| 1. |
- Alizadehheidari, Mohammadreza, 1987, et al.
(författare)
-
Nanoconfined Circular and Linear DNA: Equilibrium Conformations and Unfolding Kinetics
- 2015
-
Ingår i: Macromolecules. - : 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.
|
|
| 2. |
- Bogas, Diana, et al.
(författare)
-
Applications of optical DNA mapping in microbiology
- 2017
-
Ingår i: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 62:6, s. 255-267
-
Forskningsöversikt (refereegranskat)abstract
- Optical mapping (OM) has been used in microbiology for the past 20 years, initially as a technique to facilitate DNA sequence-based studies; however, with decreases in DNA sequencing costs and increases in sequence output from automated sequencing platforms, OM has grown into an important auxiliary tool for genome assembly and comparison. Currently, there are a number of new and exciting applications for OM in the field of microbiology, including investigation of disease outbreaks, identification of specific genes of clinical and/or epidemiological relevance, and the possibility of single-cell analysis when combined with cell-sorting approaches. In addition, designing lab-on-a-chip systems based on OM is now feasible and will allow the integrated and automated microbiological analysis of biological fluids. Here, we review the basic technology of OM, detail the current state of the art of the field, and look ahead to possible future developments in OM technology for microbiological applications.
|
|
| 3. |
- Emilsson, Gustav, 1989, et al.
(författare)
-
Identifying bacteria using DNA binding maps
- 2013
-
Ingår i: 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013; Freiburg; Germany; 27 October 2013 through 31 October 2013. - 9781632666246 ; 1, s. 473-475
-
Konferensbidrag (refereegranskat)abstract
- We have developed an assay, based on nanofluidic channels and fluorescence microscopy, for optical mapping of DNA based on competitive binding between two molecules - one fluorescent and one sequence selective. From the experimental data we can extract binding constants for the two competing DNA binders, which may be subsequently used to calculate a theoretical reference map of any DNA with known sequence. The goal is to create a method for fast identification of bacteria from single DNA molecules without the need for additional cultivation or amplification. We here demonstrate a proof-of-principle experiment on phage DNA and furthermore show that the method can be used to distinguish between two strains of E. coli DNA and to map pieces of DNA onto the full genome.
|
|
| 4. |
- Freitag, C., et al.
(författare)
-
Visualizing the entire DNA from a chromosome in a single frame
- 2015
-
Ingår i: Biomicrofluidics. - : AIP Publishing. - 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. (C) 2015 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 Unported License.
|
|
| 5. |
- Fritzsche, Joachim, 1977, et al.
(författare)
-
A lipid-based passivation scheme for nanofluidics
- 2012
-
Ingår i: 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012; Okinawa; Japan; 28 October 2012 through 1 November 2012. - 9780979806452 ; , s. 1876-1878
-
Konferensbidrag (refereegranskat)abstract
- Stretching DNA in nanochannels allows for direct, visual studies of genomic DNA at the single molecule level. In order 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 show long-term passivation of nanochannel surfaces to several relevant reagents and demonstrate that the performance of the lipid bilayer is significantly better compared to standard bovine serum albumin-based passivation. Moreover, we demonstrate how the passivated devices allow us to monitor single DNA cleavage events during enzymatic degradation.
|
|
| 6. |
- Fritzsche, Michael, et al.
(författare)
-
A Highly UV-transparent Fused Silica Biochip for Sensitive Hepatotoxicity Testing by Autofluorescence
- 2014
-
Ingår i: Biochip Journal. - : Springer Science and Business Media LLC. - 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.
|
|
| 7. |
- 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 BV. - 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.
|
|
| 8. |
- Iarko, V., 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). - 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. However, 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 experiments measuring the mean DNA extension and its standard deviation while varying the channel geometry, dye intercalation ratio, and ionic strength of the buffer. 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. In principle, our approach allows us to measure the Kuhn length and the effective width of a single DNA molecule and more generally of semiflexible polymers in solution.
|
|
| 9. |
- Jönsson, Peter, 1981, et al.
(författare)
-
Mechanical Behavior of a Supported Lipid Bilayer under External Shear Forces
- 2009
-
Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 25:11, s. 6279-6286
-
Tidskriftsartikel (refereegranskat)abstract
- Shear forces from a pressure-driven bulk flow in a microfluidic channel can be used to induce and control the motion of a supported lipid bilayer (SLB) formed on the walls of the channel. We here present a theoretical model that relates the experimentally observed drift velocities of an egg yolk phosphatidylcholine (egg PC) SLB to the hydrodynamic drag force from the bulk flow, the intermonolayer friction coefficient, b, of the bilayer, and the friction coefficient, b(ls) between the lower leaflet of the bilayer and the supporting substrate. The drift velocity and diffusivity of the lipids in the SLB were obtained by photobleaching a delimited area of fluorescently labeled lipids and subsequently monitoring the recovery and convective motion of the bleached spot. A striking observation was that the drift velocity of the lipids was observed to be nearly 6 orders of magnitude smaller than the bulk velocity at the center of the channel. This predicts a value for b(ls) that is at least 25 times as high as predicted by the traditional model with the SLB and the support spaced by a homogeneous 1 nm thick film of water. In addition, the intermonolayer friction coefficient was estimated to 2 x 10(7) Pa s/m, a value that increased after addition of glycerol to the bulk solution. This increase was accompanied by an equal decrease in the lipid diffusivity, with both observations indicating an increased viscous drag within the bilayer when glycerol was added to the bulk solution.
|
|
| 10. |
- Jönsson, Peter, 1981, et al.
(författare)
-
Shear-Driven Motion of Supported Lipid Bilayers in Microfluidic Channels
- 2009
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 131:14, s. 5294-5297
-
Tidskriftsartikel (refereegranskat)abstract
- In this work, we demonstrate how a lateral motion of a supported lipid bilayer (SLB) and its constituents can be created without relying on self-spreading forces. The force driving the SLB is instead a viscous shear force arising from a pressure-driven bulk flow acting on the SLB that is formed on a glass wall inside a microfluidic channel. In contrast to self-spreading bilayers, this method allows for accurate control of the bilayer motion by altering the bulk flow in the channel. Experiments showed that an egg yolk phosphatidylcholine SLB formed on a glass support moved in a rolling motion under these shear forces, with the lipids in the upper leaflet of the bilayer moving at twice the velocity of the bilayer front. The drift velocity of different lipid probes in the SLB was observed to be sensitive to the interactions between the lipid probe and the surrounding molecules, resulting in drift velocities that varied by up to I order of magnitude for the different lipid probes in our experiments. Since the method provides a so far unattainable control of the motion of all molecules in an SLB, we foresee great potential for this technique, alone or in combination with other methods, for studies of lipid bilayers and different membrane-associated molecules.
|
|
