| 1. |
- Nyberg, Lena, 1979, et al.
(författare)
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A single-step competitive binding assay for mapping of single DNA molecules
- 2012
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 417:1, s. 404-408
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Tidskriftsartikel (refereegranskat)abstract
- Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification cif pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.
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| 2. |
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| 3. |
- Nyberg, Lena, 1979, et al.
(författare)
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Heterogeneous staining: a tool for studies of how fluorescent dyes affect the physical properties of DNA
- 2013
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 41:19, s. articlenr e184-
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Tidskriftsartikel (refereegranskat)abstract
- The commonly used fluorescent dye YOYO-1 (YOYO) has, using bulk techniques, been demonstrated to stain DNA heterogeneously at substoichiometric concentrations. We here, using nanofluidic channels and fluorescence microscopy, investigate the heterogeneous staining on the single DNA molecule level and demonstrate that the dye distribution is continuous. The equilibration of YOYO on DNA is extremely slow but can be accelerated by increasing the ionic strength and/or the temperature. Furthermore, we demonstrate how to use the heterogeneous staining as a tool for detailed and time-efficient studies of how fluorescent dyes affect the physical properties of DNA. We show that the relative increase in extension of DNA with increasing amount of YOYO bound is higher at low ionic strengths and also extrapolate the extension of native DNA. Our study reveals important information on how YOYO affects the physical properties of DNA, but it also has broader applications. First, it reveals how cationic intercalators, such as potential DNA drugs, affect DNA under strong confinement. Second, the strategy of using heterogeneous staining is of general use for single molecule studies of DNA interacting with proteins or ligands.
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| 4. |
- Persson, Fredrik, 1979, et al.
(författare)
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Lipid-Based Passivation in Nanofluidics
- 2012
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Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6992 .- 1530-6984. ; 12:5, s. 2260-2265
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Tidskriftsartikel (refereegranskat)abstract
- Stretching DNA in nanochannels is a useful tool for direct, visual studies of genomic DNA at the single molecule level. To facilitate the study of the interaction of linear DNA with proteins in nanochannels, we have implemented a highly effective passivation scheme based on lipid bilayers. We demonstrate virtually complete long-term passivation of nanochannel surfaces to a range of relevant reagents, including streptavidin-coated quantum dots, RecA proteins, and RecA-DNA complexes. We show that the performance of the lipid bilayer is significantly better than that of standard bovine serum albumin-based passivation. Finally, we show how the passivated devices allow us to monitor single DNA cleavage events during enzymatic degradation by DNase I. We expect that our approach will open up for detailed, systematic studies of a wide range of protein-DNA interactions with high spatial and temporal resolution.
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| 5. |
- Persson, Fredrik, 1979, et al.
(författare)
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Local conformation of confined DNA studied using emission polarization anisotropy
- 2010
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Ingår i: Biophysical Society 54th Annual Meeting.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- When confined in nanochannels with dimensions smaller than the DNA radius of gyration, DNA will extend along the channel. We investigate long DNA confined in nanochannels, using fluorescence microscopy and intercalated dyes. Studies of the dynamics and statics of DNA in such nanoscale confinements as a function of e.g. degree of confinement and ionic strength have yielded new insights into the physical properties of DNA with relevance for applications in genomics as well as fundamental understanding of DNA packaging in vivo. Our work extends the field by not only studying the location of the emitting dyes along a confined DNA molecule but also monitoring the polarization of the emitted light. By measuring the emission polarized parallel and perpendicular to the extension axis of the stretched DNA, information on the local spatial distribution of the DNA backbone can be obtained. Comparing polarizations in two directions for DNA confined in channels of effective diameters of 85 nm and 170 nm reveals a striking difference. Whereas the DNA in the larger channels shows an isotropic polarization of the emitted light, the light is to a large extent polarized perpendicular to the elongation of the DNA in the smaller channels. We expect this technique to have a large impact on the studies of changes in DNA conformation induced by protein binding or during DNA compactation as well as in fundamental polymer physics studies of DNA in confined environments, for example in bacterial spores and viruses.
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| 6. |
- Persson, Fredrik, 1979, et al.
(författare)
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Local conformation of confined DNA studied using emission polarization anisotropy
- 2010
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Ingår i: NanoBioTech-Montreux 2009.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- In nanochannels with dimensions smaller than the DNA radius of gyration, DNA will extend along the channel. We investigate long DNA confined in nanochannels using fluorescence microscopy and intercalated dyes. Studies of the dynamics and statics of the DNA extension or position in such nanoscale confinements as a function of e.g. DNA contour length, degree and shape of confinement as well as ionic strength have yielded new insights in the physical properties of DNA with relevance for applications in genomics as well as fundamental understanding of DNA packaging in vivo. Our work extends the field by not only studying the location of the emitting dyes along a confined DNA molecule but also monitoring the polarization of the emitted light. We use intercalating dyes (YOYO-1) whose emission is polarized perpendicular to the DNA extension axis, and by measuring the emission polarized parallel and perpendicular to the extension axis of the stretched DNA, information on the local spatial distribution of the DNA backbone can be obtained. The results obtained are analogous to linear dichroism (LD) but on a single-molecule level, and obtained in a highly parallel fashion. We will discuss results in shallow (60 nm) and deep (180 nm) channels and describe an example of how the technique can be used to investigate non-uniform stretching of DNA on the single molecule level. Comparing polarizations in two directions for DNA confined in channels of effective diameters of 85 nm and 170 nm reveals a striking difference. Whereas the DNA in the larger channels shows an isotropic polarization of the emitted light, the light is to a large extent polarized perpendicular to the elongation of the DNA in the smaller channels. The ratio of the polarization parallel and perpendicular to the elongation direction, I|| / I⊥, is a measure of the relative local orientation of the DNA backbone. We believe that this technique will have a large impact on the studies of changes in DNA conformation induced by protein binding or during DNA compactation as well as in fundamental polymer physics studies of DNA in confined environments, for example in bacterial spores and viruses.
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| 7. |
- Persson, Marta, 1979, et al.
(författare)
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Clinically significant copy number alterations and complex rearrangements of MYB and NFIB in head and neck adenoid cystic carcinoma.
- 2012
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Ingår i: Genes, chromosomes & cancer. - : Wiley. - 1098-2264 .- 1045-2257. ; 51:8, s. 805-17
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Tidskriftsartikel (refereegranskat)abstract
- Adenoid cystic carcinoma (ACC) of the head and neck is a malignant tumor with poor long-term prognosis. Besides the recently identified MYB-NFIB fusion oncogene generated by a t(6;9) translocation, little is known about other genetic alterations in ACC. Using high-resolution, array-based comparative genomic hybridization, and massively paired-end sequencing, we explored genomic alterations in 40 frozen ACCs. Eighty-six percent of the tumors expressed MYB-NFIB fusion transcripts and 97% overexpressed MYB mRNA, indicating that MYB activation is a hallmark of ACC. Thirty-five recurrent copy number alterations (CNAs) were detected, including losses involving 12q, 6q, 9p, 11q, 14q, 1p, and 5q and gains involving 1q, 9p, and 22q. Grade III tumors had on average a significantly higher number of CNAs/tumor compared to Grade I and II tumors (P = 0.007). Losses of 1p, 6q, and 15q were associated with high-grade tumors, whereas losses of 14q were exclusively seen in Grade I tumors. The t(6;9) rearrangements were associated with a complex pattern of breakpoints, deletions, insertions, inversions, and for 9p also gains. Analyses of fusion-negative ACCs using high-resolution arrays and massively paired-end sequencing revealed that MYB may also be deregulated by other mechanisms in addition to gene fusion. Our studies also identified several down-regulated candidate tumor suppressor genes (CTNNBIP1, CASP9, PRDM2, and SFN) in 1p36.33-p35.3 that may be of clinical significance in high-grade tumors. Further, studies of these and other potential target genes may lead to the identification of novel driver genes in ACC.
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| 8. |
- Werner, Erik, et al.
(författare)
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Orientational correlations in confined DNA
- 2012
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Ingår i: Physical Review E. - 1539-3755 .- 2470-0045 .- 2470-0053. ; 86:4
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Tidskriftsartikel (refereegranskat)abstract
- We study how the orientational correlations of DNA confined to nanochannels depend on the channel diameter D by means of Monte Carlo simulations and a mean-field theory. This theory describes DNA conformations in the experimentally relevant regime where Flory-de Gennes theory does not apply. We show how local correlations determine the dependence of the end-to-end distance of the DNA molecule upon D. Tapered nanochannels provide the necessary resolution in D to study experimentally how the extension of confined DNA molecules depends upon D. Our experimental and theoretical results are in qualitative agreement.
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| 9. |
- Westerlund, Fredrik, 1978, et al.
(författare)
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Fluorescence Enhancement From Single DNA Molecules Confined In Si/SiO2 Nanochannels
- 2010
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Ingår i: Biophysical Society, 54th Annual Meeting.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- A large challenge in biophysics when studying single molecules using fluorescence microscopy is to obtain a signal that is clearly detectable above the background noise. Ways to improve or optimize the fluorescence signal is therefore of great interest. We here study DNA extended in 320 nm deep funnel-shaped SiO2 nanochannels with a width ranging from 40nm to 600nm. The DNA is stained with a fluorescent dye (YOYO-1) and we show that the total emission from the DNA varies significantly with the dimensions of the channels (Figure) and has a peak intensity at half the wavelength of the emitted light. Measurements at varying salt concentrations, where the same confinement leads to different extension of the DNA, confirm that it is solely the geometry of the channel that governs the enhancement effect, ruling out alternative explanations, such as self-quenching. By using polarizers on the emission side we can investigate the light polarized parallel and perpendicular to the channel separately and we see that they show vastly different behavior with the peak in emission only detected in the light polarized parallel to the channels. We will discuss how our data may be explained by cavity-resonance effects when the lateral dimensions of the channels coincide with half the wavelength of the emitted light. Our results suggest that it is possible to fine-tune the size and shape of the nanochannels to maximize the number of photons collected from the molecule under study, for example when studying DNA interacting with single DNA-binding proteins where maximizing the photon budget is paramount.
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| 10. |
- Westerlund, Fredrik, 1978, et al.
(författare)
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Fluorescence enhancement from single DNA molecules confined in SiO 2 nanochannels
- 2010
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Ingår i: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010; Groningen; Netherlands; 3 October 2010 through 7 October 2010. - 9781618390622
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Konferensbidrag (refereegranskat)abstract
- We demonstrate that the detected emission intensity from YOYO-labeled DNA molecules confined in 180 nm deep Si/SiO2 nanofunnels changes significantly and not monotonically with the width of the funnel, an emission enhancement that is only detected for emitted light polarized parallel to the channel. We explain the enhancement effect as being due to optical phenomena in the channels. The enhancement effect may be of importance for quantitative fluorescence microscopy and for experiments with a tight photon budget.
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| 11. |
- Westerlund, Fredrik, 1978, et al.
(författare)
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Fluorescence Enhancement of Single DNA Molecules Confined in Si/SiO2 Nanochannels
- 2010
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 10:16, s. 2049-2051
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate that the detected emission intensity from YOYO- labeled DNA molecules confined in 180 nm deep Si/SiO2 nano- funnels changes significantly and not monotonically with the width of the funnel. This effect may be of importance for quantitative fluorescence microscopy and for experiments with a tight photon budget.
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| 12. |
- 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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| 13. |
- 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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| 14. |
- Frykholm, Karolin, 1977, et al.
(författare)
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Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51
- 2013
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 443:2, s. 261-268
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Tidskriftsartikel (refereegranskat)abstract
- Low throughput is an inherent problem associated with most single-molecule biophysical techniques. We have developed a versatile tool for high-throughput analysis of DNA and DNA-binding molecules by combining microfluidic and dense DNA arrays. We use an easy-to-process microfluidic flow channel system in which dense DNA arrays are prepared for simultaneous imaging of large amounts of DNA molecules with single-molecule resolution. The Y-shaped microfluidic design, where the two inlet channels can be controlled separately and precisely, enables the creation of a concentration gradient across the microfluidic channel as well as rapid and repeated addition and removal of substances from the measurement region. A DNA array stained with the fluorescent DNA-binding dye YOYO-1 in a gradient manner illustrates the method and serves as a proof of concept. We have applied the method to studies of the repair protein Rad51 and could directly probe the concentration-dependent DNA-binding behavior of human Rad51 (HsRad51). In the low-concentration regime used (100 nM HsRad51 and below), we detected binding to double-stranded DNA (dsDNA) without positive cooperativity. (C) 2013 Elsevier Inc. All rights reserved.
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| 15. |
- Lin, Jun, et al.
(författare)
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Bandpass filtering of DNA elastic modes using confinement and tension
- 2012
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 102, s. 96-100
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Tidskriftsartikel (refereegranskat)abstract
- During a variety of biological and technological processes, biopolymers are simultaneously subject to both confinement and external forces. Although significant efforts have gone into understanding the physics of polymers that are only confined, or only under tension, little work has been done to explore the effects of the interplay of force and confinement. Here, we study the combined effects of stretching and confinement on a polymer's configurational freedom. We measure the elastic response of long double-stranded DNA molecules that are partially confined to thin, nanofabricated slits. We account for the data through a model in which the DNA's short-wavelength transverse elastic modes are cut off by applied force and the DNA's bending stiffness, whereas long-wavelength modes are cut off by confinement. Thus, we show that confinement and stretching combine to permit tunable bandpass filtering of the elastic modes of long polymers. © 2012 Biophysical Society.
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| 16. |
- Persson, Fredrik, 1979, et al.
(författare)
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DNA in nanochannels—directly visualizing genomic information
- 2010
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Ingår i: Chemical Society Reviews. - 0306-0012. ; 39, s. 985-999
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Tidskriftsartikel (refereegranskat)abstract
- The power of nanofluidic channels to analyze DNA is described along with practical experimental hints. As an introduction, a general overview is given on conventional DNA analysis tools, as well as tools under development towards the $1000 genome. The focus of this tutorial review is the stretching of DNA in nanoscale channels for coarse-grained mapping of DNA. To understand the behavior of the DNA, basic theory is discussed. Experimental details are revealed so that the reader, with the proper equipment, should be able to perform experiments. Basic approaches to the analysis of the data are discussed. Finally, potential future directions are discussed including the application of melting mapping as a simple barcode for the DNA.
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| 17. |
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| 18. |
- Shirvany, Yazdan, 1980, et al.
(författare)
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Evaluation of a finite-element reciprocity method for epileptic EEG source localization: Accuracy, computational complexity and noise robustness
- 2013
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Ingår i: Biomedical Engineering Letters. - : Springer Science and Business Media LLC. - 2093-985X .- 2093-9868. ; 3:1, s. 8-16
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSEThe aim of this paper is to evaluate the performance of an EEG source localization method that combines a finite element method (FEM) and the reciprocity theorem.METHODSThe reciprocity method is applied to solve the forward problem in a four-layer spherical head model for a large number of test dipoles. To benchmark the proposed method, the results are compared with an analytical solution and two state-of-the-art methods from the literature. Moreover, the dipole localization error resulting from utilizing the method in the inverse procedure for a realistic head model is investigated with respect to EEG signal noise and electrode misplacement.RESULTSThe results show approximately 3% relative error between numerically calculated potentials done by the reciprocity theorem and the analytical solutions. When adding EEG noise with SNR between 5 and 10, the mean localization error is approximately 4.3 mm. For the case with 10 mm electrode misplacement the localization error is 4.8 mm. The reciprocity EEG source localization speeds up the solution of the inverse problem with more than three orders of magnitude compared to the state-of-the-art methods.CONCLUSIONSThe reciprocity method has high accuracy for modeling the dipole in EEG source localization, is robust with respect to noise, and faster than alternative methods.
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| 19. |
- Svanedal, Ida, 1979-, et al.
(författare)
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Metal Ion Coordination, Conditional Stability Constants and Solution Behavior of Chelating Surfactant Metal Complexes
- 2014
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 30:16, s. 4605-4612
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Tidskriftsartikel (refereegranskat)abstract
- Coordination complexes of some divalent metal ions with the DTPA (diethylenetriaminepentaacetic acid)-based chelating surfactant 2-dodecyldiethylenetriaminepentaacetic acid (4-C12-DTPA) have been examined in terms of chelation and solution behavior. The headgroup of 4-C 12-DTPA contains eight donor atoms that can participate in the coordination of a metal ion. Conditional stability constants for five transition metal complexes with 4-C12-DTPA were determined by competition measurements between 4-C12-DTPA and DTPA, using electrospray ionization mass spectrometry (ESI-MS). Small differences in the relative strength between the coordination complexes of DTPA and 4-C12-DTPA indicated that the hydrocarbon tail only affected the chelating ability of the headgroup to a limited extent. The coordination of Cu2+ ions was investigated in particular, using UV-visible spectroscopy. By constructing Job's plots, it was found that 4-C12-DTPA could coordinate up to two Cu2+ ions. Surface tension measurements and NMR diffusometry showed that the coordination of metal ions affected the solution behavior of 4-C 12-DTPA, but there were no specific trends between the studied divalent metal complexes. Generally, the effects of the metal ion coordination could be linked to the neutralization of the headgroup charge of 4-C 12-DTPA, and the resulting reduced electrostatic repulsions between adjacent surfactants in micelles and monolayers. The pH vs concentration plots, on the other hand, showed a distinct difference between 4-C12-DTPA complexes of the alkaline earth metals and the transition metals. This was explained by the difference in coordination between the two groups of metal ions, as predicted by the hard and soft acid and base (HSAB) theory.
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| 20. |
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