the phage pursues its host through the gastrointestinal tract. Also, this feature can potentially be used to improve DNA delivery protocols applied in gene therapy. PMID:26363017

  7. DNA binding of Jun and Fos bZip domains: homodimers and heterodimers induce a DNA conformational change in solution.

    PubMed Central

    John, M; Leppik, R; Busch, S J; Granger-Schnarr, M; Schnarr, M

    1996-01-01

    We constructed plasmids encoding the sequences for the bZip modules of c-Jun and c-Fos which could then be expressed as soluble proteins in Escherichia coli. The purified bZip modules were tested for their binding capacities of synthetic oligonucleotides containing either TRE or CRE recognition sites in electrophoretic mobility shift assays and circular dichroism (CD). Electrophoretic mobility shift assays showed that bZip Jun homodimers and bZip Jun/Fos heterodimers bind a collagenase-like TRE (CTGACTCAT) with dissociation constants of respectively 1.4 x 10(-7) M and 5 x 10(-8) M. As reported earlier [Patel et al. (1990) Nature 347, 572-575], DNA binding induces a marked change of the protein structure. However, we found that the DNA also undergoes a conformational change. This is most clearly seen with small oligonucleotides of 13 or 14 bp harboring respectively a TRE (TGACTCA) or a CRE (TGACGTCA) sequence. In this case, the positive DNA CD signal at 280 nm increases almost two-fold with a concomitant blue-shift of 3-4 nm. Within experimental error the same spectral changes are observed for TRE and CRE containing DNA fragments. The spectral changes observed with a non-specific DNA fragment are weaker and the signal of free DNA is recovered upon addition of much smaller salt concentrations than required for a specific DNA fragment. Surprisingly the spectral changes induced by Jun/Jun homodimers are not identical to those induced by Jun/Fos heterodimers. However, in both cases the increase of the positive CD band and the concomitant blue shift would be compatible with a B to A-transition of part of the binding site or a DNA conformation intermediate between the canonical A and B structures. PMID:8948639

  8. Trapping of branched DNA in microfabricated structures.

    PubMed Central

    Volkmuth, W D; Duke, T; Austin, R H; Cox, E C

    1995-01-01

    We have observed electrostatic trapping of tribranched DNA molecules undergoing electrophoresis in a microfabricated pseudo-two-dimensional array of posts. Trapping occurs in a unique transport regimen in which the electrophoretic mobility is extremely sensitive to polymer topology. The arrest of branched polymers is explained by considering their center-of-mass motion; in certain conformations, owing to the constraints imposed by the obstacles a molecule cannot advance without the center of mass first moving a short distance backwards. The depth of the resulting local potential well can be much greater than the thermal energy so that escape of an immobilized molecule can be extremely slow. We summarize the expected behavior of the mobility as a function of field strength and topology and point out that the microfabricated arrays are highly suitable for detecting an extremely small number of branched molecules in a very large population of linear molecules. Images Fig. 2 PMID:7624337

  9. Electrophoresis of DNA on a disordered two-dimensional substrate

    NASA Astrophysics Data System (ADS)

    Reichhardt, C. J. Olson; Reichhardt, C.

    2006-11-01

    We propose a method for electrophoretic separation of DNA in which adsorbed polymers are driven over a disordered two-dimensional substrate which contains attractive sites for the polymers. Using simulations of a model for long polymer chains, we show that the mobility increases with polymer length, in contrast to gel electrophoresis techniques, and that separation can be achieved for a range of length scales. We demonstrate that the separation mechanism relies on steric interactions between polymer segments, which prevent substrate disorder sites from trapping more than one DNA segment each. Since thermal activation does not play a significant role in determining the polymer mobility, band broadening due to diffusion can be avoided in our separation method.

  10. Electrophoresis of DNA on a disordered two-dimensional substrate

    NASA Astrophysics Data System (ADS)

    Olson Reichhardt, Cynthia J.; Reichhardt, Charles

    2007-03-01

    We propose a method for electrophoretic separation of DNA in which adsorbed polymers are driven over a disordered two-dimensional substrate which contains attractive sites for the polymers. Using simulations of a model for long polymer chains, we show that the mobility increases with polymer length, in contrast to gel electrophoresis techniques, and that separation can be achieved for a range of length scales. We demonstrate that the separation relies on steric interactions between polymer segments, which prevent substrate disorder sites from trapping more than one DNA segment each. Since thermal activation does not play a significant role in determining the polymer mobility, band broadening due to diffusion can be avoided in our separation method. [1] Phys. Rev. E 74, 051908 (2006).

  11. Function of high-mobility group A proteins in the DNA damage signaling for the induction of apoptosis.

    PubMed

    Fujikane, Ryosuke; Komori, Kayoko; Sekiguchi, Mutsuo; Hidaka, Masumi

    2016-01-01

    O(6)-Methylguanine produced in DNA can pair with thymine during DNA replication, thus leading to a G-to-A transition mutation. To prevent such outcomes, cells harboring O(6)-methylguanine-containing mispair undergo apoptosis that requires the function of mismatch repair (MMR) protein complex. To identify the genes involved in the induction of apoptosis, we performed gene-trap mutagenesis and isolated a clone of mouse cells exhibiting an increased resistance to the killing effect of an alkylating agent, N-methyl-N-nitrosourea (MNU). The mutant carries an insertion in the Hmga2 gene, which belongs to a gene family encoding the high-mobility group A non-histone chromatin proteins. To elucidate the function of HMGA proteins in the apoptosis pathway, we introduced siRNAs for HMGA1 and/or HMGA2 into human HeLa MR cells defective in O(6)-methylguanine-DNA methyltransferase. HMGA1- and HMGA2-single knockdown cells showed an increased resistance to MNU, and HMGA1/HMGA2-double knockdown cells exhibited further increased tolerance compared to the control. The phosphorylation of ATR and CHK1, the appearance of a sub-G1 population, and caspase-9 activation were suppressed in the knockdown cells, although the formation of mismatch recognition complex was unaffected. These results suggest that HMGA family proteins function at the step following the damage recognition in the process of apoptosis triggered by O(6)-methylguanine. PMID:27538817

  12. Ku counteracts mobilization of PARP1 and MRN in chromatin damaged with DNA double-strand breaks.

    PubMed

    Cheng, Qiao; Barboule, Nadia; Frit, Philippe; Gomez, Dennis; Bombarde, Oriane; Couderc, Bettina; Ren, Guo-Sheng; Salles, Bernard; Calsou, Patrick

    2011-12-01

    In mammalian cells, the main pathway for DNA double-strand breaks (DSBs) repair is classical non-homologous end joining (C-NHEJ). An alternative or back-up NHEJ (B-NHEJ) pathway has emerged which operates preferentially under C-NHEJ defective conditions. Although B-NHEJ appears particularly relevant to genomic instability associated with cancer, its components and regulation are still largely unknown. To get insights into this pathway, we have knocked-down Ku, the main contributor to C-NHEJ. Thus, models of human cell lines have been engineered in which the expression of Ku70/80 heterodimer can be significantly lowered by the conditional induction of a shRNA against Ku70. On Ku reduction in cells, resulting NHEJ competent protein extracts showed a shift from C- to B-NHEJ that could be reversed by addition of purified Ku protein. Using a cellular fractionation protocol after treatment with a strong DSBs inducer followed by western blotting or immunostaining, we established that, among C-NHEJ factors, Ku is the main counteracting factor against mobilization of PARP1 and the MRN complex to damaged chromatin. In addition, Ku limits PAR synthesis and single-stranded DNA production in response to DSBs. These data support the involvement of PARP1 and the MRN proteins in the B-NHEJ route for the repair of DNA DSBs. PMID:21880593

  13. Function of high-mobility group A proteins in the DNA damage signaling for the induction of apoptosis

    PubMed Central

    Fujikane, Ryosuke; Komori, Kayoko; Sekiguchi, Mutsuo; Hidaka, Masumi

    2016-01-01

    O6-Methylguanine produced in DNA can pair with thymine during DNA replication, thus leading to a G-to-A transition mutation. To prevent such outcomes, cells harboring O6-methylguanine-containing mispair undergo apoptosis that requires the function of mismatch repair (MMR) protein complex. To identify the genes involved in the induction of apoptosis, we performed gene-trap mutagenesis and isolated a clone of mouse cells exhibiting an increased resistance to the killing effect of an alkylating agent, N-methyl-N-nitrosourea (MNU). The mutant carries an insertion in the Hmga2 gene, which belongs to a gene family encoding the high-mobility group A non-histone chromatin proteins. To elucidate the function of HMGA proteins in the apoptosis pathway, we introduced siRNAs for HMGA1 and/or HMGA2 into human HeLa MR cells defective in O6-methylguanine-DNA methyltransferase. HMGA1- and HMGA2-single knockdown cells showed an increased resistance to MNU, and HMGA1/HMGA2-double knockdown cells exhibited further increased tolerance compared to the control. The phosphorylation of ATR and CHK1, the appearance of a sub-G1 population, and caspase-9 activation were suppressed in the knockdown cells, although the formation of mismatch recognition complex was unaffected. These results suggest that HMGA family proteins function at the step following the damage recognition in the process of apoptosis triggered by O6-methylguanine. PMID:27538817

  14. A normalization strategy applied to HiCEP (an AFLP-based expression profiling) analysis: Toward the strict alignment of valid fragments across electrophoretic patterns

    PubMed Central

    Kadota, Koji; Fukumura, Ryutaro; Rodrigue, Joseph J; Araki, Ryoko; Abe, Masumi

    2005-01-01

    Background Gene expression analysis based on comparison of electrophoretic patterns is strongly dependent on the accuracy of DNA fragment sizing. The current normalization strategy based on molecular weight markers has limited accuracy because marker peaks are often masked by intense peaks nearby. Cumulative errors in fragment lengths cause problems in the alignment of same-length fragments across different electropherograms, especially for small fragments (< 100 bp). For accurate comparison of electrophoretic patterns, further inspection and normalization of electrophoretic data after fragment sizing by conventional strategies is needed. Results Here we describe a method for the normalization of a set of time-course electrophoretic data to be compared. The method uses Gaussian curves fitted to the complex peak mixtures in each electropherogram. It searches for target ranges for which patterns are dissimilar to the other patterns (called "dissimilar ranges") and for references (a kind of mean or typical pattern) in the set of resultant approximate patterns. It then constructs the optimal normalized pattern whose correlation coefficient against the reference in the range achieves the highest value among various combinations of candidates. We applied the procedure to time-course electrophoretic data produced by HiCEP, an AFLP-based expression profiling method which can detect a slight expression change in DNA fragments. We obtained dissimilar ranges whose electrophoretic patterns were obviously different from the reference and as expected, most of the fragments in the detected ranges were short (< 100 bp). The normalized electrophoretic patterns also agreed well with reference patterns. Conclusion The normalization strategy presented here demonstrates the importance of pre-processing before electrophoretic signal comparison, and we anticipate its usefulness especially for temporal expression analysis by the electrophoretic method. PMID:15748295

  15. Luminescent electrophoretic particles via miniemulsion polymerization for night-vision electrophoretic displays.

    PubMed

    Meng, Xianwei; Wen, Ting; Qiang, Li; Ren, Jun; Tang, Fangqiong

    2013-05-01

    A novel glowing electrophoretic display (EPD) is achieved by luminescent electrophoretic particles (EPs), which is potentially to improve the situation in which the existing EPDs disable in darkness. To combine both modes of reflective and emissive displays, a trilayer luminescence EP is designed and synthesized via an improved miniemulsion polymerization. The luminescence EP is composed of a pigment core, a polystyrene interlayer, and a fluorescent coating. The particle sizes are from 140 to 170 nm, and the size distribution is narrow. Their ζ potential value is -12.4 mV, which is enough to migrate in the electrophoretic fluid by the driving of an electric field. The display performance of the particles in an EPD cell has been characterized under the bias of 20 V. Both the reflectance (491 nm) and fluorescence (521 nm) intensities of the EPD cell remained in a constant range after 30 switches. PMID:23547950

  16. Separation of DNA in nanoscale devices with alternating channel depth.

    NASA Astrophysics Data System (ADS)

    Lau, Henry; Strychalski, Elizabeth; Craighead, Harold; Archer, Lynden

    2008-03-01

    The size-dependent separation of DNA using nanofabricated devices consisting of alternating deep and shallow regions have been the subject of numerous experimental and theoretical works. Recent Brownian dynamics simulations suggest that the separation of rigid-rod DNA can be effected at high electric fields without a loss of resolution (PRL, 2007, 98, 098106). To study the dynamics of DNA separation at high fields, electrophoresis experiments were carried out using DNA fragments up to 753 bp in size. As the transport mechanism of DNA fragments in gels has been shown to be a strong function of topology (Electrophoresis, 2004, 25, 1772), electrophoresis of branched rigid-rod DNA molecules was performed to investigate the effects of analyte architecture on mobility in nanofabricated devices. By comparing the mobility of branched and linear DNA molecules of identical total molecular weight, we exclude the influence of size and charge and focus on the effects of branch size and location, and overall analyte topology. Our results help to elucidate the electrophoretic migration mechanism of DNA molecules with complex architecture in sieving media with precisely-controlled internal structures.

  17. Heterogeneous dynamics in DNA site discrimination by the structurally homologous DNA-binding domains of ETS-family transcription factors.

    PubMed

    He, Gaofei; Tolic, Ana; Bashkin, James K; Poon, Gregory M K

    2015-04-30

    The ETS family of transcription factors exemplifies current uncertainty in how eukaryotic genetic regulators with overlapping DNA sequence preferences achieve target site specificity. PU.1 and Ets-1 represent archetypes for studying site discrimination by ETS proteins because their DNA-binding domains are the most divergent in sequence, yet they share remarkably superimposable DNA-bound structures. To gain insight into the contrasting thermodynamics and kinetics of DNA recognition by these two proteins, we investigated the structure and dynamics of site discrimination by their DNA-binding domains. Electrophoretic mobilities of complexes formed by the two homologs with circularly permuted binding sites showed significant dynamic differences only for DNA complexes of PU.1. Free solution measurements by dynamic light scattering showed PU.1 to be more dynamic than Ets-1; moreover, dynamic changes are strongly coupled to site discrimination by PU.1, but not Ets-1. Interrogation of the protein/DNA interface by DNA footprinting showed similar accessibility to dimethyl sulfate for PU.1/DNA and Ets-1/DNA complexes, indicating that the dynamics of PU.1/DNA complexes reside primarily outside that interface. An information-based analysis of the two homologs' binding motifs suggests a role for dynamic coupling in PU.1's ability to enforce a more stringent sequence preference than Ets-1 and its proximal sequence homologs. PMID:25824951

  18. A mobile group I intron from Physarum polycephalum can insert itself and induce point mutations in the nuclear ribosomal DNA of saccharomyces cerevisiae.

    PubMed Central

    Muscarella, D E; Vogt, V M

    1993-01-01

    Pp LSU3 is a mobile group I intron in the extrachromosomal nuclear ribosomal DNA (rDNA) of Physarum polycephalum. As found for other mobile introns, Pp LSU3 encodes a site-specific endonuclease, I-Ppo, which mediates "homing" to unoccupied target sites in Physarum rDNA. The recognition sequence for this enzyme is conserved in all eucaryotic nuclear rDNAs. We have introduced this intron into a heterologous species, Saccharomyces cerevisiae, in which nuclear group I introns have not been detected. The expression of Pp LSU3, under control of the inducible GAL10 promoter, was found to be lethal as a consequence of double-strand breaks in the rDNA. However, surviving colonies that are resistant to the lethal effects of I-Ppo because of alterations in the rDNA at the cleavage site were recovered readily. These survivors are of two classes. The first comprises cells that acquired one of three types of point mutations. The second comprises cells in which Pp LSU3 became inserted into the rDNA. In both cases, each resistant survivor appears to carry the same alterations in all approximately 150 rDNA repeats. When it is embedded in yeast rDNA, Pp LSU3 leads to the synthesis of I-Ppo and appears to be mobile in appropriate genetic crosses. The existence of yeast cells carrying a mobile intron should allow dissection of the steps that allow expression of the highly unusual I-Ppo gene. Images PMID:8380887

  19. Preparation and surface encapsulation of hollow TiO nanoparticles for electrophoretic displays

    NASA Astrophysics Data System (ADS)

    Zhao, Qian; Tan, Tingfeng; Qi, Peng; Wang, Shirong; Bian, Shuguang; Li, Xianggao; An, Yong; Liu, Zhaojun

    2011-02-01

    Hollow black TiO nanosparticles were obtained via deposition of inorganic coating on the surface of hollow core-shell polymer latex with Ti(OBu)4 as precursor and subsequent calcination in ammonia gas. Hollow TiO particles were characterized by scanning electron microscope, transmission electronic microscopy, X-ray diffraction, and thermogravimetric analysis. Encapsulation of TiO via dispersion polymerization was promoved by pretreating the pigments with 3-(trimethoxysilyl) propyl methacrylate, making it possible to prepare hollow TiO-polymer particles. When St and DVB were used as polymerization monomer, hollow TiO-polymer core-shell particles came into being via dispersion polymerization, and the lipophilic degree is 28.57%. Glutin-arabic gum microcapsules containing TiO-polymer particles electrophoretic liquid were prepared using via complex coacervation. It was founded that hollow TiO-polymer particles had enough electrophoretic mobility after coating with polymer.

  20. Salt dependence of DNA translocation dynamics through silicon nanopores detected by ultraviolet excitation

    NASA Astrophysics Data System (ADS)

    Ito, Shintaro; Yamazaki, Hirohito; Tsukahara, Mutsumi; Esashika, Keiko; Saiki, Toshiharu

    2016-04-01

    DNA translocation through nanopores was observed using ultraviolet excitation to investigate the effect of salt concentration and counterion species on the translocation speed. The translocation of 9.6-kbp DNA molecules was measured in an aqueous solvent containing KCl, NaCl, or LiCl. An increase in the KCl concentration from 0.5 to 2 M increased the DNA translocation time. Maintaining the salt concentration at 1.0 M but replacing KCl with NaCl or LiCl also increased the translocation time. These results suggest that the effective charge on the DNA changed due to the binding of counterions, decreasing the DNA electrophoretic mobility. Significant correlation was observed between the translocation time and the dwell time in the observation volume (time needed to move out of the observation volume), and a possible explanation for this observation is provided.

  1. Polyacrylamide medium for the electrophoretic separation of biomolecules

    DOEpatents

    Madabhushi, Ramakrishna S.; Gammon, Stuart A.

    2003-11-11

    A polyacryalmide medium for the electrophoretic separation of biomolecules. The polyacryalmide medium comprises high molecular weight polyacrylamides (PAAm) having a viscosity average molecular weight (M.sub.v) of about 675-725 kDa were synthesized by conventional red-ox polymerization technique. Using this separation medium, capillary electrophoresis of BigDye DNA sequencing standard was performed. A single base resolution of .about.725 bases was achieved in .about.60 minute in a non-covalently coated capillary of 50 .mu.m i.d., 40 cm effective length, and a filed of 160 V/cm at 40.degree. C. The resolution achieved with this formulation to separate DNA under identical conditions is much superior (725 bases vs. 625 bases) and faster (60 min. vs. 75 min.) to the commercially available PAAm, such as supplied by Amersham. The formulation method employed here to synthesize PAAm is straight-forward, simple and does not require cumbersome methods such as emulsion polymerizaiton in order to achieve very high molecular weights. Also, the formulation here does not require separation of PAAm from the reaction mixture prior to reconstituting the polymer to a final concentration. Furthermore, the formulation here is prepared from a single average mol. wt. PAAm as opposed to the mixture of two different average mo. wt. PAAm previously required to achieve high resolution.

  2. Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation.

    PubMed

    Kim, Jinho; Olsen, Timothy R; Zhu, Jing; Hilton, John P; Yang, Kyung-Ae; Pei, Renjun; Stojanovic, Milan N; Lin, Qiao

    2016-01-01

    We present a microfluidic approach to integrated isolation of DNA aptamers via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs a microbead-based protocol for the processes of affinity selection and amplification of target-binding oligonucleotides, and an electrophoretic DNA manipulation scheme for the coupling of these processes, which are required to occur in different buffers. This achieves the full microfluidic integration of SELEX, thereby enabling highly efficient isolation of aptamers in drastically reduced times and with minimized consumption of biological material. The approach as such also offers broad target applicability by allowing selection of aptamers with respect to targets that are either surface-immobilized or solution-borne, potentially allowing aptamers to be developed as readily available affinity reagents for a wide range of targets. We demonstrate the utility of this approach on two different procedures, respectively for isolating aptamers against a surface-immobilized protein (immunoglobulin E) and a solution-phase small molecule (bisboronic acid in the presence of glucose). In both cases aptamer candidates were isolated in three rounds of SELEX within a total process time of approximately 10 hours. PMID:27217242

  3. Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation

    PubMed Central

    Kim, Jinho; Olsen, Timothy R.; Zhu, Jing; Hilton, John P.; Yang, Kyung-Ae; Pei, Renjun; Stojanovic, Milan N.; Lin, Qiao

    2016-01-01

    We present a microfluidic approach to integrated isolation of DNA aptamers via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs a microbead-based protocol for the processes of affinity selection and amplification of target-binding oligonucleotides, and an electrophoretic DNA manipulation scheme for the coupling of these processes, which are required to occur in different buffers. This achieves the full microfluidic integration of SELEX, thereby enabling highly efficient isolation of aptamers in drastically reduced times and with minimized consumption of biological material. The approach as such also offers broad target applicability by allowing selection of aptamers with respect to targets that are either surface-immobilized or solution-borne, potentially allowing aptamers to be developed as readily available affinity reagents for a wide range of targets. We demonstrate the utility of this approach on two different procedures, respectively for isolating aptamers against a surface-immobilized protein (immunoglobulin E) and a solution-phase small molecule (bisboronic acid in the presence of glucose). In both cases aptamer candidates were isolated in three rounds of SELEX within a total process time of approximately 10 hours. PMID:27217242

  4. Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation

    NASA Astrophysics Data System (ADS)

    Kim, Jinho; Olsen, Timothy R.; Zhu, Jing; Hilton, John P.; Yang, Kyung-Ae; Pei, Renjun; Stojanovic, Milan N.; Lin, Qiao

    2016-05-01

    We present a microfluidic approach to integrated isolation of DNA aptamers via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs a microbead-based protocol for the processes of affinity selection and amplification of target-binding oligonucleotides, and an electrophoretic DNA manipulation scheme for the coupling of these processes, which are required to occur in different buffers. This achieves the full microfluidic integration of SELEX, thereby enabling highly efficient isolation of aptamers in drastically reduced times and with minimized consumption of biological material. The approach as such also offers broad target applicability by allowing selection of aptamers with respect to targets that are either surface-immobilized or solution-borne, potentially allowing aptamers to be developed as readily available affinity reagents for a wide range of targets. We demonstrate the utility of this approach on two different procedures, respectively for isolating aptamers against a surface-immobilized protein (immunoglobulin E) and a solution-phase small molecule (bisboronic acid in the presence of glucose). In both cases aptamer candidates were isolated in three rounds of SELEX within a total process time of approximately 10 hours.

  5. Improvements in in-plane electrophoretic displays

    NASA Astrophysics Data System (ADS)

    Henzen, Alex

    2011-03-01

    Electronic paper is now developing fast into an accepted alternative for paper. Its applications nowadays seem focused on books, documents and newspapers. Development of credible color implementations of electrophoretic displays has been initiated, focusing on multi-layer in-plane electrophoresis, but the difficulties associated with these systems (particle drift, aperture, accuracy) were so far not solved. Electro-osmotic principles lead to openings towards multi-layer color displays as well as fast switching, high reflectance grayscale displays. Drift, aperture and accuracy can be brought to the level necessary to create in-plane switching electro-osmotic displays without the need for encapsulation

  6. Scanning and storage of electrophoretic records

    DOEpatents

    McKean, Ronald A.; Stiegman, Jeff

    1990-01-01

    An electrophoretic record that includes at least one gel separation is mounted for motion laterally of the separation record. A light source is positioned to illuminate at least a portion of the record, and a linear array camera is positioned to have a field of view of the illuminated portion of the record and orthogonal to the direction of record motion. The elements of the linear array are scanned at increments of motion of the record across the field of view to develop a series of signals corresponding to intensity of light at each element at each scan increment.

  7. A hybrid molecular dynamics study of the translocation of DNA through entropic traps

    NASA Astrophysics Data System (ADS)

    Hotmar, Petr

    2010-11-01

    The interplay between thermal diffusion and electrophoretic migration of λ-phage DNA in entropic traps was studied using a hybrid molecular dynamics algorithm. The governing systems of field equations are discretized by finite differences on curvilinear overlapping grids with the solvent modeled as a continuum in unsteady creeping flow. Similar to Brownian dynamics, the polymer segments are coarse-grained into a bead-spring model that follows Langevin dynamics. The hydrodynamic interactions are captured on a semi-empirical level with localized force-transfer. We have established the non-monotonic dependence of electrophoretic mobility on chain length, which characterizes the transition from the free flowing to the trapping behavior. We further quantify the subtle effects of dielectrophoresis and induced-charge electroosmosis on the polymer dynamics.

  8. Electrophoretic Porosimetry of Sol-Gels

    NASA Technical Reports Server (NTRS)

    Snow, L. A.; Smith, D. D.; Sibille, L.; Hunt, A. J.; Ng, J.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    It has been hypothesized that gravity has an effect on the formation and resulting microstructure of sol-gels. In order to more clearly resolve the effect of gravity, pores may be non-destructively analyzed in the wet gel, circumventing the shrinkage and coarsening associated with the drying procedure. We discuss the development of an electrophoretic technique, analogous to affinity chromatography, for the determination of pore size distribution and its application to silica gels. Specifically a monodisperse charged dye is monitored by an optical densitometer as it moves through the wet gel under the influence of an electric field. The transmittance data (output) represents the convolution of the dye concentration profile at the beginning of the run (input) with the pore size distribution (transfer function), i.e. linear systems theory applies. Because of the practical difficulty in producing a delta function input dye profile we prefer instead to use a step function. Average pore size is then related to the velocity of this dye front, while the pore size distribution is related to the spreading of the front. Preliminary results of this electrophoretic porosimetry and its application to ground and space-grown samples will be discussed.

  9. Functions of the high mobility group protein, Abf2p, in mitochondrial DNA segregation, recombination and copy number in Saccharomyces cerevisiae.

    PubMed Central

    Zelenaya-Troitskaya, O; Newman, S M; Okamoto, K; Perlman, P S; Butow, R A

    1998-01-01

    Previous studies have established that the mitochondrial high mobility group (HMG) protein, Abf2p, of Saccharomyces cerevisiae influences the stability of wild-type (rho+) mitochondrial DNA (mtDNA) and plays an important role in mtDNA organization. Here we report new functions for Abf2p in mtDNA transactions. We find that in homozygous deltaabf2 crosses, the pattern of sorting of mtDNA and mitochondrial matrix protein is altered, and mtDNA recombination is suppressed relative to homozygous ABF2 crosses. Although Abf2p is known to be required for the maintenance of mtDNA in rho+ cells growing on rich dextrose medium, we find that it is not required for the maintenance of mtDNA in p cells grown on the same medium. The content of both rho+ and rho- mtDNAs is increased in cells by 50-150% by moderate (two- to threefold) increases in the ABF2 copy number, suggesting that Abf2p plays a role in mtDNA copy control. Overproduction of Abf2p by > or = 10-fold from an ABF2 gene placed under control of the GAL1 promoter, however, leads to a rapid loss of rho+ mtDNA and a quantitative conversion of rho+ cells to petites within two to four generations after a shift of the culture from glucose to galactose medium. Overexpression of Abf2p in rho- cells also leads to a loss of mtDNA, but at a slower rate than was observed for rho+ cells. The mtDNA instability phenotype is related to the DNA-binding properties of Abf2p because a mutant Abf2p that contains mutations in residues of both HMG box domains known to affect DNA binding in vitro, and that binds poorly to mtDNA in vivo, complements deltaabf2 cells only weakly and greatly lessens the effect of overproduction on mtDNA instability. In vivo binding was assessed by colocalization to mtDNA of fusions between mutant or wild-type Abf2p and green fluorescent protein.These findings are discussed in the context of a model relating mtDNA copy number control and stability to mtDNA recombination. PMID:9581629

  10. Preparation and properties of red inorganic hollow nanospheres for electrophoretic display

    NASA Astrophysics Data System (ADS)

    Fang, Yi; Wang, Shirong; Xiao, Yin; Li, Xianggao

    2014-10-01

    An effective approach had been developed for the preparation of Fe-doped TiO2 red hollow nanospheres via template method using PMMA-BA copolymers as the core template by a two-step hydrolysis process. The nanospheres were rarely displayed fragmentation and exhibited hollow structures with uniform size and shape. Then, the multicomponent Fe/Co/Al-doped TiO2 hollow nanospheres were produced with Co and Al as tinting metal ions so as to endow them with higher color saturation and brightness. The average diameter of the hollow spheres coated with a layer of α-Fe2O3 was approximately 300 nm and the thickness of the layer was roughly 50 nm. The electrophoretic mobility and zeta potential of two kinds of hollow particles were about -1.0 × 10-5 cm2 v-1 s-1 and -100 mV, respectively. Finally, the electrophoretic inks prototype device was successfully assembled using dispersion of the obtained red hollow nanospheres in a mixed dielectric solvent with TiO2 white particles as contrast. Under an applied bias voltage of 30 V, the response time of the simple EPD device was 1121 ms and the max contrast was 3.173, which had shown great potential for practical application in a vivid chromatic electrophoretic display.