Escherichia coli single-stranded DNA-binding protein,a molecular tool for improved sequence quality in pyrosequencing

Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technique based on a DNA sequencing by synthesis principle. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis. In order to expand the field for pyrosequencing, the read length needs to be improved and efforts have been made to purify reaction components as well as add single-stranded DNA-binding protein (SSB) to the pyrosequencing reaction. In this study, we have performed a systematic effort to analyze the effects of SSB by comparing the pyrosequencing result of 103 independent complementary DNA (cDNA) clones. More detailed information about the cause of low quality sequences on templates with different characteristics was achieved by thorough analysis of the pyrograms. Also, real-time biosensor analysis was performed on individual cDNA clones for investigation of primer annealing and SSB binding on these templates. Results from these studies indicate that templates with high performance in pyrosequencing without SSB possess efficient primer annealing and low SSB affinity. Alternative strategies to improve the performance in pyrosequencing by increasing the primer-annealing efficiency have also been evaluated.     

The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures

The biotin-streptavidin system is the strongest noncovalent biological interaction known, having a dissociation constant, K(d), in the order of 4x10(-14) M. The strength and specificity of the interaction has led it to be one of the most widely used affinity pairs in molecular, immunological, and cellular assays. However, it has previously been impossible to re-use any streptavidin solid support, since the conditions needed to break the interaction with biotin has led to the denaturation of the streptavidin. Here, we show that a short incubation in nonionic aqueous solutions at temperatures above 70 degrees C can efficiently break the interaction without denaturing the streptavidin tetramer. Both biotin and the streptavidin remain active after dissociation and both molecules can therefore be re-used. The efficiency of the regeneration allowed solid supports with streptavidin to be used many times, here exemplified with the multiple re-use of streptavidin beads used for sample preparation prior to automated DNA sequencing. The results suggest that streptavidin regeneration can be introduced as an improvement in existing methods and assays based on the streptavidin system as well as emerging solid phase applications in fields, such as microfluidics and nanotechnology.     

Multiple periodic solutions for a nonlinear suspension bridge equation

We investigate nonlinear oscillations in a fourth-order partialdifferential equation which models a suspension bridge. Previouswork establishes multiple periodic solutions when a parameterexceeds a certain eigenvalue. In this paper, we use Leray-Schauderdegree theory to prove that if the parameter is increased further,beyond a second eigenvalue, then additional solutions are created.     

Visual DNA as a diagnostic tool

We report on the incorporation of the Visual DNA concept in a genotyping assay as a simple and straightforward detection tool. The principle of trapping streptavidin‐coated superparamagnetic beads of micrometer size for visualization of genetic variances is used for PrASE‐based detection of a panel of mutations in the severe and common genetic disorder of cystic fibrosis. The method allows a final investigation of genotypes by the naked eye and the output is easily documented using a regular hand‐held device with an integrated digital camera. A number of samples were run through the assay, showing rapid and accurate detection using superparamagnetic beads and an off‐the‐shelf neodymium magnet. The assay emphasizes the power of Visual DNA and demonstrates the potential value of the method in future point‐of‐care tests.     

Real-time imaging of K atoms on graphite: interactions and diffusion

Scanning tunneling microscopy (STM) at liquid helium temperature is used to image potassium adsorbed on graphite at low coverage (≈0.02 monolayer). Single atoms appear as protrusions on STM topographs. A statistical analysis of the position of the atoms demonstrates repulsion between adsorbates, which is quantified by comparison with molecular dynamics simulations. This gives access to the dipole moment of a single adsorbate, found to be 10.5±1 D. Time-lapse imaging shows that long-range order is broken by thermally activated diffusion, with a 30 meV barrier to hopping between graphite lattice sites.     

Experiments with longitudinally polarized electrons in a storage ring using a siberian snake

We report on first measurements with polarized electrons stored in a medium-energy ring and with a polarized internal target. Polarized electrons were injected at 442 MeV (653 MeV), and a partial (full) Siberian snake was employed to preserve the polarization. Longitudinal polarization at the interaction point and polarization lifetime of the stored electrons were determined with laser backscattering. Spin observables were measured for electrodisintegration of polarized 3He, with simultaneous detection of scattered electrons, protons, neutrons, deuterons, and 3He nuclei, over a large phase space.     

DNA vaccine containing the mycobacterial hsp65 gene prevented insulitis in MLD-STZ diabetes

Background  

Our group previously demonstrated that a DNA plasmid encoding the mycobacterial 65-kDa heat shock protein (DNA-HSP65) displayed prophylactic and therapeutic effect in a mice model for tuberculosis. This protection was attributed to induction of a strong cellular immunity against HSP65. As specific immunity to HSP60 family has been detected in arthritis, multiple sclerosis and diabetes, the vaccination procedure with DNA-HSP65 could induce a cross-reactive immune response that could trigger or worsen these autoimmune diseases.     

Rippled graphene in an in-plane magnetic field: effects of a random vector potential

We report measurements of the effects of a random vector potential generated by applying an in-plane magnetic field to a graphene flake. Magnetic flux through the ripples cause orbital effects: Phase-coherent weak localization is suppressed, while quasirandom Lorentz forces lead to anisotropic magnetoresistance. Distinct signatures of these two effects enable the ripple size to be characterized.     

Microarray-based resequencing by apyrase-mediated allele-specific extension

We have developed an array-based resequencing method to determine genetic alterations in putative cancer genes. The method relies on that the specificity of DNA polymerase in allele-specific extensions can be enhanced by terminating the extension reactions with apyrase and that a tiling set of primers are synthesized covering the investigated gene sequence. We report on such apyrase-mediated allele-specific primer extension (AMASE) assay as a method suitable for high-throughout resequencing and mutation detection in tumor suppressor genes and oncogenes. In the experimental setup, primers complementary to codons 12, 13 and codon 61 of the N-ras proto-oncogene were spotted onto glass slides. Overlapping sense and anti-sense primers were designed so that complementary primers for all possible mutations in each base position were investigated. The extension reactions were performed in a single step following hybridization of target DNA to the immobilized primers on the array surface. Mutation detection limits and the possibility of quantifying the mutations were investigated using synthetic oligonucleotides. In addition, 64 clinical samples were sequenced and 16 of these showed mutations in the N-ras gene.