Other

Olink Ab

Navinci Diagnostics Ab

Quiatech Ab

Pharmacia Biotech Ab

Olink Bioscience Ab

Agilent Technologies, Inc.

Moleculink Ab

Ulf Landegren

Marek Kwiatkowski

Mats Nilsson

Mats Gullberg

Rachel Yuan Nong

Ola Söderberg

Ove Öhman

Simon Fredriksson

Olof John Ericsson

Henrik Johansson

Magnus Isaksson

Allan Asp

Irene Weibrecht

Peder Carstenius

Alexander Khorlin

Maritha Mendel-Hartvig

Yanling Liu

Mats Sundvall

Johan Björkesten

Irene Helbing

Arild Lagerkvist

Lei Chen

Di Wu

Yuan Nong

Caroline Gallant

Åsa Hagner-McWhirter

Daniel Ivansson

Nucleic acid probes for detection of a target nucleic acid molecule by an RCA reaction in the presence of the target nucleic acid molecule, comprise a first circular template strand which is capable of acting as a template for RCA, and is protected from RCA in the absence of the target nucleic acid molecule by at least a second protector strand which comprises a region of complementarity to the first template strand and is hybridised thereto to form a double-stranded circular structure containing the first template strand inside the protector strand(s). At least one of the second and/or any further protector strands comprises a target binding site, such that upon binding of the probe to the target nucleic acid molecule the probe is able to undergo a strand displacement reaction which allows RCA of the first template strand. Methods of detecting target analytes use such probes.

Double-stranded circle probes

The present invention relates to a method for selecting a target region of interest (ROI) in a target nucleic acid molecule using a nucleic acid probe comprising a 3' sequence capable of hybridising to a target nucleic acid molecule and acting as a primer for the production of a complement of the target ROI (i.e. by target templated extension of the primer), and a sequence capable of templating the circularisation and ligation of the extended probe comprising the reverse complement of the target ROI and a portion of the probe. The circularised molecule thus obtained contains the reverse complement of the target ROI and may be subjected to further analysis and/or amplification etc. The probe may be provided as an oligonucleotide comprising a stem-loop structure or as a partially double-stranded construct and comprises a single-stranded 3&#x2032; end region containing the target-binding site. A second binding site provided in the probe serves as the ligation template for circularisation, and the stem-loop structure, if present, is cleaved to render the second binding site available for hybridisation to the target complement. Also provided are probes and kits for carrying out such a method.

Method for selecting a target nucleic acid sequence

The present invention relates to a method of selecting a target region of interest (ROI) in a target nucleic acid molecule using a nucleic acid probe comprising sequences capable of directing the cleavage of a target nucleic acid molecule to release a fragment comprising the ROI and sequences capable of templating the circularisation and ligation of the target fragment. The circularised molecule thus obtained contains the selected ROI and may be subjected to further analysis and/or amplification etc. Also provided are probes and kits for use in such methods.

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.

Unfolding proximity probes and methods for the use thereof

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.

Multiplexed proximity ligation assay

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.

Localised RCA-based amplification method

The present invention provides a method for detecting or enriching for a target deoxyribonucleic acid (DNA) present in a nucleic acid sample, said method comprising: (a) fragmenting a nucleic acid sample to generate nucleic acid fragments including a target fragment containing said target DNA and non-specifically ligating an adaptor sequence to an end of said fragments; (b) rendering said fragments at least partially single-stranded; (c) contacting the at least partially single-stranded fragments of step (b) with oligonucleotides A and B of a single target-specific nucleic acid probe; (d) ligating oligonucleotide B of said probe to the part of the single-stranded portion of said target fragment which is hybridised to oligonucleotide A of said probe to produce a probe-target fragment hybrid; and (e) detecting or enriching for said probe-target fragment hybrid.

Probes for specific analysis of nucleic acids

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

Nucleic acid amplification method

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.

Nucleic acid enrichment

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.

Probing of specific nucleic acids

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.

Methods and kits for proximity probing

Rolling circle replication of a padlock primer is inhibited when it is hybridised to a target nucleic acid that is long or circular. The invention provides methods of addressing this problem including cutting the target nucleic acid near or preferably at the site which hybridises with the padlock probe, whereby a 3'-end of the cut target nucleic acid acts as a primer for rolling circle replication of the padlock probe. Also included is a method for assaying for a polyepitopic target by the use of two affinity probes each carrying an oligonucleotide tag and of padlock probe for rolling circle replication in association with the two affinity probes.

Growing community of inventors

Uppsala, Sweden

Ulf Landegren