Abstract: 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

Abstract: 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

Abstract: 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

Abstract: 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 subst

Abstract: The present invention relates to a proximity-probe based detection assay for detecting an analyte in a sample and in particular to a method that comprises the use of 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 directly or indirectly, wherein the nucleic acid domain of at least one of said proximity probes comprises a hairpin structure that can be unfolded by cleavage of the nucleic acid domain to generate at least one ligatable free end or region of complementarity to another nucleic acid molecule in said sample, wherein when the probes bind to said analyte unfolding said hairpin structure allows the nucleic acid domains of said at least first and second proximity probes to interact directly or indirectly.

Abstract: 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

Abstract: The invention provides a method for detecting a biomolecular feature (a protein, protein complex, or modified protein such as a phosphorylated protein) by a modified Western blot type of assay, which method either electrophoretic gel separation followed by transfer, or direct spotting of a sample containing the biomolecular feature onto a membrane; providing a proximity probe pair, each probe comprising a binding moiety with affinity for a different binding site on the bio molecular feature and a reactive oligonucleotide, coupled thereto; binding the proximity probes to their respective binding sites on the biomolecular feature through the binding moiety, adding a splint oligonucleotide and a backbone oligonucleotide which are complementary to the reactive oligonucleotide pair, and allowing hybridization among them; ligating the hybridized DNA oligonucleotides to create a circularized DNA molecule when both probes bind sufficiently close to each other on the bio molecular feature, amplifying the circularized

Abstract: The present invention relates to methods for detecting and quantifying an analyte in a sample, principally in proximity probe assays, and in particular to an improvement in such methods to extend the dynamic range of detection, which is particularly advantageous for the detection and quantification of an analyte where the concentration range of the analyte in said sample is unknown and/or the range is likely to be broad, said method comprising: (i) contacting said sample with at least a pair of proximity probes each comprising a proteinaceous target-binding domain coupled to a nucleic acid domain such that said nucleic acid domains may be allowed to interact directly or indirectly when the proximity probes have bound in proximity to their respective target, said target being either the analyte or a binding partner for the analyte; (ii) further contacting said sample with at least one set of markers which function to extend the dynamic range of detection of the method, wherein said set comprises at least two m

Abstract: A method for detecting an analyte in a sample, comprising (a) contacting the sample with at least one set of at least first, second and third proximity probes, which probes each include an analyte-binding domain and a nucleic acid domain and can simultaneously bind to the analyte, the nucleic acid domain of the third proximity probe being a splint which is capable of hybridizing at least to the nucleic acid domains of the first and second proximity probes, wherein when all of the at least three proximity probes bind to the analyte, the nucleic acid domains of the first and second proximity probes are conjugatable by means of an interaction mediated by the hybridized splint of the third proximity probe; (b) conjugating the nucleic acids, of the first and second proximity probes; and (c) detecting the conjugation. Also provided is a kit for use in such a method.

Abstract: The present invention relates to production of oligonucleotides using rolling circle replication, wherein synthesised multimeric oligonucleotides are reduced to mononucleotides using a nicking cassette. Thus, the invention provides a method for the production of oligonucleotides, enabling efficient amplification of oligonucleotides at lengths up to at least 1000 nucleotides and in high amounts contained within a nicking cassette.

Abstract: This invention relates to methods, reagents and kits for enriching nucleic acid sequences. More particularly, the present invention relates to methods, reagents and kits for sample preparation including sample modification, sample enrichment and amplification.

Abstract: The present invention relates to a method for detecting an analyte in a sample, said method comprising: (a) contacting said sample with at least one set of at least first and second proximity probes, wherein said probes each comprise an analyte-binding moiety and can simultaneously bind to the analyte, and wherein (i) said first proximity probe comprises a nucleic acid moiety attached at one end to the analyte-binding moiety, wherein a circular or circularizable oligonucleotide is hybridized to said nucleic acid moiety before, during or after said contacting step; and (ii) said second proximity probe comprises an enzyme moiety, attached to the analyte-binding moiety, capable of directly or indirectly enabling rolling circle amplification (RCA) of the circular or, when it is circularized, of the circularizable oligonucleotide hybridized to the nucleic acid moiety of the first proximity probe, wherein said RCA is primed by said nucleic acid moiety of said first proximity probe; (b) if necessary, circularizing s

Abstract: The present invention relates to sensitive, rapid and convenient assays for detection and or quantification of one or more analyte(s) in solution using multivalent proximity probes. The proximity probes each comprise several binding moieties, such as antibodies, and associated nucleic acid(s). When the binding moieties have bound to their analyte(s), the nucleic acids on opposite proximity probes interact with each other and a signal is generated based on this interaction. The multivalent proximity probes are especially valuable for highly sensitive and specific protein detection.

Abstract: Methods of detecting affinity interactions between at least two molecules of interest are provided. The method comprises: a. forming a plurality of interactors by coupling each molecule of interest with at least one nucleic acid moiety comprising an identification sequence element and at an association element; b. promoting an association between at least two nucleic acid moieties from different interactors to form a plurality of unique associated oligonucleotides, wherein each nucleic acid moiety may form more than one unique associated oligonucleotide, and wherein each unique associated oligonucleotide comprises at least two identification sequence elements derived from the at least two nucleic acid moieties; c. selecting the plurality of unique associated oligonucleotides; and d. subjecting the selected associated oligonucleotides to an analysis that permits detection of the at least two identification sequence elements.

Abstract: A method for amplifying a plurality of target sequences that minimizes amplification artefacts is provided. A sample of interest is fragmented into fragments, where each fragment that includes a target sequence has at least one defined end sequence. Selector constructs, all comprising a primer pair motif and each individual selector comprising one or two protruding ends complementary to the defined end sequences of the fragments containing the target sequences, are brought in contact with the fragments. After ligation, the selected target sequences are amplified in parallel using a primer-pair specific for the primer-pair motif common to the selectors.

Abstract: A nucleic acid amplification method, and probes for use within the method are described.

Abstract: The present invention relates to improved methods for probing of specific nucleic acids using circularizable probes designed such that they report the presence of a target sequence by allowing a detectable moiety to remain bound if an only if the probe has been cyclized in a target-dependent linking reaction. The invention may be used for distinction between sequence specific variations of nucleic acids.

Abstract: The present invention relates to sensitive, rapid and convenient assays for detection and/or quantification of one or several analyte(s) in solution using so called proximity probes. The proximity probes comprise a binding moiety and a nucleic acid. The nucleic acid from one proximity probe is only capable of interaction with the nucleic acid from the other proximity probe when these are in close proximity, i.e. have bound to the analytes for which they are specific. The present invention relates to methods and kits for proximity probing and are performed in solution without the need of a solid phase.

Abstract: A nucleic acid amplification method, and probes for use within the method are described.