Localised RCA-based amplification method
Inventors
Landegren, Ulf • Chen, Lei • Wu, Di • NONG, Yuan • GALLANT, Caroline
Assignees
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Publication Number
US-10174366-B2
Publication Date
2019-01-08
Expiration Date
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 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.
Core Innovation
The invention relates to a localised rolling circle amplification workflow using at least two rounds of RCA. A first RCA reaction forms a first RCA product comprising tandem repeat units, where each repeat unit is a complementary copy of a template circle. A second RCA reaction then produces a second RCA product that is attached to, and thereby localised on, a product of the first RCA reaction.
Localisation is enforced by directly or indirectly hybridising multiple repeat units of the first RCA product with first probes that comprise or provide a RCA primer for the second RCA reaction. The method includes constraints on priming and extension in the second RCA reaction so that the first probes and the RCA primer are not able to prime extension using the first RCA product as template, or if priming and extension occurs, the extension is unable to displace first probes hybridised to the first RCA product.
The workflow further specifies how a RCA template for the second RCA reaction is provided, either comprised in or provided by each of the first probes, or provided separately from each of the first probes. Additional probe and oligonucleotide designs inhibit unwanted processes such as 3′ exonuclease degradation, unwanted extension, or displacement of probes hybridised to the first RCA product, including modified regions, modifications at or near a 3′ end, modifications at or near a hybridised ligatable 5′ end, and blocking oligonucleotides.
The disclosed approach uses signal amplification from the second RCA in a manner localised to the first RCA product sites defined by tandem repeat units and multiple hybridised probes. It supports detection of the first product and/or the second product, and is described in assay formats including homogeneous and solid-phase or immobilised first RCA product formats.
Claims Coverage
The independent claim defines a localised two-round RCA method with at least two rounds of RCA, where attachment of a second RCA product to a first RCA product is enforced by multi-repeat hybridisation, controlled priming, extension and displacement behavior, and a second-round RCA template arrangement. The claim includes inventive-feature control over probe and RCA primer function, including conditional priming constraints and inhibition of exonuclease, extension and displacement, and over template provisioning for the second RCA reaction.
Localized two-round RCA attachment via repeat-defined probe binding
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.
First RCA product of tandem repeat units
Providing a first RCA product comprising tandem repeat units, wherein each repeat unit is a complementary copy of a template circle of a first RCA reaction.
Multiple first probes hybridise to repeat units and provide second-round RCA primer
Directly or indirectly hybridising to multiple repeat units of the first RCA product first probes which comprise or provide a RCA primer for a second RCA reaction.
Controlled priming and displacement prevention for second RCA
Performing a second RCA reaction using the RCA primer to form a second RCA product, wherein the first probes and the RCA primer are not able to prime extension using the first RCA product as template, or are able to prime extension but extension is unable to displace first probes hybridised to the first RCA product; and wherein probe modifications and/or blocking oligonucleotides inhibit 3′ exonuclease degradation, extension, or displacement.
Maintained RCA primer hybridisation ensures attachment of second RCA product
The direct or indirect hybridisation of the RCA primer to the first RCA product is maintained and, by virtue of that hybridisation, the second RCA product is attached to the first RCA product.
Second-round RCA template is provided by first probes or separately
A RCA template for the second RCA reaction is comprised in or provided by each of the first probes, or is provided separately from each of the first probes.
The claim coverage centers on a localisation-enforced two-round RCA where tandem repeat units in a first RCA product recruit multiple first probes that carry or provide a second-round RCA primer. Second-round product attachment is secured by limiting priming and preventing displacement or exonuclease-related probe loss using specified probe modifications and/or blocking oligonucleotides, while a second-round RCA template is either included in each first probe or provided separately.
Stated Advantages
Enhanced sensitivity.
Localized amplification.
Improved signal/noise ratio.
Potential low-magnification readouts due to signal amplification.
Potential digital counting / analogue to digital PCR.
Precise localization by first-product detection.
Ability to detect rare mutations, including KRAS.
Potential detection in circulating tumour cells (CTC).
Assay format options are provided, including homogeneous and solid-phase or immobilised first RCA formats.
A timing benefit is described by starting the second RCA during first RCA formation.
Unwanted priming or extension using the first RCA product as template is avoided, or extension is unable to displace probes hybridised to the first RCA product.
Unwanted displacement of probes hybridised to the first RCA product is avoided using blocking oligonucleotides.
Localization is maintained by maintaining direct or indirect hybridisation of the RCA primer to the first RCA product.
Second RCA product is localised by attachment to a product of a first RCA reaction.
Documented Applications
Detection of an analyte in a sample by generating a first RCA product via a first RCA reaction and detecting the second RCA product to detect the analyte, optionally also detecting the first RCA product.
Mutation detection, including rare mutations such as KRAS.
Detection in circulating tumour cells (CTC).
Digital counting.
Detection readouts including flow cytometry and CyTOF.
Detection modalities including fluorescence microscopy and other detection modalities described in the document.
Solid-phase immobilisation formats, including streptavidin-coated slides.
Solid-phase or immobilised assay formats in which the first RCA product is immobilised and unbound components are removed before the second RCA reaction.
Nucleic acid detection using a localized super RCA approach that detects the second RCA product and optionally uses the first RCA product.
Multiplexing using reporter or marker domains in which multiple analytes can be detected via distinct reporter features.
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