Uppsala, Sweden
Uppsala, Sweden

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Grant
Agency: Cordis | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2010.1.2-1 | Award Amount: 6.55M | Year: 2010

A new generation of molecular tools is becoming available that can digitally record numbers, identities and locations of a broad range of molecular markers for increased diagnostic accuracy. This project will for the first time combine synergistically several groundbreaking technological innovations by the partners, notably padlock and proximity probes with rolling circle amplification for single molecule detection and counting, directed self-assembly of solid phases, and advanced microfluidics and read-out techniques, bringing these from the research lab into integrated instruments useful in routine. This will enable minimally invasive diagnostics, prognostics, and follow-up of treatment of cancers. Blood samples and fine needle aspirates will be subjected to high content, multiplex and multimodal assays of nucleic acids, proteins and interacting complexes thereof in single cells as well as in cell-free bodily fluids. We will use flow cytometry to collect multi-parameter information for large populations of cells, and individual detected molecules will be recorded using a fluorescence activated molecule counter developed by one of the partners. Also, very rare cells and molecules will be targeted through enrichment techniques using novel capturing approaches of unprecedented efficiency. We will apply these diagnostic approaches to characterize biomarkers in solid tumors and in leukemia and lymphoma for minimally invasive diagnostics, monitoring disease progress and selecting optimal therapy, and the assays will be clinically validated in small-scale studies of well-characterized patient samples. This project directly addresses four of the five topics in this call.


Grant
Agency: Cordis | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2012.2.4.5-2 | Award Amount: 7.85M | Year: 2012

We propose a collaborative effort to advance our understanding of the inflammatory bowel diseases Ulcerative Colitis and Crohns Disease and to increase diagnostic precision in detection of the diseases in their early manifestation. We will utilize the largest collection of samples so far assembled of treatment nave patients recently diagnosed with inflammatory bowel disease, a total of 400 patients. The material will be extensively analyzed for DNA methylation status, RNA transcription profile, protein markers and gut microbial content in order to create a molecular snapshot of IBD in its early manifestation. As control group, material from a total of 200 recently by endoscopy diagnosed non-inflammatory but symptomatic patients (symptomatic non-IBD) as well as 200 healthy, age-matched, non-smoking, controls without any known first relatives diagnosed with IBD, will be collected and used. All patients will be diagnosed according to standardized diagnostic criterias, characterized using current known clinical markers as well as genotyped for known susceptibility loci. This large, well characterized cohort in conjunction with our proposed massive molecular profiling will yield a list of biomarkers indicative for onset of the disease. Based on the finding within the project assays capable of analyzing panels of relevant protein markers and methods for rapid profiling of gut microbial content relevant for IBD will be developed by participating SME:s, offering large commercial potential. The proposed biomarkers will form a solid ground for development of improved diagnostic assays and be a rich source to mine for novel therapeutic targets.


Grant
Agency: Cordis | Branch: FP7 | Program: MC-ITN | Phase: FP7-PEOPLE-2012-ITN | Award Amount: 4.10M | Year: 2013

This ITN is built around the biological question of O-glycosylation in gastric cancer for training in systems glycobiology. For meeting the needs of the research goal and for training for future systems glycobiology operations, cross disciplinary international institutes have been identified with top level state-of-the-art glycobioanalytical, glycotechnological and glycobioinformatics platforms. These platforms will be utilized and adopted to address the biology. The researchers will be exposed to academia and industry working together to develop resources of common use for addressing complex biological questions. This has been shown in the area of proteomics, to be a successful way for developing bioinformatics, databases, software, bioanalysis, reagents and biomolecule synthesis/production. These resources are now in use throughout the life science environment in academia, biotech and biopharma. However, there is no individual institute to our knowledge that can prepare new systems glycobiologists for the full impact of the environment they will operate in. This ITN is addressing this deficiency in training, in order to contribute to make Europe a competitive environment for the new generation of multidisciplinary research for complex glycobiological questions. The training to address the systems glycobiology in gastric cancer will provide the researchers with biological and technological workshops and courses in project management and business operations together with bidirectional secondments between academia-industry. Two dedicated training partners have been identified in order to provide efficient on-site training and self studies promoted by E-learning. In the research process we will identify and transfer technologies and biological outcomes for commercialization. A significant part of the training will also be to provide researchers with techniques peripheral to the ones available on their home base.


The present invention provides a probe for use in detecting a target analyte in a sample, wherein the probe provides or is capable of providing nucleic acid components sufficient to initiate a rolling circle amplification (RCA) reaction, said probe being a nucleic acid construct comprising: (i) one or more target binding domains capable of binding to said target analyte or an intermediate molecule bound, directly or indirectly, to the target analyte; (ii) one or more domains together comprising or capable of providing a circular or circularisable RCA template; (iii) a domain comprising or capable of providing a primer for said RCA reaction that hybridizes to a region of said circular or circularisable RCA template; and, when the probe comprises or is capable of providing a circularisable RCA template, (iv) one or more domains comprising or capable of providing a ligation template that templates the ligation of the circularisable RCA template, wherein at least part of the probe must be cleaved and/or unfolded to release said primer to enable said rolling circle amplification reaction. Also provided are methods for detecting analytes in a sample using the probe of the invention. In certain preferred embodiments of the probe cleavage of the probe into multiple parts each held in proximity by a target binding domain in each part of the probe generates a circularisable RCA template, which is circularised in a ligation reaction templated by a ligaton template domain of the probe.


Patent
Olink Ab | Date: 2012-01-30

The present invention relates to a proximity probe based detection assay (proximity assay) for an analyte in a sample, specifically a proximity probe extension assay (PEA), an in particular to an improvement in the method to reduce non-specific background signals, wherein the improvement comprises the use in such assays of a component comprising 3 exonuclease activity, said method comprising: (a) contacting said sample with at least one set of at least first and second proximity probes, which probes each comprise an analyte-binding domain and a nucleic acid domain and can simultaneously bind to the analyte; (b) allowing the nucleic acid domains of the proximity probes to interact with each other upon binding of said proximity probes to said analyte, wherein said interaction comprises the formation of a duplex; (c) contacting said sample with a component comprising 3 exonuclease activity; (d) extending the 3 end of at least one nucleic acid domain of said duplex to generate an extension product, wherein the step may occur contemporaneously with or after step (c); and (e) amplifying and detecting the extension product.


The present invention relates to a proximity probe based detection assay (proximity assay) for an analyte in a sample, specifically a proximity probe extension assay (PEA), an in particular to an improvement in the method to reduce non-specific background signals, wherein the improvement comprises the use in such assays of a hyperthermophilic polymerase, said method comprising: (a) contacting said sample with at least one set of at least first and second proximity probes, which probes each comprise an analyte-binding domain and a nucleic acid domain and can simultaneously bind to the analyte; (b) allowing the nucleic acid domains of the proximity probes to interact with each other upon binding of said proximity probes to said analyte, wherein said interaction comprises the formation of a duplex; (c) extending the 3 end of at least one nucleic acid domain of said duplex to generate an extension product, wherein the extension reaction comprises increasing the temperature of assay above room temperature and uses a polymerase enzyme which is characterised as having less than 20% of its maximal enzyme activity at 40 C. and having less than 10% of its maximal enzyme activity at 25 C., wherein the optimum temperature for maximal activity of the polymerase is more than 40 C. and wherein the polymerase is selected from Pyrococcus furiosus (Pfu) DNA polymerase and Pyrococcus woesei (Pwo) DNA polymerase or a derivative or mutant thereof, preferably wherein said derivative is a sequence-modified derivative; and (d) amplifying and detecting the extension product.


The present invention provides a method for performing a localised RCA reaction comprising at least two rounds of RCA, wherein the product of a second RCA reaction is attached, and hence localised, to a product of a first RCA reaction, said method comprising: (a) providing a concatemeric first RCA product comprising repeated monomers; (b) directly or indirectly hybridising to monomers of said first RCA product a circularisable oligonucleotide comprising target-complementary 3 and 5 end regions such that the 3 and 5 ends of said oligonucleotide hybridise in juxtaposition for ligation directly or indirectly to each other, wherein the target is a sequence in a monomer of said first RCA product or an intermediate molecule hybridised thereto, and wherein the target-complementary end regions of said circularisable oligonucleotide are 6 to 16 nucleotides in length; (c) directly or indirectly ligating the ends of said circularisable oligonucleotide to circularise the oligonucleotide, thereby to provide a template for a second RCA reaction, wherein when said ends are indirectly ligated (i) either a gap oligonucleotide is provided which hybridises to the monomers of the first RCA product in between the 3 and 5 ends of the circularisable oligonucleotide such that it may be ligated to the respective ends, or the hybridised 3 end of the circularisable oligonucleotide is extended by a polymerase such that the extended 3 end may be ligated to the hybridised 5 end, and (ii) the total length of the region of the second RCA template directly or indirectly hybridised to the monomers is no longer than 32 nucleotides in length; and (d) performing a second RCA reaction using said second RCA template of (c) and a primer for said second RCA, to form a second RCA product, wherein in said second RCA reaction the second RCA template remains attached to the first RCA product, and thereby the second RCA product is attached to the first RCA product.


Patent
Olink Ab | Date: 2012-05-23

The present invention provides a method for detecting interactions between or with any two of at least three target substrates, or any two of at least three features of a target substrate, or a combination of interactions and features of target substrates, by a multiplexed proximity ligation assay, said method comprising: a) for each of the at least three target substrates or features, providing a proximity probe comprising a binding moiety with affinity for the feature or binding site on said substrate, and a proximity probe oligonucleotide coupled on the binding moiety; wherein each of the proximity probe oligonucleotide carries a unique tag sequence; b) mixing the proximity probes with a sample, under a condition to allow binding of each proximity probe to its respective binding site or feature on each of said substrates through the binding moiety, c) simultaneous with, or following step b), forming circularized DNA molecules where any two proximity probes bind sufficiently close to each other on the substrate, wherein each of the circularized DNA molecules comprise complementary sequences to the unique tag sequences from the two proximity probes oligo-nucleotides; d) amplifying the circularized DNA; and e) characterizing the amplified DNA.


Patent
Olink Ab | Date: 2013-11-14

The present invention provides a method for performing a localised RCA reaction comprising at least two rounds of RCA, wherein the product of a second RCA reaction is attached, and hence localised, to a product of a first RCA reaction, said method comprising: (a) providing a first RCA product; (b) directly or indirectly hybridising to said first RCA product a probe which comprises or provides a primer for a second RCA reaction; and (c) performing a second RCA reaction using said RCA primer of (b) to form a second RCA product, wherein in said reaction: (i) said probe and said primer are not able to prime extension using said first RCA product as template or any such extension is limited to avoid displacement of any probe hybridised to the first RCA product; (ii) the direct or indirect hybridisation of the RCA primer of (b) to the first RCA product is maintained and, by virtue of said hybridisation, the second RCA product is attached to the first RCA product; (iii) a RCA template for said second RCA reaction is comprised in or provided by the probe, or is separately provided. The method finds particular utility in the detection of analytes, wherein the analyte is a nucleic acid or wherein a nucleic acid is used or generated as a marker for the analyte.


Grant
Agency: Cordis | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2013.0-1 | Award Amount: 7.39M | Year: 2013

Todays treatment of metastatic breast cancer is guided by characterisation of the primary tumour while 90% of deaths due to breast cancer occur as a consequence of metastases. Given important differences in molecular characteristics between metastases and primary tumour tissue, characterization of metastatic tumours instead of the primary tumour may provide better treatment guidance for patients with metastatic disease. Metastatic tumours are however difficult to assess using currently available techniques. We propose to clinically validate molecular methods and develop a clinically validated system for characterization of circulating tumour cells, used as proxies for the metastatic tumour, to guide treatment decisions in women with metastatic breast cancer. Two unique methods will be combined, single cell molecular characterization and improved circulating tumour cell sampling and identification, provided by the participating SMEs. Together with leading academic scientists and one industrial partner, the consortium is ideally suited to evaluate the clinical utility of the proposed methods and to validate the methods in order to reach the final development stage before the product will be introduced to the market, which constitutes a significant commercial potential for the SME-partners in the project.

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