| 1. |
- Ahlberg, Sebastian, et al.
(författare)
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Many-body effects on tracer particle diffusion with applications for single-protein dynamics on DNA
- 2015
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Ingår i: New Journal of Physics. - : IOP Publishing. - 1367-2630. ; 17
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Tidskriftsartikel (refereegranskat)abstract
- 30% of the DNA in E. coli bacteria is covered by proteins. Such a high degree of crowding affects the dynamics of generic biological processes (e.g. gene regulation, DNA repair, protein diffusion etc) in ways that are not yet fully understood. In this paper, we theoretically address the diffusion constant of a tracer particle in a one-dimensional system surrounded by impenetrable crowder particles. While the tracer particle always stays on the lattice, crowder particles may unbind to a surrounding bulk and rebind at another, or the same, location. In this scenario we determine how the long time diffusion constant D (after many unbinding events) depends on (i) the unbinding rate of crowder particles k(off), and (ii) crowder particle line density rho, from simulations (using the Gillespie algorithm) and analytical calculations. For small k(off), we find D similar to k(off)/rho(2) when crowder particles do not diffuse on the line, and D similar to root Dk(off)/rho when they are diffusing; D is the free particle diffusion constant. For large k(off), we find agreement with mean-field results which do not depend on k(off). From literature values of k(off) and D, we show that the small k(off) -limit is relevant for in vivo protein diffusion on crowded DNA. Our results apply to single-molecule tracking experiments.
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| 2. |
- 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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| 3. |
- 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
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 4. |
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| 5. |
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| 6. |
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| 7. |
- Dvirnas, Albertas, et al.
(författare)
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Detection of structural variations in densely-labelled optical DNA barcodes: A hidden Markov model approach
- 2021
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 16:11
-
Tidskriftsartikel (refereegranskat)abstract
- Large-scale genomic alterations play an important role in disease, gene expression, and chromosome evolution. Optical DNA mapping (ODM), commonly categorized into sparsely-labelled ODM and densely-labelled ODM, provides sequence-specific continuous intensity profiles (DNA barcodes) along single DNA molecules and is a technique well-suited for detecting such alterations. For sparsely-labelled barcodes, the possibility to detect large genomic alterations has been investigated extensively, while densely-labelled barcodes have not received as much attention. In this work, we introduce HMMSV, a hidden Markov model (HMM) based algorithm for detecting structural variations (SVs) directly in densely-labelled barcodes without access to sequence information. We evaluate our approach using simulated data-sets with 5 different types of SVs, and combinations thereof, and demonstrate that the method reaches a true positive rate greater than 80% for randomly generated barcodes with single variations of size 25 kilobases (kb). Increasing the length of the SV further leads to larger true positive rates. For a real data-set with experimental barcodes on bacterial plasmids, we successfully detect matching barcode pairs and SVs without any particular assumption of the types of SVs present. Instead, our method effectively goes through all possible combinations of SVs. Since ODM works on length scales typically not reachable with other techniques, our methodology is a promising tool for identifying arbitrary combinations of genomic alterations.
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| 8. |
- Dvirnas, Albertas, et al.
(författare)
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Facilitated sequence assembly using densely labeled optical DNA barcodes: A combinatorial auction approach
- 2018
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Ingår i: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 13:3
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Tidskriftsartikel (refereegranskat)abstract
- The output from whole genome sequencing is a set of contigs, i.e. short non-overlapping DNA sequences (sizes 1-100 kilobasepairs). Piecing the contigs together is an especially difficult task for previously unsequenced DNA, and may not be feasible due to factors such as the lack of sufficient coverage or larger repetitive regions which generate gaps in the final sequence. Here we propose a new method for scaffolding such contigs. The proposed method uses densely labeled optical DNA barcodes from competitive binding experiments as scaffolds. On these scaffolds we position theoretical barcodes which are calculated from the contig sequences. This allows us to construct longer DNA sequences from the contig sequences. This proof-of-principle study extends previous studies which use sparsely labeled DNA barcodes for scaffolding purposes. Our method applies a probabilistic approach that allows us to discard "foreign" contigs from mixed samples with contigs from different types of DNA. We satisfy the contig non-overlap constraint by formulating the contig placement challenge as a combinatorial auction problem. Our exact algorithm for solving this problem reduces computational costs compared to previous methods in the combinatorial auction field. We demonstrate the usefulness of the proposed scaffolding method both for synthetic contigs and for contigs obtained using Illumina sequencing for a mixed sample with plasmid and chromosomal DNA.
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| 9. |
- Emilsson, Gustav, 1989, et al.
(författare)
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Identifying bacteria using DNA binding maps
- 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. - 9781632666246 ; 1, s. 473-475
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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.
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| 10. |
- Fogelmark, Karl, et al.
(författare)
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Fitting a function to time-dependent ensemble averaged data
- 2018
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- Time-dependent ensemble averages, i.e., trajectory-based averages of some observable, are of importance in many fields of science. A crucial objective when interpreting such data is to fit these averages (for instance, squared displacements) with a function and extract parameters (such as diffusion constants). A commonly overlooked challenge in such function fitting procedures is that fluctuations around mean values, by construction, exhibit temporal correlations. We show that the only available general purpose function fitting methods, correlated chi-square method and the weighted least squares method (which neglects correlation), fail at either robust parameter estimation or accurate error estimation. We remedy this by deriving a new closed-form error estimation formula for weighted least square fitting. The new formula uses the full covariance matrix, i.e., rigorously includes temporal correlations, but is free of the robustness issues, inherent to the correlated chi-square method. We demonstrate its accuracy in four examples of importance in many fields: Brownian motion, damped harmonic oscillation, fractional Brownian motion and continuous time random walks. We also successfully apply our method, weighted least squares including correlation in error estimation (WLS-ICE), to particle tracking data. The WLS-ICE method is applicable to arbitrary fit functions, and we provide a publically available WLS-ICE software.
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| 11. |
- Forsling, Robin, et al.
(författare)
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Non-Markovian effects in the first-passage dynamics of obstructed tracer particle diffusion in one-dimensional systems.
- 2014
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Ingår i: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 141:9
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Tidskriftsartikel (refereegranskat)abstract
- The standard setup for single-file diffusion is diffusing particles in one dimension which cannot overtake each other, where the dynamics of a tracer (tagged) particle is of main interest. In this article, we generalize this system and investigate first-passage properties of a tracer particle when flanked by identical crowder particles which may, besides diffuse, unbind (rebind) from (to) the one-dimensional lattice with rates koff (kon). The tracer particle is restricted to diffuse with rate kD on the lattice and the density of crowders is constant (on average). The unbinding rate koff is our key parameter and it allows us to systematically study the non-trivial transition between the completely Markovian case (koff ≫ kD) to the non-Markovian case (koff ≪ kD) governed by strong memory effects. This has relevance for several quasi one-dimensional systems. One example is gene regulation where regulatory proteins are searching for specific binding sites on a crowded DNA. We quantify the first-passage time distribution, f (t) (t is time), numerically using the Gillespie algorithm, and estimate f (t) analytically. In terms of koff (keeping kD fixed), we study the transition between the two known regimes: (i) when koff ≫ kD the particles may effectively pass each other and we recover the single particle result f (t) ∼ t(-3/2), with a reduced diffusion constant; (ii) when koff ≪ kD unbinding is rare and we obtain the single-file result f (t) ∼ t(-7/4). The intermediate region displays rich dynamics where both the characteristic f (t) - peak and the long-time power-law slope are sensitive to koff.
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| 12. |
- Freitag, C., et al.
(författare)
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Visualizing the entire DNA from a chromosome in a single frame
- 2015
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Ingår i: Biomicrofluidics. - : AIP Publishing. - 1932-1058. ; 9:4
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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.
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| 13. |
- 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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| 14. |
- Genthon, Arthur, et al.
(författare)
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Equilibrium melting probabilities of a DNA molecule with a defect : An exact solution of the Poland-Scheraga model
- 2023
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Ingår i: Journal of Chemical Physics. - 0021-9606. ; 159:14
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Tidskriftsartikel (refereegranskat)abstract
- In this study we derive analytically the equilibrium melting probabilities for basepairs of a DNA molecule with a defect site. We assume that the defect is characterized by a change in the Watson-Crick basepair energy of the defect basepair, and in the associated two stacking energies for the defect, as compared to the remaining parts of the DNA. The defect site could, for instance, occur due to DNA basepair mismatching, cross-linking, or by the chemical modifications when attaching fluorescent labels, such as fluorescent-quencher pairs, to DNA. Our exact solution of the Poland-Scheraga model for DNA melting provides the probability that the labeled basepair, and its neighbors, are open at different temperatures. Our work is of direct importance, for instance, for studies where fluorophore-quencher pairs are used for studying single basepair fluctuations of designed DNA molecules.
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| 15. |
- Goyal, Gaurav, 1983, et al.
(författare)
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A simple cut and stretch assay to detect antimicrobial resistance genes on bacterial plasmids by single-molecule fluorescence microscopy
- 2022
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Antimicrobial resistance (AMR) is a fast-growing threat to global health. The genes conferring AMR to bacteria are often located on plasmids, circular extrachromosomal DNA molecules that can be transferred between bacterial strains and species. Therefore, effective methods to characterize bacterial plasmids and detect the presence of resistance genes can assist in managing AMR, for example, during outbreaks in hospitals. However, existing methods for plasmid analysis either provide limited information or are expensive and challenging to implement in low-resource settings. Herein, we present a simple assay based on CRISPR/Cas9 excision and DNA combing to detect antimicrobial resistance genes on bacterial plasmids. Cas9 recognizes the gene of interest and makes a double-stranded DNA cut, causing the circular plasmid to linearize. The change in plasmid configuration from circular to linear, and hence the presence of the AMR gene, is detected by stretching the plasmids on a glass surface and visualizing by fluorescence microscopy. This single-molecule imaging based assay is inexpensive, fast, and in addition to detecting the presence of AMR genes, it provides detailed information on the number and size of plasmids in the sample. We demonstrate the detection of several beta-lactamase-encoding genes on plasmids isolated from clinical samples. Furthermore, we demonstrate that the assay can be performed using standard microbiology and clinical laboratory equipment, making it suitable for low-resource settings.
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| 16. |
- Goyal, Gaurav, 1983, et al.
(författare)
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CRISPR/CAS9 BASED DNA-COMBING ASSAY FOR DETECTING ANTIMICROBIAL RESISTANCE GENES ON PLASMIDS
- 2021
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Ingår i: MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. ; , s. 801-802
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Konferensbidrag (refereegranskat)abstract
- We present a method based on CRISPR/Cas9 excision and DNA combing to detect anti-microbial resistance (AMR) genes on bacterial plasmids. The assay is inexpensive, simple, fast, and also provides information on the number and size of plasmids in a sample. We demonstrate detection of the gene encoding for the New Delhi metallobeta-lactamase 1 (blaNDM-1) enzyme, known to make bacteria resistant to a broad range of beta-lactam antibiotics.
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| 17. |
- Grunwald, Assaf, et al.
(författare)
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Bacteriophage strain typing by rapid single molecule analysis.
- 2015
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 43:18, s. 117-117
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Tidskriftsartikel (refereegranskat)abstract
- Rapid characterization of unknown biological samples is under the focus of many current studies. Here we report a method for screening of biological samples by optical mapping of their DNA. We use a novel, one-step chemo-enzymatic reaction to covalently bind fluorophores to DNA at the four-base recognition sites of a DNA methyltransferase. Due to the diffraction limit of light, the dense distribution of labels results in a continuous fluorescent signal along the DNA. The amplitude modulations (AM) of the fluorescence intensity along the stretched DNA molecules exhibit a unique molecular fingerprint that can be used for identification. We show that this labelling scheme is highly informative, allowing accurate genotyping. We demonstrate the method by labelling the genomes of λ and T7 bacteriophages, resulting in a consistent, unique AM profile for each genome. These profiles are also successfully used for identification of the phages from a background phage library. Our method may provide a facile route for screening and typing of various organisms and has potential applications in metagenomics studies of various ecosystems.
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| 18. |
- 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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| 19. |
- Jeffet, Jonathan, et al.
(författare)
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Super-Resolution Genome Mapping in Silicon Nanochannels
- 2016
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Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-0851 .- 1936-086X. ; 10:11, s. 9823-9830
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Tidskriftsartikel (refereegranskat)abstract
- Optical genome mapping in nanochannels is a powerful genetic analysis method, complementary to deoxyribonucleic acid (DNA) sequencing. The method is based on detecting a pattern of fluorescent labels attached along individual DNA molecules. When such molecules are extended in nanochannels, the labels create a fluorescent genetic barcode that is used for mapping the DNA molecule to its genomic locus and identifying large-scale variation from the genome reference. Mapping resolution is currently limited by two main factors: the optical diffraction limit and the thermal fluctuations of DNA molecules suspended in the nanochannels. Here, we utilize single-molecule tracking and super-resolution localization in order to improve the mapping accuracy and resolving power of this genome mapping technique and achieve a 15-fold increase in resolving power compared to currently practiced methods. We took advantage of a naturally occurring genetic repeat array and labeled each repeat with custom-designed Trolox conjugated fluorophores for enhanced photostability. This model system allowed us to acquire extremely long image sequences of the equally spaced fluorescent markers along DNA molecules, enabling detailed characterization of nanoconfined DNA dynamics and quantitative comparison to the Odijk theory for confined polymer chains. We present a simple method to overcome the thermal fluctuations in the nanochannels and exploit single-step photobleaching to resolve subdiffraction spaced fluorescent markers along fluctuating DNA molecules with ∼100 bp resolution. In addition, we show how time-averaging over just ∼50 frames of 40 ms enhances mapping accuracy, improves mapping P-value scores by 3 orders of magnitude compared to nonaveraged alignment, and provides a significant advantage for analyzing structural variations between DNA molecules with similar sequence composition.
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| 20. |
- Johnning, Anna, 1985, et al.
(författare)
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The resistomes of six carbapenem-resistant pathogens - a critical genotype-phenotype analysis
- 2018
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Ingår i: Microbial Genomics. - : Microbiology Society. - 2057-5858. ; 4:11
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Tidskriftsartikel (refereegranskat)abstract
- Carbapenem resistance is a rapidly growing threat to our ability to treat refractory bacterial infections. To understand how carbapenem resistance is mobilized and spread between pathogens, it is important to study the genetic context of the underlying resistance mechanisms. In this study, the resistomes of six clinical carbapenem-resistant isolates of five different species - Acinetobacter baumannii, Escherichia colt, two Klebsiella pneumoniae, Proteus mirabilis and Pseudomonas aeruginosa - were characterized using whole genome sequencing. All Enterobacteriaceae isolates and the A. baumannii isolate had acquired a large number of antimicrobial resistance genes (7-18 different genes per isolate), including the following encoding carbapenemases: bla(KPC-2), bla(OXA-48), bla(OXA-72), bla(NDM-1), bla(NDm-7) and bla(VIM-1). In addition, a novel version of bla(SHv) was discovered. Four new resistance plasmids were identified and their fully assembled sequences were verified using optical DNA mapping. Most of the resistance genes were colocalized on these and other plasmids, suggesting a risk for coselection. In contrast, five out of six carbapenemase genes were present on plasmids with no or few other resistance genes. The expected level of resistance - based on acquired resistance determinants - was concordant with measured levels in most cases. There were, however, several important discrepancies for four of the six isolates concerning multiple classes of antibiotics. In conclusion, our results further elucidate the diversity of carbapenemases, their mechanisms of horizontal transfer and possible patterns of co-selection. The study also emphasizes the difficulty of using whole genome sequencing for antimicrobial susceptibility testing of pathogens with complex genotypes.
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| 21. |
- Krog, Jens, et al.
(författare)
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Photophysical image analysis : Unsupervised probabilistic thresholding for images from electron-multiplying charge-coupled devices
- 2024
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Ingår i: PLoS ONE. - 1932-6203. ; 19:4, s. 0300122-0300122
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Tidskriftsartikel (refereegranskat)abstract
- We introduce the concept photophysical image analysis (PIA) and an associated pipeline for unsupervised probabilistic image thresholding for images recorded by electron-multiplying charge-coupled device (EMCCD) cameras. We base our approach on a closed-form analytic expression for the characteristic function (Fourier-transform of the probability mass function) for the image counts recorded in an EMCCD camera, which takes into account both stochasticity in the arrival of photons at the imaging camera and subsequent noise induced by the detection system of the camera. The only assumption in our method is that the background photon arrival to the imaging system is described by a stationary Poisson process (we make no assumption about the photon statistics for the signal). We estimate the background photon statistics parameter, λbg, from an image which contains both background and signal pixels by use of a novel truncated fit procedure with an automatically determined image count threshold. Prior to this, the camera noise model parameters are estimated using a calibration step. Utilizing the estimates for the camera parameters and λbg, we then introduce a probabilistic thresholding method, where, for the first time, the fraction of misclassified pixels can be determined a priori for a general image in an unsupervised way. We use synthetic images to validate our a priori estimates and to benchmark against the Otsu method, which is a popular unsupervised non-probabilistic image thresholding method (no a priori estimates for the error rates are provided). For completeness, we lastly present a simple heuristic general-purpose segmentation method based on the thresholding results, which we apply to segmentation of synthetic images and experimental images of fluorescent beads and lung cell nuclei. Our publicly available software opens up for fully automated, unsupervised, probabilistic photophysical image analysis.
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| 22. |
- Krog, Jens, et al.
(författare)
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Stochastic unfolding of nanoconfined DNA: Experiments, model and Bayesian analysis
- 2018
-
Ingår i: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 149:21
-
Tidskriftsartikel (refereegranskat)abstract
- Nanochannels provide a means for detailed experiments on the effect of confinement on biomacro-molecules, such as DNA. Here we introduce a model for the complete unfolding of DNA from the circular to linear configuration. Two main ingredients are the entropic unfolding force and the friction coefficient for the unfolding process, and we describe the associated dynamics by a non-linear Langevin equation. By analyzing experimental data where DNA molecules are photo-cut and unfolded inside a nanochannel, our model allows us to extract values for the unfolding force as well as the friction coefficient for the first time. In order to extract numerical values for these physical quantities, we employ a recently introduced Bayesian inference framework. We find that the determined unfolding force is in agreement with estimates from a simple Flory-type argument. The estimated friction coefficient is in agreement with theoretical estimates for motion of a cylinder in a channel. We further validate the estimated friction constant by extracting this parameter from DNA's center-of -mass motion before and after unfolding, yielding decent agreement. We provide publically available software for performing the required image and Bayesian analysis. Published by AIP Publishing.
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| 23. |
- Kumar Bikarolla, Santosh, 1985, et al.
(författare)
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Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak
- 2019
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Ingår i: mBio. - : AMER SOC MICROBIOLOGY. - 2161-2129 .- 2150-7511. ; 10:4
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Tidskriftsartikel (refereegranskat)abstract
- The global spread of antibiotic resistance among Enterobacteriaceae is largely due to multidrug resistance plasmids that can transfer between different bacterial strains and species. Horizontal gene transfer of resistance plasmids can complicate hospital outbreaks and cause problems in epidemiological tracing, since tracing is usually based on bacterial clonality. We have developed a method, based on optical DNA mapping combined with Cas9-assisted identification of resistance genes, which is used here to characterize plasmids during an extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae outbreak at a Swedish neonatal intensive care unit. The outbreak included 17 neonates initially colonized with ESBL-producing Klebsiella pneumoniae (ESBL-KP), some of which were found to carry additional ESBL-producing Escherichia coli (ESBL-EC) in follow-up samples. We demonstrate that all ESBL-KP isolates contained two plasmids with the blaCTX-M-15 gene located on the smaller one (~80 kbp). The same ESBL-KP clone was present in follow-up samples for up to 2 years in some patients, and the plasmid carrying the blaCTX-M-15 gene was stable throughout this time period. However, extensive genetic rearrangements within the second plasmid were observed in the optical DNA maps for several of the ESBL-KP isolates. Optical mapping also demonstrated that even though other bacterial clones and species carrying blaCTX-M group 1 genes were found in some neonates, no transfer of resistance plasmids had occurred. The data instead pointed toward unrelated acquisition of ESBL-producing Enterobacteriaceae (EPE). In addition to revealing important information about the specific outbreak, the method presented is a promising tool for surveillance and infection control in clinical settings.IMPORTANCE This study presents how a novel method, based on visualizing single plasmids using sequence-specific fluorescent labeling, could be used to analyze the genetic dynamics of an outbreak of resistant bacteria in a neonatal intensive care unit at a Swedish hospital. Plasmids are a central reason for the rapid global spread of bacterial resistance to antibiotics. In a single experimental procedure, this method replaces many traditional plasmid analysis techniques that together provide limited details and are slow to perform. The method is much faster than long-read whole-genome sequencing and offers direct genetic comparison of patient samples. We could conclude that no transfer of resistance plasmids had occurred between different bacteria during the outbreak and that secondary cases of ESBL-producing Enterobacteriaceae carriage were instead likely due to influx of new strains. We believe that the method offers potential in improving surveillance and infection control of resistant bacteria in hospitals.
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| 24. |
- Kumra Ahnlide, Vibha, et al.
(författare)
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A Predictive Model of Antibody Binding in the Presence of IgG-Interacting Bacterial Surface Proteins
- 2021
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 12
-
Tidskriftsartikel (refereegranskat)abstract
- Many bacteria can interfere with how antibodies bind to their surfaces. This bacterial antibody targeting makes it challenging to predict the immunological function of bacteria-associated antibodies. The M and M-like proteins of group A streptococci (GAS) exhibit IgGFc-binding regions, which they use to reverse IgG binding orientation depending on the host environment. Unraveling the mechanism behind these binding characteristics may identify conditions under which bound IgG can drive an efficient immune response. Here, we have developed a biophysical model for describing these complex protein-antibody interactions. We show how the model can be used as a tool for studying the binding behavior of various IgG samples to M protein by performing in silico simulations and correlating this data with experimental measurements. Besides its use for mechanistic understanding, this model could potentially be used as a tool to aid in the development of antibody treatments. We illustrate this by simulating how IgG binding to GAS in serum is altered as specified amounts of monoclonal or pooled IgG is added. Phagocytosis experiments link this altered antibody binding to a physiological function and demonstrate that it is possible to predict the effect of an IgG treatment with our model. Our study gives a mechanistic understanding of bacterial antibody targeting and provides a tool for predicting the effect of antibody treatments in the presence of bacteria with IgG-modulating surface proteins.
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| 25. |
- Lizana, L., et al.
(författare)
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Diffusion of finite-sized hard-core interacting particles in a one-dimensional box: Tagged particle dynamics
- 2009
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Ingår i: Physical Review E (Statistical, Nonlinear, and Soft Matter Physics). - 1539-3755. ; 80:5
-
Tidskriftsartikel (refereegranskat)abstract
- We solve a nonequilibrium statistical-mechanics problem exactly, namely, the single-file dynamics of N hard-core interacting particles (the particles cannot pass each other) of size Delta diffusing in a one-dimensional system of finite length L with reflecting boundaries at the ends. We obtain an exact expression for the conditional probability density function rho T(yT,t vertical bar yT,0) that a tagged particle T (T=1,...,N) is at position yT at time t given that it at time t=0 was at position yT,0. Using a Bethe ansatz we obtain the N-particle probability density function and, by integrating out the coordinates (and averaging over initial positions) of all particles but particle T, we arrive at an exact expression for rho T(yT,t vertical bar yT,0) in terms of Jacobi polynomials or hypergeometric functions. Going beyond previous studies, we consider the asymptotic limit of large N, maintaining L finite, using a nonstandard asymptotic technique. We derive an exact expression for rho T(yT,t vertical bar yT,0) for a tagged particle located roughly in the middle of the system, from which we find that there are three time regimes of interest for finite-sized systems: (A) for times much smaller than the collision time t
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| 26. |
- Lizana, Ludvig, et al.
(författare)
-
Foundation of fractional Langevin equation: Harmonization of a many-body problem
- 2010
-
Ingår i: Physical Review E (Statistical, Nonlinear, and Soft Matter Physics). - 1539-3755. ; 81:5
-
Tidskriftsartikel (refereegranskat)abstract
- In this study we derive a single-particle equation of motion, from first principles, starting out with a microscopic description of a tracer particle in a one-dimensional many-particle system with a general two-body interaction potential. Using a harmonization technique, we show that the resulting dynamical equation belongs to the class of fractional Langevin equations, a stochastic framework which has been proposed in a large body of works as a means of describing anomalous dynamics. Our work sheds light on the fundamental assumptions of these phenomenological models and a relation derived by Kollmann.
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| 27. |
- Lizana, Ludvig, 1977-, et al.
(författare)
-
Single-file diffusion with non-thermal initial conditions
- 2014
-
Ingår i: Physica A: Statistical Mechanics and its Applications. - : Elsevier BV. - 0378-4371 .- 1873-2119. ; 395, s. 148-153
-
Tidskriftsartikel (refereegranskat)abstract
- Single-file diffusion is a theoretically challenging many-body problem where the calculation of even the simplest observables, e.g. mean square displacement, for a tracer particle requires an elaborate mathematical machinery. There is therefore a need for simple approaches which provide intuitive understandings and predict qualitatively correct behaviours. Here we put forward a scaling-type method which we use to investigate the influence of non-thermal initial conditions on the dynamics of a tracer particle. With our new approach we reproduce, up to scaling, several known long and short time asymptotic results for the tracer particle mean square displacement. (C) 2013 Elsevier B.V. All rights reserved.
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| 28. |
- Lizana, Ludvig, et al.
(författare)
-
Tracer particles in two-dimensional elastic networks diffuse logarithmically slow
- 2017
-
Ingår i: Journal of Physics A: Mathematical and Theoretical. - : IOP Publishing. - 1751-8113 .- 1751-8121. ; 50:3
-
Tidskriftsartikel (refereegranskat)abstract
- Several experiments on tagged molecules or particles in living systems suggest that they move anomalously slow - their mean squared displacement (MSD) increase slower than linearly with time. Leading models aimed at understanding these experiments predict that the MSD grows as a power law with a growth exponent that is smaller than unity. However, in some experiments the growth is so slow (fitted exponent ∼0.1-0.2) that they hint towards other mechanisms at play. In this paper, we theoretically demonstrate how in-plane collective modes excited by thermal fluctuations in a two dimensional membrane lead to logarithmic time dependence for the the tracer particle's MSD.
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| 29. |
- Lomholt, Michael A, et al.
(författare)
-
Dissimilar bouncy walkers
- 2011
-
Ingår i: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 134:4
-
Tidskriftsartikel (refereegranskat)abstract
- We consider the dynamics of a one-dimensional system consisting of dissimilar hardcore interacting (bouncy) random walkers. The walkers' (diffusing particles') friction constants ξ(n), where n labels different bouncy walkers, are drawn from a distribution ϱ(ξ(n)). We provide an approximate analytic solution to this recent single-file problem by combining harmonization and effective medium techniques. Two classes of systems are identified: when ϱ(ξ(n)) is heavy-tailed, ϱ(ξ(n))≃ξ(n) (-1-α) (0<α<1) for large ξ(n), we identify a new universality class in which density relaxations, characterized by the dynamic structure factor S(Q, t), follows a Mittag-Leffler relaxation, and the mean square displacement (MSD) of a tracer particle grows as t(δ) with time t, where δ = α∕(1 + α). If instead ϱ is light-tailed such that the mean friction constant exist, S(Q, t) decays exponentially and the MSD scales as t(1/2). We also derive tracer particle force response relations. All results are corroborated by simulations and explained in a simplified model.
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| 30. |
- Lomholt, Michael A., et al.
(författare)
-
Microscopic Origin of the Logarithmic Time Evolution of Aging Processes in Complex Systems
- 2013
-
Ingår i: Physical Review Letters. - : American Physical Society. - 1079-7114 .- 0031-9007. ; 110:20
-
Tidskriftsartikel (refereegranskat)abstract
- There exists compelling experimental evidence in numerous systems for logarithmically slow time evolution, yet its full theoretical understanding remains elusive. We here introduce and study a generic transition process in complex systems, based on nonrenewal, aging waiting times. Each state n of the system follows a local clock initiated at t = 0. The random time tau between clock ticks follows the waiting time density psi (tau). Transitions between states occur only at local clock ticks and are hence triggered by the local forward waiting time, rather than by psi (tau). For power-law forms psi (tau) similar or equal to tau(-1-alpha) (0 < alpha < 1) we obtain a logarithmic time evolution of the state number < n(t)> similar or equal to log(t/t(0)), while for alpha > 2 the process becomes normal in the sense that < n(t)> similar or equal to t. In the intermediate range 1 < alpha < 2 we find the power-law growth < n(t)> similar or equal to t(alpha-1). Our model provides a universal description for transition dynamics between aging and nonaging states.
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| 31. |
- Lomholt, Michael A., et al.
(författare)
-
Universality and nonuniversality of mobility in heterogeneous single-file systems and Rouse chains
- 2014
-
Ingår i: Physical Review E (Statistical, Nonlinear, and Soft Matter Physics). - 1539-3755. ; 89:3
-
Tidskriftsartikel (refereegranskat)abstract
- We study analytically the tracer particle mobility in single-file systems with distributed friction constants. Our system serves as a prototype for nonequilibrium, heterogeneous, strongly interacting Brownian systems. The long time dynamics for such a single-file setup belongs to the same universality class as the Rouse model with dissimilar beads. The friction constants are drawn from a density rho(xi), and we derive an asymptotically exact solution for the mobility distribution P[mu(0)(s)], where mu(0)(s) is the Laplace-space mobility. If rho is light tailed (first moment exists), we find a self-averaging behavior: P[mu(0)(s)] = delta[mu(0)(s) - mu(s)], with mu(s) alpha s(1/2). When rho(xi) is heavy tailed, rho(xi) similar or equal to xi(-1-alpha) (0 < alpha < 1) for large xi, we obtain moments <[mu(s)(0)(n)]> alpha s(beta n), where beta = 1/(1 + alpha) and there is no self-averaging. The results are corroborated by simulations.
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| 32. |
- Metzler, R., et al.
(författare)
-
Ageing single file motion
- 2014
-
Ingår i: The European Physical Journal. Special Topics. - : Springer Science and Business Media LLC. - 1951-6355 .- 1951-6401. ; 223:14, s. 3287-3293
-
Tidskriftsartikel (refereegranskat)abstract
- The mean squared displacement of a tracer particle in a single file of identical particles with excluded volume interactions shows the famed Harris scaling aEurox (2)(t)aEuro parts per thousand a parts per thousand integral K (1/2) t (1/2) as function of time. Here we study what happens to this law when each particle of the single file interacts with the environment such that it is transiently immobilised for times tau with a power-law distribution psi(tau) a parts per thousand integral (tau(a similar to...))(alpha), and different ranges of the exponent alpha are considered. We find a dramatic slow-down of the motion of a tracer particle from Harris' law to an ultraslow, logarithmic time evolution aEurox (2)(t)aEuro parts per thousand a parts per thousand integral K (0) log (1/2)(t) when 0 < alpha < 1. In the intermediate case 1 < alpha < 2, we observe a power-law form for the mean squared displacement, with a modified scaling exponent as compared to Harris' law. Once alpha is larger than two, the Brownian single file behaviour and thus Harris' law are restored. We also point out that this process is weakly non-ergodic in the sense that the time and ensemble averaged mean squared displacements are disparate.
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| 33. |
- Müller, Vilhelm, 1990, et al.
(författare)
-
Cultivation-Free Typing of Bacteria Using Optical DNA Mapping
- 2020
-
Ingår i: Acs Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 6:5, s. 1076-1084
-
Tidskriftsartikel (refereegranskat)abstract
- A variety of pathogenic bacteria can infect humans, and rapid species identification is crucial for the correct treatment. However, the identification process can often be time-consuming and depend on the cultivation of the bacterial pathogen(s). Here, we present a stand-alone, enzyme-free, optical DNA mapping assay capable of species identification by matching the intensity profiles of large DNA molecules to a database of fully assembled bacterial genomes (>10 000). The assay includes a new data analysis strategy as well as a general DNA extraction protocol for both Gram-negative and Gram-positive bacteria. We demonstrate that the assay is capable of identifying bacteria directly from uncultured clinical urine samples, as well as in mixtures, with the potential to be discriminative even at the subspecies level. We foresee that the assay has applications both within research laboratories and in clinical settings, where the time-consuming step of cultivation can be minimized or even completely avoided.
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| 34. |
- Müller, Vilhelm, 1990, et al.
(författare)
-
Detailed characterization of plasmids carrying resistance genes using optical DNA mapping
- 2016
-
Ingår i: 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016. - 9780979806490 ; , s. 1561-1562
-
Konferensbidrag (refereegranskat)abstract
- We present an assay, based on optical DNA mapping in nanochannels that is capable of characterizing the plasmid content of bacterial isolates resistant to antibiotics in a fast an detailed way. In a single experiment we determine the number of different plasmids in each sample, their size, as well as a barcode that can be used for plasmid identification and tracing. In addition we demonstrate that we can identify resistance genes on individual plasmids using CRISPR/Cas9. We foresee that the assay can be a useful tool all the way from fundamental plasmid biology to diagnostics and surveillance of resistant infections.
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| 35. |
- Müller, Vilhelm, 1990, et al.
(författare)
-
Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping.
- 2016
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 6, s. 37938-
-
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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| 36. |
- Müller, Vilhelm, 1990, et al.
(författare)
-
Enzyme-free optical DNA mapping of the human genome using competitive binding
- 2019
-
Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 47:15
-
Tidskriftsartikel (refereegranskat)abstract
- Optical DNA mapping (ODM) allows visualization of long-range sequence information along single DNA molecules. The data can for example be used for detecting long range structural variations, for aiding DNA sequence assembly of complex genomes and for mapping epigenetic marks and DNA damage across the genome. ODM traditionally utilizes sequence specific marks based on nicking enzymes, combined with a DNA stain, YOYO-1, for detection of the DNA contour. Here we use a competitive binding approach, based on YOYO-1 and netropsin, which highlights the contour of the DNA molecules, while simultaneously creating a continuous sequence specific pattern, based on the AT/GC variation along the detected molecule. We demonstrate and validate competitive-binding-based ODM using bacterial artificial chromosomes (BACs) derived from the human genome and then turn to DNA extracted from white blood cells. We generalize our findings with in-silico simulations that show that we can map a vast majority of the human genome. Finally, we demonstrate the possibility of combining competitive binding with enzymatic labeling by mapping DNA damage sites induced by the cytotoxic drug etoposide to the human genome. Overall, we demonstrate that competitive-binding-based ODM has the potential to be used both as a standalone assay for studies of the human genome, as well as in combination with enzymatic approaches, some of which are already commercialized.
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| 37. |
- Müller, Vilhelm, 1990, et al.
(författare)
-
Rapid Tracing of Resistance Plasmids in a Nosocomial Outbreak Using Optical DNA Mapping
- 2016
-
Ingår i: Acs Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 2:5, s. 322-328
-
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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| 38. |
- Müller, Vilhelm, 1990, et al.
(författare)
-
Rapid Tracing of Resistance Plasmids in a Nosocomial Outbreak Using Optical DNA Mapping
- 2016
-
Ingår i: ACS Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 2:5, s. 322-328
-
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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| 39. |
- Ni, Weihai, et al.
(författare)
-
Observing Plasmonic-Molecular Resonance Coupling on Single Gold Nanorods
- 2010
-
Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6992 .- 1530-6984. ; 10:1, s. 77-84
-
Tidskriftsartikel (refereegranskat)abstract
- Strong plasmonic-molecular resonance coupling occurs between noble metal nanocrystals and organic adsorbates when the plasmonic resonance is degenerate with the molecular one. This interaction forms the basis for many fundamental studies and practical applications. We describe here the first direct measurement of the resonance coupling on single gold nanorods. The dark-field scattering technique is employed. The nanorods are embedded in hydrogel to facilitate uniform dye adsorption. The adsorbed dye molecules exhibit both monomer and H-aggregate absorption bands. The same gold nanorods are measured before and after the dye adsorption. Both strong and weak Coupling are investigated by selecting nanorods with different longitudinal plasmon bands. Excellent agreement between the experiments and an analytic theory is obtained. The resonance coupling reveals a unique three-band structure, The tunability of the coupling on individual nanorods is further demonstrated by photodecomposing the adsorbed dye molecules.
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| 40. |
- Nilsson, Adam, et al.
(författare)
-
Competitive binding-based optical DNA mapping for fast identification of bacteria - multi-ligand transfer matrix theory and experimental applications on Escherichia coli.
- 2014
-
Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 42:15, s. 118-118
-
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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| 41. |
- Noble, Charleston, et al.
(författare)
-
A fast and scalable kymograph alignment algorithm for nanochannel-based optical DNA mappings.
- 2015
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:4
-
Tidskriftsartikel (refereegranskat)abstract
- Optical mapping by direct visualization of individual DNA molecules, stretched in nanochannels with sequence-specific fluorescent labeling, represents a promising tool for disease diagnostics and genomics. An important challenge for this technique is thermal motion of the DNA as it undergoes imaging; this blurs fluorescent patterns along the DNA and results in information loss. Correcting for this effect (a process referred to as kymograph alignment) is a common preprocessing step in nanochannel-based optical mapping workflows, and we present here a highly efficient algorithm to accomplish this via pattern recognition. We compare our method with the one previous approach, and we find that our method is orders of magnitude faster while producing data of similar quality. We demonstrate proof of principle of our approach on experimental data consisting of melt mapped bacteriophage DNA.
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| 42. |
- Nyberg, Lena, 1979, et al.
(författare)
-
Rapid identification of intact bacterial resistance plasmids via optical mapping of single DNA molecules
- 2016
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 6
-
Tidskriftsartikel (refereegranskat)abstract
- The rapid spread of antibiotic resistance - currently one of the greatest threats to human health according to WHO - is to a large extent enabled by plasmid-mediated horizontal transfer of resistance genes. Rapid identification and characterization of plasmids is thus important both for individual clinical outcomes and for epidemiological monitoring of antibiotic resistance. Toward this aim, we have developed an optical DNA mapping procedure where individual intact plasmids are elongated within nanofluidic channels and visualized through fluorescence microscopy, yielding barcodes that reflect the underlying sequence. The assay rapidly identifies plasmids through statistical comparisons with barcodes based on publicly available sequence repositories and also enables detection of structural variations. Since the assay yields holistic sequence information for individual intact plasmids, it is an ideal complement to next generation sequencing efforts which involve reassembly of sequence reads from fragmented DNA molecules. The assay should be applicable in microbiology labs around the world in applications ranging from fundamental plasmid biology to clinical epidemiology and diagnostics.
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| 43. |
- Nyberg, Markus, et al.
(författare)
-
A simple method to calculate first-passage time densities with arbitrary initial conditions
- 2016
-
Ingår i: New Journal of Physics. - : IOP Publishing. - 1367-2630. ; 18:6
-
Tidskriftsartikel (refereegranskat)abstract
- Numerous applications all the way from biology and physics to economics depend on the density of first crossings over a boundary. Motivated by the lack of general purpose analytical tools for computing first-passage time densities (FPTDs) for complex problems, we propose a new simple method based on the independent interval approximation (IIA). We generalise previous formulations of the IIA to include arbitrary initial conditions as well as to deal with discrete time and non-smooth continuous time processes. We derive a closed form expression for the FPTD in z and Laplace-transform space to a boundary in one dimension. Two classes of problems are analysed in detail: discrete time symmetric random walks (Markovian) and continuous time Gaussian stationary processes (Markovian and non-Markovian). Our results are in good agreement with Langevin dynamics simulations.
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| 44. |
- Nyberg, Markus, et al.
(författare)
-
Modeling protein target search in human chromosomes
- 2021
-
Ingår i: Physical Review Research. - : American Physical Society. - 2643-1564. ; 3:1
-
Tidskriftsartikel (refereegranskat)abstract
- Several processes in the cell, such as gene regulation, start when key proteins recognize and bind to short DNA sequences. However, as these sequences can be hundreds of million times shorter than the genome, they are hard to find by simple diffusion: diffusion-limited association rates may underestimate in vitro measurements up to several orders of magnitude. Moreover, the rates increase if the DNA is coiled rather than straight. Here we model how this works in vivo in mammalian cells. We use chromatin-chromatin contact data from Hi-C experiments to map the protein target-search onto a network problem. The nodes represent DNA segments and the weight of the links are proportional to measured contact probabilities. We then put forward a diffusion-reaction equation for the density of searching protein that allows us to calculate the association rates across the genome analytically. For segments where the rates are high, we find that they are enriched with active gene starts and have high RNA expression levels. This paper suggests that the DNA's 3D conformation is important for protein search times in vivo and offers a method to interpret protein-binding profiles in eukaryotes that cannot be explained by the DNA sequence itself.
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| 45. |
- Nyberg, Markus, et al.
(författare)
-
Zero-crossing statistics for non-Markovian time series
- 2018
-
Ingår i: Physical Review E. - : American Physical Society. - 2470-0045 .- 2470-0053. ; 97:3
-
Tidskriftsartikel (refereegranskat)abstract
- In applications spanning from image analysis and speech recognition to energy dissipation in turbulence and time-to failure of fatigued materials, researchers and engineers want to calculate how often a stochastic observable crosses a specific level, such as zero. At first glance this problem looks simple, but it is in fact theoretically very challenging, and therefore few exact results exist. One exception is the celebrated Rice formula that gives the mean number of zero crossings in a fixed time interval of a zero-mean Gaussian stationary process. In this study we use the so-called independent interval approximation to go beyond Rice's result and derive analytic expressions for all higher-order zero-crossing cumulants and moments. Our results agree well with simulations for the non-Markovian autoregressive model.
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| 46. |
- Nyblom, My, 1995, et al.
(författare)
-
Bacterial identification by optical mapping of genomic DNA in nanofluidic channels
- 2019
-
Ingår i: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. - 9781733419000 ; , s. 821-822
-
Konferensbidrag (refereegranskat)abstract
- A variety of pathogenic bacteria can infect humans and the increase in bacteria resistant to common antibiotics is a large threat to human health worldwide. This work presents a method, based on optical DNA mapping (ODM) in nanofluidic channels, that can detect the type of bacterial present in a sample by matching the obtained maps of large DNA molecules to a database of fully assembled bacterial genomes. The extraction and labelling protocol has been designed to work for both Gram-positive and Gram-negative bacteria, not requiring any prior knowledge about the sample content.
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| 47. |
- Pedersen, Jonas, et al.
(författare)
-
Bubble merging in breathing DNA as a vicious walker problem in opposite potentials.
- 2009
-
Ingår i: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 130:16
-
Tidskriftsartikel (refereegranskat)abstract
- We investigate the coalescence of two DNA bubbles initially located at weak domains and separated by a more stable barrier region in a designed construct of double-stranded DNA. In a continuum Fokker-Planck approach, the characteristic time for bubble coalescence and the corresponding distribution are derived, as well as the distribution of coalescence positions along the barrier. Below the melting temperature, we find a Kramers-type barrier crossing behavior, while at high temperatures, the bubble corners perform drift diffusion toward coalescence. In the calculations, we map the bubble dynamics on the problem of two vicious walkers in opposite potentials. We also present a discrete master equation approach to the bubble coalescence problem. Numerical evaluation and stochastic simulation of the master equation show excellent agreement with the results from the continuum approach. Given that the coalesced state is thermodynamically stabilized against a state where only one or a few of the base pairs of the barrier region are re-established, it appears likely that this type of setup could be useful for the quantitative investigation of thermodynamic DNA stability data as well as the rate constants involved in the unzipping and zipping dynamics of DNA in single molecule fluorescence experiments.
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| 48. |
- Pigeon, Simon, et al.
(författare)
-
Tracer particle diffusion in a system with hardcore interacting particles
- 2017
-
Ingår i: Journal of Statistical Mechanics: Theory and Experiment. - : IOP Publishing. - 1742-5468. ; 2017:12
-
Tidskriftsartikel (refereegranskat)abstract
- In this study, inspired by the work of Nakazato and Kitahara (1980 Prog. Theor. Phys. 64 2261), we consider the theoretical problem of tracer particle diffusion in an environment of diffusing hardcore interacting crowder particles. The tracer particle has a different diffusion constant from the crowder particles. Based on a transformation of the generating function, we provide an exact formal expansion for the tracer particle probability density, valid for any lattice in the thermodynamic limit. By applying this formal solution to dynamics on a regular Bravais lattice we provide a closed form approximation for the tracer particle diffusion constant which extends the Nakazato and Kitahara results to include also b.c.c. and f.c.c. lattices. Finally, we compare our analytical results to simulations in two and three dimensions.
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| 49. |
- Reiter-Schad, Michaela, et al.
(författare)
-
How nanochannel confinement affects the DNA melting transition within the Poland-Scheraga model.
- 2015
-
Ingår i: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 143:11
-
Tidskriftsartikel (refereegranskat)abstract
- When double-stranded DNA molecules are heated, or exposed to denaturing agents, the two strands are separated. The statistical physics of this process has a long history and is commonly described in terms of the Poland-Scheraga (PS) model. Crucial to this model is the configurational entropy for a melted region (compared to the entropy of an intact region of the same size), quantified by the loop factor. In this study, we investigate how confinement affects the DNA melting transition, by using the loop factor for an ideal Gaussian chain. By subsequent numerical solutions of the PS model, we demonstrate that the melting temperature depends on the persistence lengths of single-stranded and double-stranded DNA. For realistic values of the persistence lengths, the melting temperature is predicted to decrease with decreasing channel diameter. We also demonstrate that confinement broadens the melting transition. These general findings hold for the three scenarios investigated: 1. homo-DNA, i.e., identical basepairs along the DNA molecule, 2. random sequence DNA, and 3. "real" DNA, here T4 phage DNA. We show that cases 2 and 3 in general give rise to broader transitions than case 1. Case 3 exhibits a similar phase transition as case 2 provided the random sequence DNA has the same ratio of AT to GC basepairs (A - adenine, T - thymine, G - guanine, C - cytosine). A simple analytical estimate for the shift in melting temperature is provided as a function of nanochannel diameter. For homo-DNA, we also present an analytical prediction of the melting probability as a function of temperature.
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| 50. |
- Sanders, Lloyd, et al.
(författare)
-
First passage times for a tracer particle in single file diffusion and fractional Brownian motion.
- 2012
-
Ingår i: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 136:17
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Tidskriftsartikel (refereegranskat)abstract
- We investigate the full functional form of the first passage time density (FPTD) of a tracer particle in a single-file diffusion (SFD) system whose population is: (i) homogeneous, i.e., all particles having the same diffusion constant and (ii) heterogeneous, with diffusion constants drawn from a heavy-tailed power-law distribution. In parallel, the full FPTD for fractional Brownian motion [fBm-defined by the Hurst parameter, H ∈ (0, 1)] is studied, of interest here as fBm and SFD systems belong to the same universality class. Extensive stochastic (non-Markovian) SFD and fBm simulations are performed and compared to two analytical Markovian techniques: the method of images approximation (MIA) and the Willemski-Fixman approximation (WFA). We find that the MIA cannot approximate well any temporal scale of the SFD FPTD. Our exact inversion of the Willemski-Fixman integral equation captures the long-time power-law exponent, when H ≥ 1∕3, as predicted by Molchan [Commun. Math. Phys. 205, 97 (1999)] for fBm. When H < 1∕3, which includes homogeneous SFD (H = 1∕4), and heterogeneous SFD (H < 1∕4), the WFA fails to agree with any temporal scale of the simulations and Molchan's long-time result. SFD systems are compared to their fBm counter parts; and in the homogeneous system both scaled FPTDs agree on all temporal scales including also, the result by Molchan, thus affirming that SFD and fBm dynamics belong to the same universality class. In the heterogeneous case SFD and fBm results for heterogeneity-averaged FPTDs agree in the asymptotic time limit. The non-averaged heterogeneous SFD systems display a lack of self-averaging. An exponential with a power-law argument, multiplied by a power-law pre-factor is shown to describe well the FPTD for all times for homogeneous SFD and sub-diffusive fBm systems.
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