DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 10/30/2023, 04/01/2026 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Status of Claims
This office action is in response to an amendment filed on May 11, 2026.
Improper Amendment to the Claims: Claim 25 has been improperly amended because it is identified as both cancelled and as an original claim. The cancelled status designation appears to be a clerical error, as Applicant’s remarks indicate that claim 25 remains pending (Remarks, page 7).
In the interest of compact prosecution, however, the examiner will consider the claim listing on its merits. MPEP 714.03 (“Where an amendment substantially responds to the rejections, objections, or requirements in a non-final Office action (and is a bona fide attempt to advance the application to final action) but contains a minor deficiency . . . , the examiner may simply act on the amendment and issue a new (non-final or final) Office action.”) The examiner reserves the right to send a PTOL-324 Notice of Noncompliant Amendment in the event of more severe deficiencies, which may result in loss of patent term.
Claims 1-8, 10, 12-13, 15-16, 18, 20-21, 25, 30 and 32-33 were previously pending. Applicant cancelled claims 20-21 and 30; claims 37-39 are newly added.
Claims 1-8, 10, 12-13, 15-16, 18, 25, 32-33 and 37-39 are currently pending, with claims 15-16, 18, 25, and 32-33 withdrawn.
Claims 1-8, 10, 12-13 and 37-39 are under consideration. This is the first action on the merits.
Election/Restrictions
Applicant’s election without traverse of Group I (claims 1-8, 10, 12-13) in the reply filed on May 11, 2026 is acknowledged 1.
Applicant’s election without traverse of the following species in the reply filed on May 11, 2026 is acknowledged:
Species of Amplification reaction composition: A) The polymerase comprised by the composition is an RT polymerase for reverse transcription of a RNA target nucleic acid (claim 4, 5; see spec [0032] lines 3-4).
Claims 15-16, 18, 25, and 32-33 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention.
Examination on the merits commences on claims 1-8, 10, 12-13 and 37-39.
Priority
The priority date of the instant claims 1-8, 10, 12-13 and 37-39 is 04/29/2021, filling date of the US provisional application NO. 63/181,769.
Claim Interpretation
In evaluating the patentability of the claims presented in this application, claim terms have been given their broadest reasonable interpretation (BRI) consistent with the specification, as understood by one of ordinary skill in the art, as outlined in MPEP§ 2111.
Regarding all claims, terms such as "first" and "second" are interpreted as adjectives for identification purposes to distinguish between repeated instances of an element or limitation, and do not impose any additional features, such as any specific temporal limitation, any sequential order of steps, or any structural or composition differences. See 3M Innovative Props. Co. v. Avery Dennison Corp., 350 F.3d 1365 (Fed. Cir. 2003).
For the purpose of applying prior art, claim 1 recites the terms "5' primer sequence" and "3' primer sequence," which are not expressly defined with any structural features or characteristics by the application's disclosure.
Thus, because the application's disclosure does not define these terms with any structural features that distinguishes it from sequences known in the art, the terms "5' primer sequence" and "3' primer sequence" are interpreted under BRI as encompassing any oligonucleotide sequence.
For the purpose of applying prior art, claim 7 recites the term "rolling circle amplification primer," which is not expressly defined in the application's disclosure.
Therefore, in the absence of any clear structural feature that distinguishes a "rolling circle amplification primer" from oligonucleotides known in the art, this term is interpreted to encompass any oligonucleotide.
For the purpose of applying prior art, claim 39 recites the term "index sequence," which is not expressly defined in the application's disclosure. Therefore, in the absence of any clear structural feature that distinguishes an "index sequence" from sequences known in the art, this term is interpreted to encompass any sequence.
Claim Rejections - 35 USC § 102
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 (i.e., changing from AIA to pre-AIA ) 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.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(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.
Claims 1-6, 8, 12-13 and 38-39 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Glezer (WO2022032195A2 - Spatial sequencing; effective filing date Aug 6, 2020), evidenced by Baker (Baker et al. Review and re-analysis of domain-specific 16S primers. J Microbiol Methods. 2003 Dec;55(3):541-55. doi: 10.1016/j.mimet.2003.08.009. PMID: 14607398).
Regarding claim 1, Glezer teaches a nucleic acid composition (FIGs 1, 3; example 3 [0180-0182] targeted oligonucleotide probes hybridizes to regions which flank the target nucleic acid sequence, for sequencing variable regions in 16s RNA ), comprising:
a first oligonucleotide (FIG. 3A, V2 probe ) comprising
a first 5' primer sequence (FIG. 3A, the first complementary region of V2 probe; [0182]lines 6-8, describes probes design illustrated in FIG. 3 is shown in FIG. 1: “As shown in FIG. 1B, the oligonucleotide probe hybridizes to regions which flank the target nucleic acid sequence or a portion thereof, referred to as the first and the second complementary regions” ; see also FIG. 2),
a first 3' primer sequence(FIG. 3A; [0182]lines 6-8, “second complementary region”; see also FIG. 2), and
a first intervening region disposed between the first 5' primer sequence and the first 3' primer sequence (FIG. 3A, the loop region not hybridized to target RNA);
a second oligonucleotide (FIG. 3A, V4 probe ) comprising
a second 5' primer sequence (FIG. 3A, the first complementary region of V4 probe),
a second 3' primer sequence (FIG. 3A, the second complementary region of V4 probe), and
a second intervening region disposed between the second 5' primer sequence and the second 3' primer sequence (FIG. 3A, the loop region not hybridized to target RNA); and
a target nucleic acid (FIG. 3, 16S RNA),
wherein the first 5' primer sequence and the first 3' primer sequence are complementary to first regions flanking a first target sequence of the target nucleic acid (FIG. 3A; [0182]lines 6-8) and
wherein the second 5' primer sequence and the second 3' primer sequence are complementary to second regions flanking a second target sequence of the target nucleic acid (FIG. 3A; [0182]lines 6-8) such that
the first oligonucleotide, when bound to the target nucleic acid, forms a first looped structure about the first target sequence and
the second oligonucleotide, when bound to the target nucleic acid, forms a second looped structure around the second target sequence.
Regarding claim 2, Glezer teaches the first target sequence and the second target sequence are between 50-350 bases in length by teaching 16S RNA V2 and V4 being the target sequences each flanked by the first and the second complementary regions of a corresponding probe (FIG. 3; [0182]lines 6-8).
Baker, cited by Glezer ([0181] lines 4-5), teaches length information of the 16S rRNA variable regions (Fig. 1). Specifically, Baker teaches that the length of V2 and V4 region are both within the range of 50-350 bases.
Regarding claim 3, Glezer teaches wherein the target nucleic acid is a single- stranded RNA(FIG. 3, 16S RNA).
Regarding claim 4, Glezer teaches a polymerase that is capable of extending the first looped structure between the first 5' primer sequence and the first 3' primer sequence and extending the second looped structure between the second 5' primer sequence and the second 3' primer sequence (FIG. 3A; [0182] lines 8-10 “In the presence of a polymerase (e.g., a non-strand displacing polymerase), the complement to the target sequence is generated by extending from the first complementary region and is ligated (not shown) to the second complementary region to form a circularized oligonucleotide, as found in FIG. IC.”).
Regarding claim 5, Glezer teaches an RT polymerase ([0066] lines 22-24 ; [0131] Thermus thermophilus (Tth) DNA polymerase).
Regarding claim 6, Glezer teaches a ligase ([0131] lines 9-14) that is capable of closing the first looped structure by ligating an extended first 3' end to the first 5' primer sequence and the second looped structure by ligating an extended second 3' end to the second 5' primer sequence .
Regarding claim 8, Glezer teaches a first single-stranded concatenated nucleic acid comprising repeating units, the repeating units comprising the first target sequence and complements of the first 5' primer sequence, the first 3' primer sequence, and the first intervening region ([0182] lines 11-14 “The resulting circularized oligonucleotide is primed with an amplification primer and extended with a strand-displacing polymerase to generate a concatemer containing multiple copies of the target nucleic acid sequence, as shown in FIG. ID.”).
Regarding claim 12, Glezer teaches wherein the first 5' primer sequence binds the target nucleic acid 5' of the first target sequence and wherein the first 3' primer sequence binds 3' of the first target sequence (FIG 1C; FIG. 3; [0182]).
Regarding claim 13, Glezer teaches wherein the second 5' primer sequence binds the target nucleic acid 5' of the second target sequence and wherein the second 3' primer sequence binds 3' of the second target sequence(FIG 1C; FIG. 3; [0182]).
Regarding claim 38, Glezer teaches the first target sequence and the second target sequence are spaced apart on the target nucleic acid (FIG. 3A, V2 and V4 are spaced apart).
Regarding claim 39, Glezer teaches the first intervening region and the second intervening region comprise an index sequence (FIG. 3A, FIG. 2, oligonucleotides comprising sequencing priming site).
Claim Rejections - 35 USC § 103
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 (i.e., changing from AIA to pre-AIA ) 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.
Claims 7, 10 and 37 are rejected under 35 U.S.C. 103 as being unpatentable over Glezer (WO2022032195A2 - Spatial sequencing; effective filing date Aug 6, 2020), in view of Stuelpnagel (US20050214825A1 - Multiplex sample analysis on universal arrays; Published 2005-09-29), as evidenced by
Gong (Gong et al. Multiplex real-time PCR assay combined with rolling circle amplification (MPRP) using universal primers for non-invasive detection of tumor-related mutations. RSC Adv. 2018 Aug 1;8(48):27375-27381. doi: 10.1039/c8ra05259j. PMID: 35540013; PMCID: PMC9083282);
Carrascosa (Carrascosa, et al. "Molecular inversion probe-based SPR biosensing for specific, label-free and real-time detection of regional DNA methylation." Chemical communications 50.27 (2014): 3585-3588).
The teachings of Glezer are recited above and applied as for base claim 1.
Glezer teaches methods of using gap-filling padlock probes (also known as molecular inversion probes) for analysis of nucleic acid targets (see FIGs. 1, 3, 4 for examples). The probe hybridizes to regions which flank the target nucleic acid sequence. In the presence of a polymerase, the complement to the target sequence is generated by extending from the 3' end of probe, and is then ligated to the 5' end to form a circularized oligonucleotide (FIG. 1). A plurality of circularized oligonucleotide probes can be generated on the same target RNA molecule (FIG 4A, 3A).
Claims 7, 10, and 37 recite the following:
7. The composition of claim 1, comprising a rolling circle amplification primer that is specific for a common sequence in the first intervening region and the second intervening region.
10. The composition of claim 1, wherein the first intervening region and the second intervening region have a same sequence.
37. The composition of claim 1, wherein the first intervening region and the second intervening region comprise a common primer binding site.
Regarding claims 7, 10 and 37, Glezer teaches an amplification primer, and each oligonucleotide probe comprises an amplification priming site in the region between the 3' and 5 primer sequence (i.e., intervening region) for rolling circle amplification (RCA) (FIG. 2;
[00182] “The resulting circularized oligonucleotide is primed with an amplification primer and extended with a strand-displacing polymerase to generate a concatemer containing multiple copies of the target nucleic acid sequence, as shown in FIG. ID.”).
While Glezer does not explicitly teach the intervening regions of the first and second oligonucleotide probes comprise a common sequence, this feature is obvious in view of the knowledge in the prior art.
The use of universal primer for amplifying circularized padlock probes is well known in the art. See Gong (Fig. 1), see also Carrascosa2.
Specifically, Stuelpnagel teaches specific advantages of using identical primer site across different padlock probes for RCA reaction, such as the ability to use only a single primer oligonucleotide to prime the RCA assay with a plurality of different hybridization complexes, thereby simplifying the amplification process ([0327]):
"The padlock probe also contains a priming site for priming the RCA reaction. That is, each padlock probe comprises a sequence to which a primer nucleic acid hybridizes forming a template for the polymerase. The primer can be found in any portion of the circular probe. In a preferred embodiment, the primer is located at a discrete site in the probe. In this embodiment, the primer site in each distinct padlock probe is identical, although this is not required. Advantages of using primer sites with identical sequences include the ability to use only a single primer oligonucleotide to prime the RCA assay with a plurality of different hybridization complexes. That is, the padlock probe hybridizes uniquely to the target nucleic acid to which it is designed. A single primer hybridizes to all of the unique hybridization complexes forming a priming site for the polymerase. RCA then proceeds from an identical locus within each unique padlock probe of the hybridization complexes."
Accordingly, It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the padlock probes in Glezer to comprise primer sites in their intervening regions with identical, common sequences, as taught by Stuelpnagel.
The skilled artisan would have been motivated to do so, in order to gain the ability to use a single primer to prime the RCA reaction with different hybridization complexes, as suggested by Stuelpnagel. This potential benefit is particularly relevant to the teachings of Glezer, which describes the formation of different hybridization complexes with different target sequence regions using different padlock probes (e.g., FIG. 3A). Accordingly, the ability to use a single primer to amplify all of the circularized probes would have provided a clear benefit by simplifying the amplification process and eliminating the need to design individual primer for each specific probe.
The person of ordinary skill would have had a reasonable expectation of success in making this modification because the use of a universal primer for amplification of padlock probes was well-known in the art. Thus, a skilled artisan would have had the knowledge and skills to design and use the probes accordingly.
Prior Art
Below are relevant prior art not used in rejection but pertinent to the claims or disclosure.
The use of padlock probes followed by RCA for analysis of RNA splicing is known in the art. See Ren, Xiaojun, et al. "RNA splicing analysis: from in vitro testing to single-cell imaging." Chem 5.10 (2019): 2571-2592. (Figure 2)
The following references also teach padlock probes binding to different regions on a target nucleic acid:
Carlson et al., MIPSTR: a method for multiplex genotyping of germline and somatic STR variation across many individuals. Genome Res. 2015 May;25(5):750-61. doi: 10.1101/gr.182212.114. Epub 2015 Feb 6. Erratum in: Genome Res. 2015 Aug;25(8):1244. PMID: 25659649; PMCID: PMC4417122. (Figure 1);
Glezer2 (Glezer et al. ; WO2022032194A1 - Methods for in situ transcriptomics and proteomics ; effective filing date Aug 6, 2020) ; FIG. 6, 11;
Landegren (US20160289750A1 - Localised rca-based amplification method using a padlock-probe; 2016-10-06); Figure 1;
Fan (US20150368704A1- Methods and compositions for single cell genomics; Published 2015-12-24) : FIG. 1.
Conclusion
No claims are allowed.
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/TIAN NMN YU/Examiner , Art Unit 1681 /AARON A PRIEST/Primary Examiner, Art Unit 1681
1 Claims 15-16, 18, 25, and 32-33 are withdrawn as being drawn to non-elected groups II and IV-V.
2 Carrascosa, page 3585, right-hand col, para 2, lines 17-21:
"The superior specificity and accuracy of MIP based approaches over conventional primer binding approaches can be attributed to the enzymatic digestion of all non-circularized probes and the use of the universal primers for amplifying all MIPs."
Prosecution Insights