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1.
  • Forskningsöversikt (refereegranskat)
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2.
  • Alizadehheidari, Mohammadreza, et al. (författare)
  • Nanoconfined Circular and Linear DNA: Equilibrium Conformations and Unfolding Kinetics
  • 2015
  • Ingår i: Macromolecules. - : The American Chemical Society (ACS). - 0024-9297 .- 1520-5835. ; 48:3, s. 871-878
  • Tidskriftsartikel (refereegranskat)abstract
    • Studies of circular DNA confined to nanofluidic channels are relevant both from a fundamental polymer-physics perspective and due to the importance of circular DNA molecules in vivo. We here observe the unfolding of confined DNA from the circular to linear configuration as a light-induced double-strand break occurs, characterize the dynamics, and compare the equilibrium conformational statistics of linear and circular configurations. This is important because it allows us to determine to what extent existing statistical theories describe the extension of confined circular DNA. We find that the ratio of the extensions of confined linear and circular DNA configurations increases as the buffer concentration decreases. The experimental results fall between theoretical predictions for the extended de Gennes regime at weaker confinement and the Odijk regime at stronger confinement. We show that it is possible to directly distinguish between circular and linear DNA molecules by measuring the emission intensity from the DNA. Finally, we determine the rate of unfolding and show that this rate is larger for more confined DNA, possibly reflecting the corresponding larger difference in entropy between the circular and linear configurations.
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3.
  • Freitag, Camilla, et al. (författare)
  • Visualizing the entire DNA from a chromosome in a single frame
  • 2015
  • Ingår i: Biomicrofluidics. - : American Institute of Physics (AIP). - 1932-1058. ; 9:4
  • Tidskriftsartikel (refereegranskat)abstract
    • The contiguity and phase of sequence information are intrinsic to obtain complete understanding of the genome and its relationship to phenotype. We report the fabrication and application of a novel nanochannel design that folds megabase lengths of genomic DNA into a systematic back-and-forth meandering path. Such meandering nanochannels enabled us to visualize the complete 5.7 Mbp (1mm) stained DNA length of a Schizosaccharomyces pombe chromosome in a single frame of a CCD. We were able to hold the DNA in situ while implementing partial denaturation to obtain a barcode pattern that we could match to a reference map using the Poland-Scheraga model for DNA melting. The facility to compose such long linear lengths of genomic DNA in one field of view enabled us to directly visualize a repeat motif, count the repeat unit number, and chart its location in the genome by reference to unique barcode motifs found at measurable distances from the repeat. Meandering nanochannel dimensions can easily be tailored to human chromosome scales, which would enable the whole genome to be visualized in seconds.
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4.
  • Fritzsche, Michael, et al. (författare)
  • A Highly UV-transparent Fused Silica Biochip for Sensitive Hepatotoxicity Testing by Autofluorescence
  • 2014
  • Ingår i: Biochip Journal. - : Korean BioChip Society (KBCS). - 2092-7843 .- 1976-0280. ; 8:2, s. 115-121
  • Tidskriftsartikel (refereegranskat)abstract
    • Fabrication and application of a non-fluorescent and UV-transparent microfluidic biochip in fused silica that allows sensitive autofluorescence detection are described. The biochip is particularly useful in cell-based assays where the most informative autofluorescence signals from the cells reside in the ultraviolet spectral range and where plastic labware materials commonly used in cell culture work severely disturb such measurements. In this study the fused silica biochip was used for measuring intrinsic autofluorescence from liver cells in order to assess hepatotoxic effects of drugs. The assessment assay was carried out with the human liver cell line HepG2 under perfusion conditions in the microfluidics of the biochip. The autofluorescence from the.liver cells exposed to quinidine was readily recorded without background disturbance and correlated well with reference toxicity data.
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5.
  • Frykholm, Karolin, 1977, et al. (författare)
  • Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51
  • 2013
  • Ingår i: Analytical Biochemistry. - : Elsevier. - 0003-2697 .- 1096-0309. ; 443:2, s. 261-268
  • Tidskriftsartikel (refereegranskat)abstract
    • Low throughput is an inherent problem associated with most single-molecule biophysical techniques. We have developed a versatile tool for high-throughput analysis of DNA and DNA-binding molecules by combining microfluidic and dense DNA arrays. We use an easy-to-process microfluidic flow channel system in which dense DNA arrays are prepared for simultaneous imaging of large amounts of DNA molecules with single-molecule resolution. The Y-shaped microfluidic design, where the two inlet channels can be controlled separately and precisely, enables the creation of a concentration gradient across the microfluidic channel as well as rapid and repeated addition and removal of substances from the measurement region. A DNA array stained with the fluorescent DNA-binding dye YOYO-1 in a gradient manner illustrates the method and serves as a proof of concept. We have applied the method to studies of the repair protein Rad51 and could directly probe the concentration-dependent DNA-binding behavior of human Rad51 (HsRad51). In the low-concentration regime used (100 nM HsRad51 and below), we detected binding to double-stranded DNA (dsDNA) without positive cooperativity. (C) 2013 Elsevier Inc. All rights reserved.
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6.
  • Iarko, Vitalii, 1993, et al. (författare)
  • Extension of nanoconfined DNA: quantitative comparison between experiment and theory
  • 2015
  • Ingår i: Physical Review E. Statistical, Nonlinear, and Soft Matter Physics. - : American Physical Society. - 1539-3755 .- 1550-2376. ; 92:6, s. Art. Nr. 062701-
  • Tidskriftsartikel (refereegranskat)abstract
    • The extension of DNA confined to nanochannels has been studied intensively and in detail. Yet quantitative comparisons between experiments and model calculations are difficult because most theoretical predictions involve undetermined prefactors, and because the model parameters (contour length, Kuhn length, effective width) are difficult to compute reliably, leading to substantial uncertainties. Here we use a recent asymptotically exact theory for the DNA extension in the "extended de Gennes regime" that allows us to compare experimental results with theory. For this purpose we performed new experiments, measuring the mean DNA extension and its standard deviation while varying the channel geometry, dye intercalation ratio, and ionic buffer strength. The experimental results agree very well with theory at high ionic strengths, indicating that the model parameters are reliable. At low ionic strengths the agreement is less good. We discuss possible reasons. Our approach allows, in principle, to measure the Kuhn length and effective width of a single DNA molecule and more generally of semiflexible polymers in solution.
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7.
  • McGinn, Steven, et al. (författare)
  • New Technologies for DNA analysis-A review of the READNA Project.
  • Ingår i: New Biotechnology. - : Elsevier. - 1876-4347 .- 1871-6784. ; 33:3, s. 311-330
  • Forskningsöversikt (refereegranskat)abstract
    • The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 4 1/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.
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8.
  • Nyberg, Lena, 1979, et al. (författare)
  • A single-step competitive binding assay for mapping of single DNA molecules
  • 2012
  • Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier. - 0006-291X .- 1090-2104. ; 417:1, s. 404-408
  • Tidskriftsartikel (refereegranskat)abstract
    • Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification cif pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.
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9.
  • Ohlsson, Gabriel, 1982, et al. (författare)
  • Solute transport on the sub 100 ms scale across the lipid bilayer membrane of individual proteoliposomes
  • 2012
  • Ingår i: Lab on a Chip. - : Royal Society of Chemistry. - 1473-0197 .- 1473-0189. ; 12:22, s. 4635-4643
  • Tidskriftsartikel (refereegranskat)abstract
    • Screening assays designed to probe ligand and drug-candidate regulation of membrane proteins responsible for ion-translocation across the cell membrane are wide spread, while efficient means to screen membrane-protein facilitated transport of uncharged solutes are sparse. We report on a microfluidic-based system to monitor transport of uncharged solutes across the membrane of multiple (>100) individually resolved surface-immobilized liposomes. This was accomplished by rapidly switching (<10 ms) the solution above dye-containing liposomes immobilized on the floor of a microfluidic channel. With liposomes encapsulating the pH-sensitive dye carboxyfluorescein (CF), internal changes in pH induced by transport of a weak acid (acetic acid) could be measured at time scales down to 25 ms. The applicability of the set up to study biological transport reactions was demonstrated by examining the osmotic water permeability of human aquaporin (AQP5) reconstituted in proteoliposomes. In this case, the rate of osmotic-induced volume changes of individual proteoliposomes was time resolved by imaging the self quenching of encapsulated calcein in response to an osmotic gradient. Single-liposome analysis of both pure and AQP5-containing liposomes revealed a relatively large heterogeneity in osmotic permeability. Still, in the case of AQP5-containing liposomes, the single liposome data suggest that the membrane-protein incorporation efficiency depends on liposome size, with higher incorporation efficiency for larger liposomes. The benefit of low sample consumption and automated liquid handling is discussed in terms of pharmaceutical screening applications.
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10.
  • Persson, Fredrik, 1979, et al. (författare)
  • Lipid-Based Passivation in Nanofluidics
  • 2012
  • Ingår i: Nano Letters. - : The American Chemical Society (ACS). - 1530-6992 .- 1530-6984. ; 12:5, s. 2260-2265
  • Tidskriftsartikel (refereegranskat)abstract
    • Stretching DNA in nanochannels is a useful tool for direct, visual studies of genomic DNA at the single molecule level. To facilitate the study of the interaction of linear DNA with proteins in nanochannels, we have implemented a highly effective passivation scheme based on lipid bilayers. We demonstrate virtually complete long-term passivation of nanochannel surfaces to a range of relevant reagents, including streptavidin-coated quantum dots, RecA proteins, and RecA-DNA complexes. We show that the performance of the lipid bilayer is significantly better than that of standard bovine serum albumin-based passivation. Finally, we show how the passivated devices allow us to monitor single DNA cleavage events during enzymatic degradation by DNase I. We expect that our approach will open up for detailed, systematic studies of a wide range of protein-DNA interactions with high spatial and temporal resolution.
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