| 1. |
- Alizadehheidari, Mohammadreza, 1987, et al.
(författare)
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Nanoconfined Circular and Linear DNA: Equilibrium Conformations and Unfolding Kinetics
- 2015
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Ingår i: Macromolecules. - : American Chemical Society (ACS). - 0024-9297 .- 1520-5835. ; 48:3, s. 871-878
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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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| 2. |
- Alizadehheidari, Mohammadreza, 1987, et al.
(författare)
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Nanoconfined circular DNA
- 2014
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Ingår i: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014. - 9780979806476 ; , s. 1353-1355
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Konferensbidrag (refereegranskat)abstract
- Studies of nanoconfined circular DNA are of interest both from a biological as well as a fundamental polymer physics perspective. We here present the use of nanofluidic channels as a tool for comparing statics and dynamics of the linear and circular configuration of the same DNA molecule.
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| 3. |
- Alizadehheidari, Mohammadreza, 1987, et al.
(författare)
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Nanoconfined Circular DNA
- 2014
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)
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| 4. |
- Alizadehheidari, Mohammadreza, 1987, et al.
(författare)
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Unfolding of nanoconfined circular DNA
- 2015
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Ingår i: BIOPHYSICAL JOURNAL. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 108:2 Supplement 1, s. 231A-231A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 5. |
- Beech, J. P., et al.
(författare)
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What do photons do to fluorescently stained DNA in confinement?
- 2013
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Ingår i: 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013; Freiburg; Germany; 27 October 2013 through 31 October 2013. ; 1, s. 5-7
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Konferensbidrag (refereegranskat)abstract
- We have studied a selection of factors influencing the damage of DNA in nanochannels during fluorescence imaging. For cutting and nicking of DNA we show that the DNA is shortened during imaging. To avoid photodamage over the course of several hours of a typical experiment, we demonstrate the importance of an oxygen free gas to propel the buffer solution through the device. Finally, by varying the size of the channels, we show indications that higher DNA concentrations lead to higher rates of photodamage necessitating a balance between needs for highly stretched DNA and needs for long measurement times.
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| 6. |
- Frykholm, Karolin, 1977, et al.
(författare)
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Fast size-determination of intact bacterial plasmids using nanofluidic channels
- 2015
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 15:13, s. 2739-2743
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate how nanofluidic channels can be used as a tool to rapidly determine the number and sizes of plasmids in bacterial isolates. Each step can be automated at low cost, opening up opportunities for general use in microbiology labs.
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| 7. |
- Iarko, V., et al.
(författare)
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Extension of nanoconfined DNA: Quantitative comparison between experiment and theory
- 2015
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Ingår i: Physical Review E (Statistical, Nonlinear, and Soft Matter Physics). - 1539-3755 .- 1550-2376. ; 92:6, s. Art. Nr. 062701-
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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.
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| 8. |
- Müller, Vilhelm, 1990, et al.
(författare)
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Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping.
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 6, s. 37938-
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples.
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| 9. |
- Müller, Vilhelm, 1990, et al.
(författare)
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Rapid Tracing of Resistance Plasmids in a Nosocomial Outbreak Using Optical DNA Mapping
- 2016
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Ingår i: Acs Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 2:5, s. 322-328
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Tidskriftsartikel (refereegranskat)abstract
- Resistance to life-saving antibiotics increases rapidly worldwide, and multiresistant bacteria have become a global threat to human health. Presently, the most serious threat is the increasing spread of Enterobacteriaceae carrying genes coding for extended spectrum beta-lactamases (ESBL) and carbapenemases on highly mobile plasmids. We here demonstrate how optical DNA maps of single plasmids can be used as fingerprints to trace plasmids, for example, during resistance outbreaks. We use the assay to demonstrate a potential transmission route of an ESBL-carrying plasmid between bacterial strains/species and between patients, during a polyclonal outbreak at a neonatal ward at Sahlgrenska University Hospital (Gothenburg, Sweden). Our results demonstrate that optical DNA mapping is an easy and rapid method for detecting the spread of plasmids mediating resistance. With the increasing prevalence of multiresistant bacteria, diagnostic tools that can aid in solving ongoing routes of transmission, in particular in hospital settings, will be of paramount importance.
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| 10. |
- Nilsson, Adam, et al.
(författare)
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Competitive binding-based optical DNA mapping for fast identification of bacteria - multi-ligand transfer matrix theory and experimental applications on Escherichia coli.
- 2014
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 42:15, s. 118-118
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a single DNA molecule optical mapping assay able to resolve a specific Escherichia coli strain from other strains. The assay is based on competitive binding of the fluorescent dye YOYO-1 and the AT-specific antibiotic netropsin. The optical map is visualized by stretching the DNA molecules in nanofluidic channels. We optimize the experimental conditions to obtain reproducible barcodes containing as much information as possible. We implement a multi-ligand transfer matrix method for calculating theoretical barcodes from known DNA sequences. Our method extends previous theoretical approaches for competitive binding of two types of ligands to many types of ligands and introduces a recursive approach that allows long barcodes to be calculated with standard computer floating point formats. The identification of a specific E. coli strain (CCUG 10979) is based on mapping of 50-160 kilobasepair experimental DNA fragments onto the theoretical genome using the developed theory. Our identification protocol introduces two theoretical constructs: a P-value for a best experiment-theory match and an information score threshold. The developed methods provide a novel optical mapping toolbox for identification of bacterial species and strains. The protocol does not require cultivation of bacteria or DNA amplification, which allows for ultra-fast identification of bacterial pathogens.
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