Office Action 1: Non-Final Rejection (2024-09-16)
Rejection types: 102 - Lack of Novelty, 103 - Obviousness, 112 - Written Description/Enablement

CLAIMS:
AMENDMENTS TO THE CLAIMS

This listing of claims will replace all prior versions, and listing, of claims in the application.

LISTING OF CLAIMS:

1-98. (Cancelled)297

99\. (New) A method of analyzing a biological sample, comprising:a) hybridizing a nucleic acid oligonucleotide to an oligonucleotide hybridization region in a target ribonucleic acid (RNA) in the biological sample;
b) cleaving the target RNA with an RNase H in the oligonucleotide hybridization region to generate a cleaved target RNA;
c) hybridizing a circular probe or a circularizable probe or probe set comprising a target recognition sequence complementary to a target sequence in the target RNA;
wherein the oligonucleotide hybridization region is adjacent to the 3' end of the target sequence or is overlapping with the 3' end of the target sequence;d) performing rolling circle amplification of the circular probe or of a circularized probe generated from the circularizable probe or probe set to generate a rolling circle amplification product (RCP) using the cleaved target RNA as a primer; and
e) detecting the RCP in the biological sample.

100\. (New) The method of claim 99, wherein the biological sample is contacted with the nucleic acid oligonucleotide and with the RNase H simultaneously before contacting the biological sample with the circular probe or the circularizable probe or probe set.

101\. (New) The method of claim 99, wherein the method comprises washing the biological sample after cleaving the target RNA with the RNase H and before contacting the biological sample with the circular probe or the circularizable probe or probe set.

102\. (New) The method of claim 99, wherein the oligonucleotide hybridization region and the target sequence overlap by between 1 and 20 nucleotides.

103\. (New) The method of claim 99, wherein the method does not comprise contacting the biological sample with a DNA primer that hybridizes to the circular probe or the circularized probe.

104\. (New) The method of claim 99, wherein the target recognition sequence of the circularizable probe or probe set is a split recognition sequence comprising a first hybridization region having a first ligatable end and a second hybridization region having a second ligatable end,wherein the first hybridization region hybridizes to a 5' portion of the target sequence, and the second hybridization region hybridizes to a 3' portion of the target sequence, and the method comprises ligating the first ligatable end to the second ligatable end to generate the circularized probe.

105\. (New) The method of claim 99, wherein the RNase H comprises an RNase H1 and/or an RNase H2.

106\. (New) The method of claim 99, wherein contacting the biological sample with the RNase H comprises contacting the biological sample with between 0.5 enzyme units (U) and 50 U of the RNase H.

107\. (New) The method of claim 99, wherein the method comprises imaging the biological sample to detect the RCP.

108\. (New) The method of claim 107, wherein the imaging comprises detecting a signal associated with a fluorescently labeled probe that directly or indirectly binds to the RCP.

109\. (New) The method of claim 108, wherein a sequence of the RCP is analyzed by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof.

110\. (New) The method of claim 99, wherein the oligonucleotide hybridization region and the 3' portion of the target sequence overlap by about 6 to about 10 nucleotides.

111\. (New) The method of claim 99, wherein the method comprises contacting the biological sample with a polymerase in a first reaction mixture comprising a non-catalytic cofactor of the polymerase, and then contacting the biological sample with a second reaction mixture comprising a catalytic cofactor of the polymerase to perform the rolling circle amplification.

112\. (New) The method of claim 111, wherein the di-cation that is not a co-factor of the polymerase is barium, strontium, iron, cobalt, nickel, tin, zinc, or europium.

113\. (New) The method of claim 111, wherein the non-catalytic cofactor of the polymerase is calcium or strontium.

114\. (New) The method of claim 111, wherein the non-catalytic cofactor of the polymerase is strontium.

115\. (New) The method of claim 99, wherein a sequence of the RCP is analyzed at a location in the biological sample or a matrix embedding the biological sample.

116\. (New) The method of claim 99, comprising reacting at least one RNA in the biological sample with a polynucleotide kinase (PNK).

117\. (New) The method of claim 116, wherein the method comprises reacting at least one RNA in the biological sample with the PNK after contacting the biological sample with the nucleic acid oligonucleotide and the RNase H.

118\. (New) The method of claim 117, wherein the PNK is a T4 Polynucleotide Kinase (T4 PNK) or a T7 Polynucleotide Kinase (T7-PNK).

119\. (New) The method of claim 99, wherein the nucleic acid oligonucleotide is 20 to 35 nucleotides in length.

120\. (New) A method of analyzing a biological sample, comprising:a) contacting the biological sample with a plurality of nucleic acid oligonucleotides, wherein a first oligonucleotide of the plurality hybridizes to a first oligonucleotide hybridization region in a first target ribonucleic acid (RNA) in the biological sample, and a second

oligonucleotide of the plurality hybridizes to a second oligonucleotide hybridization region in a second target RNA in the biological sample;b) cleaving the first and second target RNAs with an RNase H in their respective oligonucleotide hybridization regions to generate a first cleaved target RNA and a second cleaved target RNA;c) contacting the biological sample with a plurality of circular probes or circularizable probes or probe sets,
wherein a first circular probe or first circularizable probe or probe set of the plurality comprises a first target recognition sequence complementary to a first target sequence in the first target RNA,
wherein a second circular probe or second circularizable probe or probe set of the plurality comprises a second target recognition sequence complementary to a second target sequence in the second target RNA,
wherein the first and second circular probe or the first and second circularizable probe or probe set hybridize to their respective target RNAs,
wherein the first oligonucleotide hybridization region is overlapping with the 3' end of the first target sequence, and
wherein the second oligonucleotide hybridization region is overlapping with the 3' end of the second target sequence;
d) performing rolling circle amplification of the first and second circular probe or of a first and second circularized probe generated from the first and second circularizable probes or probe sets to generate a first and second rolling circle amplification product (RCP) using the first cleaved target RNA and the second cleaved target RNA as primers; ande) detecting the first and second RCPs in the biological sample.

121\. (New) The method of claim 120, wherein the method comprises imaging the biological sample to detect the first and second RCPs.

122\. (New) The method of claim 120, wherein a sequence of the first and second RCPs is analyzed by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof.

123\. (New) The method of claim 120, wherein the first oligonucleotide hybridization region and the 3' portion of the first target RNA overlap by about 6 to about 10 nucleotides and the second oligonucleotide hybridization region and the 3' portion of the second target RNA overlap by about 6 to about 10 nucleotides.

124\. (New) The method of claim 120, wherein the first oligonucleotide and the second oligonucleotide are each 20 to 35 nucleotides in length.

125\. (New) The method of claim 120, wherein the method comprises contacting the biological sample with a polymerase in a first reaction mixture comprising a non-catalytic cofactor of the polymerase, and then contacting the biological sample with a second reaction mixture comprising a catalytic cofactor of the polymerase to perform the rolling circle amplification.

OFFICE ACTION:
Detailed Action
►	The applicant's Preliminary Amendment filed 30 JUL 2024 has been entered.  Following the entry of the Preliminary Amendment, Claim(s) 99-125 is/are pending.
►  	The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . In the event the determination of the status of the application as subject to AIA  35 U.S.C. 102 and 103 (or as subject to pre-AIA  35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
35 U.S.C. 112(b)/ 112 (pre-AIA ), second paragraph
►	The following is a quotation of 35 U.S.C. 112(b):
(b)  CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim Rejection(s) under 35 U.S.C. 112(b)
►	Claim(s) 112 is/are rejected under 35 U.S.C. 112(b), as being indefinite in that it fails to point out what is included or excluded by the claim language.
Claim 112 is indefinite because the phrase “the di-cation” on line 1 lacks proper antecedent basis in Claim 99 and/or Claim 111.
35 U.S.C. 102
►	The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that may form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
35 U.S.C. 103
►	The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
►	This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary.  Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim Rejection(s) under 35 U.S.C. 102
►	Claim 99, 103-106 is/are rejected under 35 U.S.C. 102(a)(1)  as being anticipated by Takahashi et al. [Science Reports 8:7770(2018) – hereinafter “Takahashi”].
Claim 99 is drawn to a method of analyzing a biological sample, comprising: a) hybridizing a nucleic acid oligonucleotide to an oligonucleotide hybridization region in a target ribonucleic acid (RNA) in the biological sample; b) cleaving the target RNA with an RNase H in the oligonucleotide hybridization region to generate a cleaved target RNA; c) hybridizing a circular probe or a circularizable probe or probe set comprising a target recognition sequence complementary to a target sequence in the target RNA; wherein the oligonucleotide hybridization region is adjacent to the 3’ end of the target sequence or is overlapping with the 3’ end of the target sequence; d) performing rolling circle amplification of the circular probe or of a circularized probe generated from the circularizable probe or probe set to generate a rolling circle amplification product (RCP) using the cleaved target RNA as a primer; and e) detecting the RCP in the biological sample.
Regarding Instant Claim 99, Takahashi teach a method of detecting  a target RNA (e.g. mRNA) they term “RNaseH-assisted RNA-primed rolling circle amplification (hereinafter - RNase Assisted RPRCA) which comprises all of the limitations of Claim 99. For example consider Fig. 1 and Fig. 3A. There, Takahashi teach “hybridizing a circular probe or a circularizable probe or probe set comprising a target recognition sequence complementary to a target sequence in the target RNA; wherein the oligonucleotide hybridization region is adjacent to the 3’ end of the target sequence or is overlapping with the 3’ end of the target sequence (i.e. step “c” of Claim 99); Takahashi further teach teach d) performing rolling circle amplification of the circular probe or of a circularized probe generated from the circularizable probe or probe set to generate a rolling circle amplification product (RCP) using the cleaved target RNA as a primer; and e) detecting the RCP in the biological sample.  As regards step “a)”, note that Takahashi teach using multiple different probes  targeting different regions of target mRNA (e.g. P1 and P5 of Fig. 3A) wherein P5 targets the 3’ end while P1 hybridizes upstream (i.e. torwards the 5’ end of the target RNA. P1 constitutes the nucleic acid oligonucleotide of step “a)”
Claim 103 is drawn to an embodiment of the method of Claim 99,  wherein the method does not comprise contacting the biological sample with a DNA primer that hybridizes to the circular probe or the circularized probe.
No DNA primer is used in Fig. 1 of Takahashi.
Regarding Claim 104 Takahashi teaches the limitation(s) recited by Claim 104, see especially Fig.1.
Regarding Claim 106.  Takahashi teaches the use of RNase H are very low concentrations e.g. 0.6 units/reaction, the para bridging pp. 4-5, thereby meeting the limitation of Claim 106 which reads  “between 0.5 U -50U”.
Claim Rejection(s) under 35 U.S.C. 103
►	Claim(s_100-101, 105-106 and 119 is/are  rejected under 35 U.S.C. 103 as being unpatentable over Takahashi.
Regarding Claims 100-101, the selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results.
Regarding Claim 105 Takahashi teaches the use of RNaseH (presumably RNase H1). Regardless, several RNase H molecules  were known including RNase H1 and RNase H2 (?Official Notice – see for example Hyjek et al. [DNA Repair 84 (2019)102672].
Regarding Claim 106.  Takahashi teaches the use of RNase H are very low concentrations e.g. 0.6 units, the para bridging pp. 4-5. Regardless,  where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
As regards Claim 119, Takahashi teach 70-mer circularizable probes but do not teach  don’t teach that P1, for example, (i.e. the oligo nucleotide of Claim 99) should be between 20-35 nucleotides in length. However, where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
►	Claim(s) 107-109 and 115 is/are  rejected under 35 U.S.C. 103 as being unpatentable over Takahashi as applied above against Claim 99 and further in view of Kűhnmund et al.[ US 2021/0198727 – hereinafter “Kűhnmund”].
Claim 107 is drawn to an embodiment of the method of Claim 99,  wherein the method further comprises imaging the biological same  to detect the RCP.
Takahashi teach detection  of their RCPs by fluorescence detection, see especially Fig. 1, but fail to teach detecting RCP by imaging their biological sample. However,  it was known to perform RCA-type reaction in situ (i.e. within biological sample/tissue) with subsequent detection of the RCP using fluorescently labeled probes which detection comprises imaging of said sample. Consider for example Fig.1 and para 34-37 and 141 in  Kűhnmund.
As regards Claims 108-109 and 115 see Kűhnmund  and the para(s) cited above.
►	Claim(s) 116-118 is/are  rejected under 35 U.S.C. 103 as being unpatentable over Takahashi as applied above against Claim 99 and further in view of Wang et al. [EMBO J 14:2873-3880(2002) – hereinafter “Wang”].
Regarding Claims 116-117, Takahashi  does not teach the use of PNKs However, PNKs were known as was the use PNKs in nucleic acid based assays, see at least the abstract in Wang. Absent an unexpected result, it would have been prima facie obvious to the PHOSITA to modify the method of  Takahashi  wherein a PNK treatment steps is included in said assay following the RNase H step. The   PHOSITA would have been motivated to make this modification in order to repair any damaged ends resulting from the RNaseH nicking step.
As regards Claim 118, Wang teach T4 PNK. See at least the abstract.
Claim Objections
►	Claim(s) 102 and 110-114 is /are objected to as being dependent upon a rejected base claim, but would be allowable over the prior art of record if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Allowable Subject Matter
►	Claim(s) 120-125 are novel and unobvious over the prior art now of record. In particular Takahashi does not teach the limitation recited in “c) of Claim 120 which reads  “wherein the first oligonucleotide hybridization region is overlapping with the 3’ end of the first target sequence, and wherein the second oligonucleotide hybridization region is overlapping with the 3’ end of the second target sequence”.
Conclusion
C.


NEXT OFFICE ACTION: Notice of Allowability
►	Claim(s) 99-125  as amended in the paper(s) filed  13 DEC 2024 is/are pending in this application.
►	The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . In the event the determination of the status of the application as subject to AIA  35 U.S.C. 102 and 103 (or as subject to pre-AIA  35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
Reason(s) for Allowance
►	Claim(s) 99-125 is/are deemed to be allowable in light of the paper(s) filed 13 DEC 2024 and the persuasive argument(s) therein. The Claims have now been renumbered as Claims 1-27 in the same order as presented by the applicant.
Conclusion
C.

CITED REFERENCE SUMMARIES: Ref US20210198727A1: Claims:
A method for detecting an analyte in a sample, comprising: (i) providing a rolling circle amplification product (RCP), wherein said RCP comprises multiple repeat copies of a marker sequence indicative of said analyte; (ii) performing a hybridization chain reaction (HCR), wherein said HCR reaction is initiated by an HCR initiator which directly or indirectly hybridizes to said marker sequence or which is comprised in said marker sequence; and (iii) detecting the product of the HCR reaction, thereby detecting the analyte.
(i) providing a rolling circle amplification product (RCP), wherein said RCP comprises multiple repeat copies of a marker sequence indicative of said analyte;
(ii) performing a hybridization chain reaction (HCR), wherein said HCR reaction is initiated by an HCR initiator which directly or indirectly hybridizes to said marker sequence or which is comprised in said marker sequence; and
(iii) detecting the product of the HCR reaction, thereby detecting the analyte.
The method of claim 1 , wherein the analyte is a nucleic acid molecule, a lipid or a protein.
The method of claim 2 , wherein the nucleic acid molecule is DNA or RNA.
The method of claim 1 , wherein the sample comprises cells.
The method of claim 4 , wherein the cells are immobilized, fixed or in suspension.
The method of claim 1 , wherein the analyte is detected in situ in a tissue sample.
The method of claim 1 , wherein the analyte is detected in single cells.
The method of claim 1 , wherein a padlock probe comprising a complement of the marker 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.
The method of claim 1 , wherein the method is used to detect multiple analytes in a sample, and at least one RCP is provided for each analyte.
The method of claim 1 , wherein HCR is performed using HCR monomers in the form of hairpins, 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.
The method of claim 1 , wherein the HCR initiator is provided separately to the RCP and comprises a marker-binding domain capable of hybridizing to the marker sequence in the RCP, and wherein the HCR initiator is hybridized to the RCP before the HCR reaction is performed.
The method of claim 1 , wherein at least a fraction of at least one of the HCR monomers in an HCR reaction is labelled with a detectable label.
The method of claim 9 , wherein for each RCP, multiple sequential HCR reactions are performed, and the respective HCR products are detected together to provide for detection of the analyte.
The method of claim 15 , wherein sequential HCR reactions are performed using differentially labelled HCR monomer sets, such that each RCP is combinatorially labelled to detect the analyte.
The method of claim 1 , for detecting multiple analytes in sample, wherein a RCP is provided for each analyte, and said method is performed sequentially in cycles and a different set of RCPs is detected in each cycle, to detect a different set of analytes.
The method of claim 17 , 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.
The method of claim 18 , wherein in each cycle the same sets of HCR monomers are used.
The method of claim 1 , wherein the marker sequence is between 5 and 50 nucleotides in length.
- 23 . (canceled)
The method of claim 1 , wherein the rolling circle amplification for generating the RCP is performed for no more than 3 hours.
The method of claim 1 , wherein the rolling circle amplification for generating the RCP is performed for no more than 2 hours.
Ref US20230374573A1: Claims:
A method for analyzing a biological sample, comprising: (a) contacting the biological sample with a circular or circularizable probe, wherein the circular or circularizable probe comprises a hybridization region that hybridizes to a target RNA sequence in a target RNA in the biological sample to form a duplex, (b) contacting the biological sample with a primer oligonucleotide, a ribonuclease H (RNase H), and a polymerase, wherein the primer oligonucleotide hybridizes to the circular or circularizable probe, and wherein the RNase H cleaves the target RNA in the duplex at one or more sites in the target RNA sequence; (c) performing rolling circle amplification (RCA) using the circular probe or a circularized probe formed by circularizing the circularizable probe as template; and (d) detecting one or more products of the RCA, thereby detecting the target RNA or a sequence thereof in the biological sample.
(a) contacting the biological sample with a circular or circularizable probe,
wherein the circular or circularizable probe comprises a hybridization region that hybridizes to a target RNA sequence in a target RNA in the biological sample to form a duplex,
(b) contacting the biological sample with a primer oligonucleotide, a ribonuclease H (RNase H), and a polymerase, wherein the primer oligonucleotide hybridizes to the circular or circularizable probe, and wherein the RNase H cleaves the target RNA in the duplex at one or more sites in the target RNA sequence;
(c) performing rolling circle amplification (RCA) using the circular probe or a circularized probe formed by circularizing the circularizable probe as template; and
(d) detecting one or more products of the RCA, thereby detecting the target RNA or a sequence thereof in the biological sample.
The method of claim 1 , wherein the RCA is performed using the circularized probe as a template, and wherein the method comprises ligating the circularizable probe to form the circularized probe.
The method of claim 2 , wherein the ligating is performed using the target RNA as a template.
- 6 . (canceled)
The method of claim 1 , wherein the ligating is performed prior to contacting the biological sample with the RNase H.
- 10 . (canceled)
The method of claim 1 , wherein the circular or circularizable probe comprises deoxyribonucleotide residues.
- 13 . (canceled)
The method of claim 1 , wherein the circular or circularizable probe comprises two, three, four, five, six, or more consecutive ribonucleotide residues.
The method of claim 1 , wherein the biological sample is contacted with the primer oligonucleotide, the RNase H, and the polymerase simultaneously.
The method of claim 1 , wherein the biological sample is contacted with the RNase H prior to being contacted with the primer oligonucleotide and the polymerase.
The method of claim 1 , wherein the biological sample is contacted with the RNase H prior to being contacted with the polymerase.
The method of claim 1 , wherein the polymerase is a Phi29 DNA polymerase or a Bst DNA polymerase.
The method of claim 1 , wherein the RNase is RNase H1 or an RNase H2.
- 24 . (canceled)
The method of claim 1 , wherein the contacting with the RNase H comprises: a first step performed in a first buffer that is free or substantially free of a cofactor for the RNase H, and a second step performed in a second buffer comprising the cofactor.
a first step performed in a first buffer that is free or substantially free of a cofactor for the RNase H, and
a second step performed in a second buffer comprising the cofactor.
The method of claim 25 , further comprising: incubating the biological sample with the RNase H in the first buffer, thereby allowing the RNase H to bind the target RNA, the circular or circularizable probe, and/or the duplex; removing excess RNase H from the biological sample; and incubating the biological sample in the second buffer, thereby allowing the RNase H to cleave at one or more sites in the target RNA sequence.
incubating the biological sample with the RNase H in the first buffer, thereby allowing the RNase H to bind the target RNA, the circular or circularizable probe, and/or the duplex;
removing excess RNase H from the biological sample; and
incubating the biological sample in the second buffer, thereby allowing the RNase H to cleave at one or more sites in the target RNA sequence.
The method of any one of claim 1 , wherein the one or more products of the RCA comprise an RCA product generated by using the primer oligonucleotide as a primer.
- 32 . (canceled)
The method of claim 1 , wherein the biological sample is contacted with the RNase H at a concentration of between about 1Ã10 â3 U/Î¼L and about 2Ã10 â2 U/Î¼L.
- 40 . (canceled)
The method of claim 1 , wherein the detecting step comprises contacting the one or more RCA products with a plurality of detectable probes and/or a plurality of intermediate probes in sequential cycles.
- 64 . (canceled)
A method for analyzing a biological sample, comprising: (a) contacting the biological sample with a circularizable probe comprising a hybridization region that hybridizes to a target RNA sequence in a target RNA in the biological sample to form a duplex, (b) ligating the circularizable probe to form a circularized probe; (c) contacting the biological sample with (i) a primer oligonucleotide that hybridizes to the circularized probe, (ii) a ribonuclease H (RNase H), and (iii) a Phi29 DNA polymerase, wherein the contacting with the RNase H comprises: (i) a first step that is performed in a first buffer that is free or substantially free of a cofactor for the RNase H, and (ii) a second step that is performed in a second buffer comprising the cofactor, (d) performing rolling circle amplification (RCA) using the circularized probe as template; and (e) detecting one or more products of the RCA, thereby detecting the target RNA or a sequence thereof in the biological sample.
(a) contacting the biological sample with a circularizable probe comprising a hybridization region that hybridizes to a target RNA sequence in a target RNA in the biological sample to form a duplex,
(b) ligating the circularizable probe to form a circularized probe;
(c) contacting the biological sample with (i) a primer oligonucleotide that hybridizes to the circularized probe, (ii) a ribonuclease H (RNase H), and (iii) a Phi29 DNA polymerase,
wherein the contacting with the RNase H comprises: (i) a first step that is performed in a first buffer that is free or substantially free of a cofactor for the RNase H, and (ii) a second step that is performed in a second buffer comprising the cofactor,
(i) a first step that is performed in a first buffer that is free or substantially free of a cofactor for the RNase H, and
(ii) a second step that is performed in a second buffer comprising the cofactor,
(d) performing rolling circle amplification (RCA) using the circularized probe as template; and
(e) detecting one or more products of the RCA, thereby detecting the target RNA or a sequence thereof in the biological sample.
- 69 . (canceled)
The method of claim 25 , wherein the cofactor for the RNase H is Mg 2+ .
The method of claim 1 , wherein the biological sample is a cell or tissue sample.
The method of claim 71 , wherein the one or more products of the RCA are detected at location(s) in the biological sample immobilized on a solid support.
- 80 . (canceled)
Ref US20210198727: Claims:
1. A method for detecting an analyte in a sample, comprising:
(i) providing a rolling circle amplification product (RCP), wherein said RCP comprises multiple repeat copies of a marker sequence indicative of said analyte; (ii) performing a hybridization chain reaction (HCR), wherein said HCR reaction is initiated by an HCR initiator which directly or indirectly hybridizes to said marker sequence or which is comprised in said marker sequence; and (iii) detecting the product of the HCR reaction, thereby detecting the analyte.
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, fixed or in suspension.
6. The method of claim 1, wherein the analyte is detected in situ in a tissue sample.
7. The method of claim 1, wherein the analyte is detected in single cells.
8. The method of claim 1, wherein a padlock probe comprising a complement of the marker 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 a sample, and at least one RCP is provided for each analyte.
10. (canceled)
11. The method of claim 1, wherein HCR is performed using HCR monomers in the form of hairpins, 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.
12. The method of claim 1, wherein the HCR initiator is provided separately to the RCP and comprises a marker-binding domain capable of hybridizing to the marker sequence in the RCP, and wherein the HCR initiator is hybridized to the RCP before the HCR reaction is performed.
13. The method of claim 1, wherein at least a fraction of at least one of the HCR monomers in an HCR reaction is labelled with a detectable label.
14. (canceled)
15. The method of claim 9, wherein for each RCP, multiple sequential HCR reactions are performed, and the respective HCR products are detected together to provide for detection of the analyte.
16. The method of claim 15, wherein sequential HCR reactions are performed using differentially labelled HCR monomer sets, such that each RCP is combinatorially labelled to detect the analyte.
17. The method of claim 1, for detecting multiple analytes in sample, wherein a RCP is provided for each analyte, and said method is performed sequentially in cycles and a different set of RCPs is detected in each cycle, to detect a different set of analytes.
18. The method of claim 17, 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.
19. The method of claim 18, wherein in each cycle the same sets of HCR monomers are used.
20. The method of claim 1, wherein the marker sequence is between 5 and 50 nucleotides in length.
21-23. (canceled)
24. The method of claim 1, wherein the rolling circle amplification for generating the RCP is performed for no more than 3 hours.
25. The method of claim 1, wherein the rolling circle amplification for generating the RCP is performed for no more than 2 hours.