| 1. |
|
|
| 2. |
|
|
| 3. |
- Jarvius, Jonas, et al.
(author)
-
Digital Quantification using Amplified Single-Molecule Detection
- 2006
-
In: Nature Methods. - 1548-7091 .- 1548-7105. ; 3:9, s. 725-727
-
Journal article (peer-reviewed)abstract
- We describe a scheme for biomolecule enumeration by converting nanometer-scale specific molecular recognition events mediated by rolling-circle amplification to fluorescent micrometer-sized DNA molecules amenable to discrete optical detection. Our amplified single-molecule detection (SMD) approach preserves the discrete nature of the molecular population, allowing multiplex detection and highly precise quantification of molecules over a dynamic range of seven orders of magnitude. We apply the method for sensitive detection and quantification of the bacterial pathogen Vibrio cholerae.
|
|
| 4. |
- Leuchowius, Karl-Johan, et al.
(author)
-
High content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation
- 2010
-
In: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 9:1, s. 178-183
-
Journal article (peer-reviewed)abstract
- The cost of developing new drugs is a major obstacle for pharmaceutical companies and academia with many drugs identified in the drug discovery process failing approval for clinical use due to lack of intended effect or because of severe side effects. Since the early 1990 s, high throughput screening of drug compounds has increased enormously in capacity but has not resulted in a higher success rate of the identified drugs. Thus, there is a need for methods that can identify biologically relevant compounds and more accurately predict in vivo effects early in the drug discovery process. To address this, we developed a proximity ligation-based assay for high content screening of drug effects on signaling pathways. As a proof of concept, we used the assay to screen through a library of previously identified kinase inhibitors, including six clinically used tyrosine kinase inhibitors, to identify compounds that inhibited the platelet-derived growth factor (PDGF) receptor beta signaling pathway in stimulated primary human fibroblasts. Thirteen of the 80 compounds were identified as hits, and the dose responses of these compounds were measured. The assay exhibited a very high Z' factor (0.71) and signal to noise ratio (11.7), demonstrating excellent ability to identify compounds interfering with the specific signaling event. A comparison with regular immunofluorescence detection of phosphorylated PDGF receptor demonstrated a far superior ability by the in situ proximity ligation assay to reveal inhibition of receptor phosphorylation. In addition, inhibitor-induced perturbation of protein-protein interactions of the PDGF signaling pathway could be quantified, further demonstrating the usefulness of the assay in drug discovery.
|
|
| 5. |
|
|
| 6. |
|
|
| 7. |
- Melin, Jonas, et al.
(author)
-
Homogeneous amplified single-molecule detection : Characterization of key parameters
- 2007
-
In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 368:2, s. 230-238
-
Journal article (peer-reviewed)abstract
- We recently presented a method that enables single-molecule enumeration by transforming specific molecular recognition events at nanometer dimensions to micrometer-sized DNA macromolecules. This transformation process is mediated by target-specific padlock probe ligation, followed by rolling circle amplification (RCA), resulting in the creation of one rolling circle product (RCP) for each recognized target. The transformation makes optical detection and quantification possible using standard fluorescence microscopy by counting the number of generated RCPs in a sample pumped through a microfluidic channel. In this study, we demonstrate that confocal volume definition is crucial to achieve high-precision measurements in the microfluidic quantification (coefficient of variance typically 3%). We further demonstrate that complementary sequence motifs between RCPs is only a weak inducer of aggregates and that all detection sites of the RCPs are occupied at detection oligonucleotide concentrations greater than 5 nM if hybridized in the proper buffer conditions. Therefore, the signal/noise ratio is limited by the number of detection sites. By increasing the density of detection sites in the RCP by a factor of 1.9, we show that the optical signal/noise level can be increased from 42 to 75.
|
|
| 8. |
|
|
| 9. |
- Melin, Jonas, et al.
(author)
-
Ligation-based molecular tools for lab-on-a-chip devices
- 2008
-
In: New Biotechnology. - : Elsevier BV. - 1871-6784. ; 25:1, s. 42-8
-
Journal article (peer-reviewed)abstract
- Molecular diagnostics can offer early detection of disease, improved diagnostic accuracy, and qualified follow-up. Moreover, the use of microfluidic devices can in principle render these analyses quickly and user-friendly, placing them within the reach of the general practitioner and maybe even in households. However, the progress launching such devices has been limited so far. We propose that an important limiting factor has been the difficulty of establishing molecular assays suitable for microfabricated formats. The assays should be capable of monitoring a wide range of molecules, including genomic DNA, RNA and proteins with secondary modifications and interaction partners, and they must exhibit excellent sensitivity and specificity. We discuss these problems and describe a series of molecular tools that may present new opportunities for lab-on-a-chip devices at the point-of-care.
|
|
| 10. |
|
|
| 11. |
- Melin, Jonas, et al.
(author)
-
Thermoplastic microfluidic platform for single-molecule detection, cell culture and actuation
- 2005
-
In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 77:22, s. 7122-30
-
Journal article (peer-reviewed)abstract
- We have developed a multipurpose microfluidic platform that allows for sensitive fluorescence detection on inexpensive disposable chips. The fabrication scheme involves rapid injection molding of thermoplastics, followed by silica deposition and covalent attachment of an unstructured flexible lid. This combines the virtues of elastomer technology with high-throughput compact disk injection molding. Using this technique, the time to produce 100 chips using a single master can be lowered from more than 1 week by standard PDMS technologies to only a couple hours. The optical properties of the fabricated chips were evaluated by studying individual fluorescence-labeled DNA molecules in a microchannel. Concatemeric DNA molecules were generated through rolling circle replication of circular DNA molecules, which were labeled by hybridization of fluorescence-tagged oligonucleotides. Rolling circle products (RCPs) were detected after as little as 5 min of DNA polymerization, and the RCPs in solution showed no tendency for aggregation. To illustrate the versatility of the platform, we demonstrate two additional applications: The flexible property of the lid was used to create a peristaltic pump generating a flow rate of 9 nL/s. Biocompatibility of the platform was illustrated by culturing Chinese hamster ovary cells for 7 days in the microfluidic channels.
|
|
| 12. |
- Pinidiyaarachchi, Amalka, et al.
(author)
-
Digital image processing for multiplexing of single molecule detection
- 2005
-
In: Medicinteknikdagarna.
-
Conference paper (other academic/artistic)abstract
- Using padlock and proximity probing techniques, individual molecular identification events are converted to long DNA molecules, carrying repeated sequence motifs used for identification of the detected molecules. We show that identification events can be amplified using rolling circle replication, and randomly attached to a surface for repeated access by identification probes. Repeated hybridization with detection probes carrying fluorescing nano-crystals (quantum dots) of varying spectral properties opens the possibility to search for large numbers of different identification events simultaneously. Methods for digital image processing of the resulting multi-spectral data include spatial as well as spectral data clustering. Spatial data processing includes registration of images from repeated hybridization events as well as delineation of clustered reporter events. Spectral data processing and analysis includes classification of spectral data into groups of either pre-defined or unknown patterns representing different molecular identification events.
|
|
| 13. |
- Söderberg, Ola, et al.
(author)
-
Direct observation of individual endogenous protein complexes in situ by proximity ligation
- 2006
-
In: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 3:12, s. 995-1000
-
Journal article (peer-reviewed)abstract
- Cellular processes can only be understood as the dynamic interplay of molecules. There is a need for techniques to monitor interactions of endogenous proteins directly in individual cells and tissues to reveal the cellular and molecular architecture and its responses to perturbations. Here we report our adaptation of the recently developed proximity ligation method to examine the subcellular localization of protein-protein interactions at single-molecule resolution. Proximity probes-oligonucleotides attached to antibodies against the two target proteins-guided the formation of circular DNA strands when bound in close proximity. The DNA circles in turn served as templates for localized rolling-circle amplification (RCA), allowing individual interacting pairs of protein molecules to be visualized and counted in human cell lines and clinical specimens. We used this method to show specific regulation of protein-protein interactions between endogenous Myc and Max oncogenic transcription factors in response to interferon-gamma (IFN-gamma) signaling and low-molecular-weight inhibitors.
|
|
| 14. |
|
|
| 15. |
|
|
| 16. |
- Banér, Johan, et al.
(author)
-
Parallel gene analysis with allele-specific padlock probes and tag microarrays
- 2003
-
In: Nucleic Acids Research. - 0305-1048 .- 1362-4962. ; 31:17, s. e103-
-
Journal article (peer-reviewed)abstract
- Parallel, highly specific analysis methods are required to take advantage of the extensive information about DNA sequence variation and of expressed sequences. We present a scalable laboratory technique suitable to analyze numerous target sequences in multiplexed assays. Sets of padlock probes were applied to analyze single nucleotide variation directly in total genomic DNA or cDNA for parallel genotyping or gene expression analysis. All reacted probes were then co-amplified and identified by hybridization to a standard tag oligonucleotide array. The technique was illustrated by analyzing normal and pathogenic variation within the Wilson disease-related ATP7B gene, both at the level of DNA and RNA, using allele-specific padlock probes.
|
|
| 17. |
- Barkefors, Irmeli, et al.
(author)
-
Endothelial cell migration in stable gradients of vascular endothelial growth factor A and fibroblast growth factor 2 : effects on chemotaxis and chemokinesis
- 2008
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 283:20, s. 13905-13912
-
Journal article (peer-reviewed)abstract
- Gradients of secreted signaling proteins guide growing blood vessels during both normal and pathological angiogenesis. However, the mechanisms by which endothelial cells integrate and respond to graded distributions of chemotactic factors are still poorly understood. We have in this study investigated endothelial cell migration in response to hill-shaped gradients of vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2) using a novel microfluidic chemotaxis chamber (MCC). Cell migration was scored at the level of individual cells using time-lapse microscopy. A stable gradient of VEGFA165 ranging from 0 to 50 ng/ml over a distance of 400 microm was shown to strongly induce chemotaxis of endothelial cells of different vascular origin. VEGFA121, unable to bind proteoglycan and neuropilin coreceptors, was also shown to induce chemotaxis in this setup. Furthermore, a gradient of FGF2 was able to attract venular but not arterial endothelial cells, albeit less efficiently than VEGFA165. Notably, constant levels of VEGFA165, but not of FGF2, were shown to efficiently reduce chemokinesis. Systematic exploration of different gradient shapes led to the identification of a minimal gradient steepness required for efficient cell guidance. Finally, analysis of cell migration in different regions of the applied gradients showed that chemotaxis is reduced when cells reach the high end of the gradient. Our findings suggest that chemotactic growth factor gradients may instruct endothelial cells to shift toward a nonmigratory phenotype when approaching the growth factor source.
|
|
| 18. |
- Ericsson, Olle, et al.
(author)
-
A dual-tag microarray platform for high-performance nucleic acid and protein analyses
- 2008
-
In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 36:8, s. e45-
-
Journal article (peer-reviewed)abstract
- DNA microarrays serve to monitor a wide range of molecular events, but emerging applications like measurements of weakly expressed genes or of proteins and their interaction patterns will require enhanced performance to improve specificity of detection and dynamic range. To further extend the utility of DNA microarray-based approaches we present a high-performance tag microarray procedure that enables probe-based analysis of as little as 100 target cDNA molecules, and with a linear dynamic range close to 10(5). Furthermore, the protocol radically decreases the risk of cross-hybridization on microarrays compared to current approaches, and it also allows for quantification by single-molecule analysis and real-time on-chip monitoring of rolling-circle amplification. We provide proof of concept for microarray-based measurement of both mRNA molecules and of proteins, converted to tag DNA sequences by padlock and proximity probe ligation, respectively.
|
|
| 19. |
|
|
| 20. |
|
|
| 21. |
- Fredriksson, Simon, et al.
(author)
-
Protein detection using proximity-dependent DNA ligation assays
- 2002
-
In: Nature Biotechnology. - : Springer Science and Business Media LLC. - 1087-0156 .- 1546-1696. ; 20:5, s. 473-477
-
Journal article (peer-reviewed)abstract
- The advent of in vitro DNA amplification has enabled rapid acquisition of genomic information. We present here an analogous technique for protein detection, in which the coordinated and proximal binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to each aptamer affinity probe. The ligation of two such proximity probes gives rise to an amplifiable DNA sequence that reflects the identity and amount of the target protein. This proximity ligation assay detects zeptomole (40 x 10(-21) mol) amounts of the cytokine platelet-derived growth factor (PDGF) without washes or separations, and the mechanism can be generalized to other forms of protein analysis.
|
|
| 22. |
|
|
| 23. |
- Gullberg, Mats, et al.
(author)
-
Cytokine detection by antibody-based proximity ligation
- 2004
-
In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 101:22, s. 8420-4
-
Journal article (other academic/artistic)abstract
- Efficient and precise detection techniques, along with extensive repertoires of specific binding reagents, will be needed to meet the challenges of proteome analyses. The recently established proximity ligation mechanism enables sensitive high-capacity protein measurements by converting the detection of specific proteins to the analysis of DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins and to form amplifiable tag sequences by ligation when brought in proximity. In our previous report, both the ligatable arms and the protein binders were DNA molecules. We now generalize the method by providing simple and convenient protocols to convert any polyclonal antibodies or matched pair of monoclonal antibodies to proximity probe sets through the attachment of oligonucleotide sequences. Sufficient reagent for >100,000 proximity ligation assays can be prepared from 1 microg of antibody. The technique is applied to measure cytokines in a homogenous test format with femtomolar detection sensitivities in 1-microl samples, and we exemplify its utility in situations when only minute sample amounts are available.
|
|
| 24. |
|
|
| 25. |
- Göransson, Jenny, 1978-, et al.
(author)
-
A single molecule array for digital targeted molecular analyses
- 2009
-
In: Nucleic Acids Research. - England : Oxford University Press. - 0305-1048 .- 1362-4962. ; 37:1, s. e7-
-
Journal article (peer-reviewed)abstract
- We present a new random array format together with a decoding scheme for targeted multiplex digital molecular analyses. DNA samples are analyzed using multiplex sets of padlock or selector probes that create circular DNA molecules upon target recognition. The circularized DNA molecules are amplified through rolling-circle amplification (RCA) to generate amplified single molecules (ASMs). A random array is generated by immobilizing all ASMs on a microscopy glass slide. The ASMs are identified and counted through serial hybridizations of small sets of tag probes, according to a combinatorial decoding scheme. We show that random array format permits at least 10 iterations of hybridization, imaging and dehybridization, a process required for the combinatorial decoding scheme. We further investigated the quantitative dynamic range and precision of the random array format. Finally, as a demonstration, the decoding scheme was applied for multiplex quantitative analysis of genomic loci in samples having verified copy-number variations. Of 31 analyzed loci, all but one were correctly identified and responded according to the known copy-number variations. The decoding strategy is generic in that the target can be any biomolecule which has been encoded into a DNA circle via a molecular probing reaction.
|
|
| 26. |
- Göransson, Jenny, et al.
(author)
-
Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification
- 2012
-
In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:2, s. e31068-
-
Journal article (peer-reviewed)abstract
- Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field.
|
|
| 27. |
|
|
| 28. |
- Jarvius, Jonas, 1971-
(author)
-
DNA Tools and Microfluidic Systems for Molecular Analysis
- 2006
-
Doctoral thesis (other academic/artistic)abstract
- Improved methods are needed to interrogate the genome and the proteome. Methods with high selectivity, wide dynamic range, and excellent precision, capable of simultaneously analyzing many biomolecules are required to decipher cellular function. This thesis describes a molecular and microfluidic toolbox designed with those criteria in mind. It also presents a tool for graphical representation of nucleic acid sequences.Proximity ligation is a novel protein detection method that requires dual and proximate binding of two oligonucleotide-tagged affinity reagents to a protein or protein complex in order to elicit a signal. The responses from such recognition reactions are the formation of specific nucleic acid reporter molecules that are subsequently amplified and quantitatively detected.A scalable microfluidic platform suitable for fluorescence detection, cell culture, and actuation is also described. The platform uses rapid injection molding to produce microstructures in thermoplastic materials. By applying a thin layer of silica to the structures, a lid made of silicone rubber coated onto a thermoplastic support can be covalently bonded to generate enclosed channels.A method is presented for precise biomolecule counting, termed “amplified single-molecule detection”. The method preserves the discrete nature of biomolecules, converting specific molecular recognition events to fluorescence-labeled micrometer-sized objects that are enumerated in microfluidic channels.I also present a novel microarray-based detection method. To attain high selectivity and a wide dynamic range, the method is based on dual recognition with enzymatic discrimination and amplification. Upon target recognition in solution, DNA probes are subjected to thousand-fold amplification in solution, followed by selective detection on arrays and another hundred-fold amplification of reporter molecule created from the first amplification reaction.Lastly, I describe a novel graphical representation of nucleic acid sequences using TrueType fonts that can be of value for visual inspection of DNA sequences and for teaching purposes
|
|
| 29. |
- Jarvius, Jonas, et al.
(author)
-
Oligonucleotide ligation assay
- 2003
-
In: Methods in Molecular Biology. - 1064-3745 .- 1940-6029. ; 212, s. 215-28
-
Journal article (peer-reviewed)
|
|
| 30. |
- Landegren, Ulf, et al.
(author)
-
Molecular tools for a molecular medicine : analyzing genes, transcripts and proteins using padlock and proximity probes
- 2004
-
In: Journal of Molecular Recognition. - : Wiley. - 0952-3499 .- 1099-1352. ; 17:3, s. 194-7
-
Journal article (peer-reviewed)abstract
- Procedures and reagents are needed to specifically detect all the macromolecules that are being identified in the course of genome projects. We discuss how this challenge may be met using a set of ligation-based reagents termed padlock probes and proximity ligation probes. These probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules. The information content of DNA strands that form in the detection reactions are recorded after amplification, allowing the recognized target molecules to be identified. The procedures permit highly specific solution-phase or localized analyses of large sets of target molecules as required in future molecular analyses.
|
|
| 31. |
- Landegren, Ulf, et al.
(author)
-
Padlock and proximity probes for in situ and array-based analyses : tools for the post genomic era
- 2003
-
In: Comparative and functional genomics. - : Hindawi Limited. - 1531-6912 .- 1532-6268. ; 4:5, s. 525-30
-
Journal article (peer-reviewed)abstract
- Highly specific high-throughput assays will be required to take full advantage of the accumulating information about the macromolecular composition of cells and tissues, in order to characterize biological systems in health and disease. We discuss the general problem of detection specificity and present the approach our group has taken, involving the reformatting of analogue biological information to digital reporter segments of genetic information via a series of DNA ligation assays. The assays enable extensive, coordinated analyses of the numbers and locations of genes, transcripts and protein.
|
|
| 32. |
|
|
| 33. |
- Russell, Camilla, et al.
(author)
-
Gold Nanowire Based Electrical DNA Detection Using Rolling Circle Amplification
- 2014
-
In: ACS Nano. - : American Chemical Society (ACS). - 1936-0851 .- 1936-086X. ; 8:2, s. 1147-1153
-
Journal article (peer-reviewed)abstract
- We present an electrical sensor that uses rolling circle amplification (RCA) of DNA to stretch across the gap between two electrodes, interact with metal nanoparticle seeds to generate an electrically conductive nanowire, and produce electrical signals upon detection of specific target DNA sequences. RCA is a highly specific molecular detection mechanism based on DNA probe circularization. With this technique, long single-stranded DNA with simple repetitive sequences are produced. Here we show that stretched RCA products can be metalized using silver or gold solutions to form metal wires. Upon metallization, the resistance drops from T Omega to k Omega for silver and to Omega for gold. Metallization is seeded by gold nanoparticles aligned along the single-stranded DNA product through hybridization of functionalized oligonucleotides. We show that combining RCA with electrical DNA detection produces results in readout with very high signal-to-noise ratio, an essential feature for sensitive and specific detection assays. Finally, we demonstrate detection of 10 ng of Escherichia coli genomic DNA using the sensor concept.
|
|
| 34. |
- Sato, Kae, et al.
(author)
-
Microbead-based rolling circle amplification in a microchip for sensitive DNA detection
- 2010
-
In: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 10:10, s. 1262-1266
-
Journal article (peer-reviewed)abstract
- The sensitive detection and quantification of DNA targets in the food industry and in environmental and clinical settings are issues of utmost importance in ensuring contamination-free food, monitoring the environment, and battling disease. Selective probes coupled with powerful amplification techniques are therefore of major interest. In this study, we set out to create an integrated microchemical chip that benefits from microfluidic chip technology in terms of sensitivity and a strong detection methodology provided jointly by padlock probes and rolling circle amplification (RCA). Here, we have integrated padlock probes and RCA into a microchip. The chip uses solid phase capture in a microchannel to enable washing cycles and decrease analytical area, and employs on-bead RCA for single-molecule amplification and detection. We investigated the effects of reagent concentration and amount of padlock probes, and demonstrated the feasibility of detecting Salmonella.
|
|
