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- Ericsson, Olle, et al.
(författare)
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A dual-tag microarray platform for high-performance nucleic acid and protein analyses
- 2008
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 36:8, s. e45-
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Tidskriftsartikel (refereegranskat)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.
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| 2. |
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| 3. |
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| 4. |
- Göransson, Jenny, 1978-, et al.
(författare)
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A single molecule array for digital targeted molecular analyses
- 2009
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Ingår i: Nucleic Acids Research. - England : Oxford University Press. - 0305-1048 .- 1362-4962. ; 37:1, s. e7-
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Tidskriftsartikel (refereegranskat)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.
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| 5. |
- Göransson, Jenny, et al.
(författare)
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Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:2, s. e31068-
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Tidskriftsartikel (refereegranskat)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.
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| 6. |
- Howell, Aaron, et al.
(författare)
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Black Liquor Falling Film Evaporation: Computational Model and Pilot Experiments
- 2014
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Ingår i: Proceedings of International Chemical Recovery Conference, Tampere,Finland, June 8 – 13 2014. ; , s. 389-401
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Konferensbidrag (refereegranskat)abstract
- The focus of this work is to improve our understanding of the black liquor evaporator fouling process. Theinvestigation method is two pronged, consisting of the development of a computational model paired withexperiments conducted in a pilot evaporator. Experiments for black liquor at solids up to 45% dry solids wereconducted and used to validate the computational model. It was found that for fixed Reynolds number, loweringpump frequency induces waves closer to the inlet. And, for fixed pumping frequency, increasing the Reynoldsnumber has the effect of inducing waves closer to the inlet. The numerical model reproduces the complex wavesstructures present in black liquor flow. The model and pilot plant are to be used to investigate the effect of relevantparameters for the hydrodynamics, heat transfer, and soluble scale fouling of black liquor, which is of specificconcern to the pulping industry.
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| 7. |
- Howell, W. Mathias, et al.
(författare)
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Glycosylases and AP-cleaving enzymes as a general tool for probe-directed cleavage of ssDNA targets
- 2010
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 38:7, s. e99-
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Tidskriftsartikel (refereegranskat)abstract
- The current arsenal of molecular tools for site-directed cleavage of single-stranded DNA (ssDNA) is limited. Here, we describe a method for targeted DNA cleavage that requires only the presence of an A nucleotide at the target position. The procedure involves hybridization of a complementary oligonucleotide probe to the target sequence. The probe is designed to create a deliberate G:A mismatch at the desired position of cleavage. The DNA repair enzyme MutY glycosylase recognizes the mismatch structure and selectively removes the mispaired A from the duplex to create an abasic site in the target strand. Addition of an AP-endonuclease, such as Endonuclease IV, subsequently cleaves the backbone dividing the DNA strand into two fragments. With an appropriate choice of an AP-cleaving enzyme, the 3'- and 5'-ends of the cleaved DNA are suitable to take part in subsequent enzymatic reactions such as priming for polymerization or joining by DNA ligation. We define suitable standard reaction conditions for glycosylase/AP-cleaving enzyme (G/AP) cleavage, and demonstrate the use of the method in an improved scheme for in situ detection using target-primed rolling-circle amplification of padlock probes.
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| 8. |
- Howell, Walter Mathias
(författare)
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SNP technology and Alzheimer’s disease
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- One major goal of genetic research is to understand the role of genetic variation. By far the most common type of such variation in humans involves single DNA bases, and is termed single nucleotide polymorphism (SNP). With sufficient technological solutions, one strong belief is that SNPs can enable the mapping of disease genes involved in complex genetic disorders. Alzheimer’s disease (AD) is a complex disorder characterized by progressive cognitive decline and memory impairment. Some individuals acquire this form of dementia before the age of 65 (referred to as early-onset or familial AD) but most often AD occurs late in life. It is in the early- onset form, however, where causative mutations have been found in three different genes; APP, PSEN1, & PSEN2. Other than these, the only additional risk factor identified for AD is the epsilon 4 allele of the APOE gene. Together these only account for a fraction of AD, leaving room for studies to identify additional AD susceptibility genes. In the initial investigation of this thesis, polymorphisms in the PSEN1, PSEN2, APOE and VLDL-R genes were tested for association with early-onset Alzheimer's disease (EOAD). Aside from confirming the well-established APOE-epsilon4 association, an allele of the PSEN2 showed a significant disease association. In an attempt to verify the PSEN2 association and localize the potential pathogenic variant, a series of eight additional SNPs, located throughout the PSEN2 gene, were tested for association with EOAD. None of the tested markers showed significant disease association upon replication in a second set of cases and controls. The remainder of the thesis describes the invention and development of a novel technique for scoring SNPs. The method, called Dynamic Allele Specific Hybridization (DASH), is a great improvement in both throughput and reliability compared to traditional techniques. The crucial step in the procedure is the heating of the DNA duplex (formed from hybridization of the PCR- amplified target with an allele-specific probe) whilst measuring the fluorescence of a double-strand specific intercalating dye. SNP alleles are detected and scored by comparative analysis of the melting profiles. Improvements to the initial DASH format are detailed in the final two papers of this thesis. A novel alternative in fluorescence detection, termed induced fluorescent resonance energy transfer (iFRET), is introduced. IFRET employs energy transfer between a generic intercalating dye and an FRET acceptor attached to the allele-specific probe. This system retains the spectral-multiplex potential offered by traditional FRET systems, while reducing costs and improving fluorescence signal intensities. Aside from detection, DASH was converted from a microtiter-plate format to an array format which greatly improved flexibility and simplified the assay procedure. The complete DASH-2 system is examined in terms of multiplex options, throughput, cost and accuracy.
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| 9. |
- Pinidiyaarachchi, Amalka, et al.
(författare)
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Digital image processing for multiplexing of single molecule detection
- 2005
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Ingår i: Medicinteknikdagarna.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)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.
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| 10. |
- Weibrecht, Irene, et al.
(författare)
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Proximity ligation assays : a recent addition to the proteomics toolbox
- 2010
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Ingår i: Expert Reviews of Proteomics. - 1478-9450. ; 7:3, s. 401-409
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Forskningsöversikt (refereegranskat)abstract
- An essential skill for every researcher is to learn how to select and apply the most appropriate methods for the questions they are trying to answer. With the extensive variety of methods available, it is increasingly important to scrutinize the advantages and disadvantages of these techniques prior to making a decision on which to use. In this article, we describe an approach to evaluate methods by reducing them into subcomponents. This is exemplified by a brief description of some commonly used proteomics methods. The same approach can also be used in method development by rearranging subcomponents in order to create new methods, as demonstrated with the development of proximity ligation assays (PLAs). PLA is a method as designed in our laboratory for detection of proteins, protein-protein interactions and post-translational modifications. Fundamentally, protein-recognition events are converted into detectable DNA molecules. The technique uses protein DNA conjugates as binders for the targets of interest. Binding of two or more conjugates to the target results in assembly of an assay-specific DNA molecule. Subsequent amplification of the DNA molecule generates a signal that can be detected using PCR, for detection of minute amounts of proteins in serum, or standard fluorescence microscopy for detection of protein protein interactions in tissue sections. Lastly, we apply the approach of recombining subcomponents to develop a few novel hypothetical methods hoping this might stimulate the readers to utilize this approach themselves.
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