Method of detecting an analyte
The present disclosure in some aspects relates to methods for detecting a target analyte in a sample by a method comprising rolling circle amplification (RCA) and hybridisation chain reaction (HCR).
1 . A method for detecting an analyte in a sample, comprising:
(i) providing a rolling circle amplification product (RCP) in the sample, wherein the RCP comprises multiple repeat copies of a barcode sequence indicative of the analyte;
(ii) performing a hybridization chain reaction (HCR) wherein the HCR is performed using HCR monomers in the form of hairpins, wherein the HCR is initiated by an HCR initiator which hybridizes to the RCP at the barcode sequence; and
(iii) detecting the product of the HCR at a location in the sample, thereby detecting the analyte at the location in the sample.
2 . The method of claim 1 , wherein the analyte is a nucleic acid molecule, a lipid or a protein.
3 . The method of claim 2 , wherein the nucleic acid molecule is DNA or RNA.
4 . The method of claim 1 , wherein the sample comprises cells.
5 . The method of claim 4 , wherein the cells are immobilized and/or fixed.
6 . The method of claim 1 , wherein the sample is a tissue sample and the analyte is detected in situ in the tissue sample.
7 . The method of claim 1 , wherein the analyte is detected in single cells.
8 . The method of claim 1 , wherein the analyte is a nucleic acid molecule, and wherein a circularizable probe comprising a complement of the barcode sequence is hybridized to the analyte and is circularized to form a rolling circle amplification (RCA) template, which is subjected to RCA to form the RCP.
9 . The method of claim 1 , wherein the method is used to detect multiple analytes in the sample, and at least one RCP is provided for each analyte.
10 . The method of claim 1 , wherein the HCR monomers comprise first HCR monomers and second HCR monomers, wherein the first HCR monomers comprise a toehold region which is complementary to the HCR initiator and to the interacting region in the second HCR monomers, and the toehold region in the second HCR monomers is complementary to the interacting region in the first HCR monomers.
11 . The method of claim 10 , wherein at least a fraction of at least one of the HCR monomers is labelled with a detectable label.
12 . The method of claim 9 , wherein for each RCP, multiple sequential HCRs are performed, and the respective HCR products are detected together to provide for detection of the analyte.
13 . The method of claim 12 , wherein the sequential HCRs are performed using differentially labelled HCR monomer sets, such that each RCP is combinatorially labelled to detect the analyte.
14 . The method of claim 1 , wherein the analyte is one of multiple analytes in the sample, wherein the multiple analytes are detected by providing an RCP for each analyte, and wherein the method is performed sequentially in cycles and a different set of RCPs is detected in each cycle, to detect a different set of analytes.
15 . The method of claim 14 , wherein in a first cycle, a first set of RCPs is detected using a first set of HCR initiators each specific for a different member of the first set of RCPs and the HCR products are removed after the detection, and in a second or subsequent cycle, a second or subsequent set of RCPs is detected using a second or subsequent set of HCR initiators each specific for a different member of the first or subsequent set of RCPs, and the HCR products are removed after the detection.
16 . The method of claim 15 , wherein in each cycle the same sets of HCR monomers are used.
17 . The method of claim 1 , wherein the barcode sequence is between 5 and 50 nucleotides in length.
18 . The method of claim 1 , wherein the rolling circle amplification for generating the RCP is performed for no more than 3 hours.
19 . The method of claim 1 , wherein the rolling circle amplification for generating the RCP is performed for no more than 2 hours.
20 . The method of claim 1 , wherein the rolling circle amplification for generating the RCP is performed for between 2 hours and 3 hours, and the HCR is performed for at least 30 minutes.
21 . The method of claim 1 , wherein the rolling circle amplification for generating the RCP is performed for about 2 hours, and the HCR is performed for about 1 hour.