ECHO AMPLIFICATION: A COMPREHENSIVE SYSTEM OF CHEMISTRY AND METHODS FOR AMPLIFICATION AND DETECTION OF SPECIFIC NUCLEIC ACID SEQUENCES

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 . Art Unit Designation The art unit designation for correspondence to this Office Action has changed to 1681. This application has been transferred from Examiner Jehanne Sitton to Examiner Tian Yu. All future correspondence should be directed to Examiner Tian Yu whose contact information appears at the end of this Office Action. Information Disclosure Statement The information disclosure statements (IDS) submitted on 07/11/2024, 08/26/2025 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner. Objection to Specification The disclosure is objected to because paragraph [0223] of the specification contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01. Nucleotide and/or Amino Acid Sequence Disclosures Summary of Requirements for Patent Applications Filed On Or After July 1, 2022, That Have Sequence Disclosures 37 CFR 1.831(a) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.831(b) must contain a “Sequence Listing XML”, as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.831-1.835. This “Sequence Listing XML” part of the disclosure may be submitted: 1. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter “Legal Framework”) in XML format, together with an incorporation by reference statement of the material in the XML file in a separate paragraph of the specification (an incorporation by reference paragraph) as required by 37 CFR 1.835(a)(2) or 1.835(b)(2) identifying: a. the name of the XML file b. the date of creation; and c. the size of the XML file in bytes; or 2. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation by reference statement of the material in the XML format according to 37 CFR 1.52(e)(8) and 37 CFR 1.835(a)(2) or 1.835(b)(2) in a separate paragraph of the specification identifying: a. the name of the XML file; b. the date of creation; and c. the size of the XML file in bytes. SPECIFIC DEFICIENCIES AND THE REQUIRED RESPONSE TO THIS NOTICE ARE AS FOLLOWS: Specific deficiency - This application fails to comply with the requirements of 37 CFR 1.831-1.834 because it does not contain a “Sequence Listing XML” as a separate part of the disclosure. A “Sequence Listing XML” is required because the drawings (FIGs. 18, 19, 20, 21, 22, 23) and specification ([0091]-[0096]; [0205]; [0208]-[0209]; [0213]; [0216]; [0217]; [0218]; [0219]) references specific SEQ ID NOs, which appear to correspond to specific amino acid and nucleotide sequences disclosed in pages 68-72 in the specification. However, no sequence listing has been filed in this application. Required response - Applicant must provide: • A “Sequence Listing XML” part of the disclosure, as described above in item 1. or 2.; together with o A statement that indicates the basis for the amendment, with specific references to particular parts of the application as originally filed, as required by 37 CFR 1.835(a)(3); o A statement that the “Sequence Listing XML” includes no new matter as required by 37 CFR 1.835(a)(4) AND • A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 inserting the required incorporation by reference paragraph as required by 37 CFR 1.835(a)(2), consisting of: o A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version); o A copy of the amended specification without markings (clean version); and o A statement that the substitute specification contains no new matter. Status of Claims This office action is in response to Applicant's Response to Election / Restriction filed on July 02, 2026. Claims 1-5, 7-17, 19-21 and 23 are currently pending, with claims 10-15, 20 and 23 withdrawn. Claims 1-5, 7-9, 16-17, 19 and 21 are under examination. This is the first action on the merits. Election/Restrictions Applicant’s election without traverse of Group I (claims 1-5, 7-9, 16-17, and 19-21) in the reply filed on July 02, 2026 is acknowledged1. The requirement for "Species election 1" for a particular non-natural nucleotide base recited in claims 7-9, set forth in the office action mailed on May 04, 2026 has been withdrawn. Applicant’s election without traverse of the following species in the reply filed on July 02, 2026 is acknowledged: Species election 2: elect from a) the non-natural nucleotide base of the forward primer is the same as the non-natural nucleotide base of the reverse primer, or b) the non-natural nucleotide base of the forward primer is different from the non-natural nucleotide base of the reverse primer. Applicant elects "a) the non-natural nucleotide base of the forward primer is the same as the non-natural nucleotide base of the reverse primer." 2 Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)). Claims 10-15, 20 and 23 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 1-5, 7-9, 16-17, 19 and 21. Priority The priority date of the instant claims 1-5, 7-9, 16-17, 19 and 21 is 09/16/2021, filling date of the US provisional application NO. 63/245,149. 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. For the purpose of applying prior art, claim 1 recites a "nucleic acid construct, " which is not expressly defined in the application's disclosure. The application's disclosure does not define this term by any structural feature that distinguishes a "nucleic acid construct" from nucleic acids known in the art. Accordingly, under BRI, "nucleic acid construct" is interpreted as encompassing any nucleic acid structure. For the purpose of applying prior art, claim 1 recites a nucleic acid construct comprising a "target-specific region," which is not expressly defined in the application's disclosure. The specification provides the following relevant description in para. [0109]: "A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template. A primer generally contains a sequence that is complementary to a sequence (termed the “target sequence”) present in sample nucleic acids. The sequence in the primer is said to be “target-specific.”" ([0109]lines 12-16) Accordingly, under BRI and in light of the specification, "target-specific region" is interpreted as encompassing a sequence region capable of hybridizing to a target sequence. For the purpose of applying prior art, claim 1 recites a "universal adapter sequence," located 5' of the target-specific region. The term "universal adapter sequence" is defined in the specification as "a nucleotide sequence that is, or becomes, linked to a target nucleotide sequence." ([00130]). Accordingly, under BRI and in light of the specification, the claimed "universal adapter sequence" is interpreted to encompass a nucleotide sequence that is linked to, and 5' of a target-specific region in a nucleic acid construct. For the purpose of applying prior art, claim 1 recites a "non-natural nucleotide base," which is defined by the specification as follows: "Individual nucleotides in DNA or RNA are described herein as “bases” generally; as “deoxyribonucleotides” or “deoxybases” for nucleotides in DNA; or as “ribonucleotides” or “ribobases in RNA. Nucleotides can be natural or non-natural. “Natural” nucleotides are those known to occur in nature as of the original filing date of the present disclosure. “Non-natural” nucleotides are those not known to occur in nature as of the original filing date of the present disclosure. " ([0104]) For the purpose of applying prior art, claim 2 recites the nucleic acid construct comprises a “terminal 3' cap.” The specification defines the term "cap" as follows: "With reference to an element of a primer, the term “cap” refers to a structure (typically, at the terminus of the primer) that cannot be extended by a polymerase. The primer bearing a cap, e.g., a 3′ terminal cap is said to be “blocked.”" ([0111]) 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. 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. Claims 1-5, 16-17, 19 and 21 are rejected under 35 U.S.C. 103 as being unpatentable over Jung (Jung et al., ; Isothermal target and signaling probe amplification method, based on a combination of an isothermal chain amplification technique and a fluorescence resonance energy transfer cycling probe technology Anal Chem, 82 (2010), pp. 5937-5943) , in view of Walder (US20150307917A1- Rnase h-based assays utilizing modified rna monomers; published 2015-10-29), as evidenced by Collingwood (Collingwood et al. Chemical modification patterns compatible with high potency dicer-substrate small interfering RNAs. Oligonucleotides. 2008 Jun;18(2):187-200. doi: 10.1089/oli.2008.0123. PMID: 18637735; PMCID: PMC2582043.). A) Jung teaches an isothermal chain amplification method, utilizing a DNA-RNA-DNA chimeric inner primer set, wherein each of the chimeric inner primer comprises a 5’ region not hybridized to the target (corresponding to the claimed “universal adapter sequence” region according to the claim interpretation), comprising ribonucleic acid bases 5’ to target-specific region, (Figure 1, ribonucleic acid region indicated by red-dotted box). After liner amplification via extension of the inner primer, the RNA region at the 5’ end of the inner primer-containing strand of the double stranded DNA, formed in the previous step, is degraded by RNase H, thereby allowing for further strand displacement amplification. Regarding claim 1, Jung teaches a nucleic acid construct (Figure 1; Table 1) comprising: a target-specific region (Figure 1, Inner primer 3' portion that hybridize to target nucleic acid; Table 1) and, a universal adapter sequence located 5' of the target-specific region (Figure 1, Inner primer 5' portion that is linked to target-specific region). Although Jung does not explicitly teach its inner primer 5’ region (universal adapter sequence) comprises a non-natural nucleotide base, this feature is obvious in view of Walder. Walder similarly teaches chimeric primers comprising RNase H cleavage sites (see Abstract; [0053]; FIG. 18 for examples). Walder further teaches that in addition to the naturally occurring RNA bases, other non-naturally occurring analogs can also be recognized by RNase H, such as 2'-fluoro modified bases ([0203] lines 11-14, “Alternatively, any analog recognized by RNase H2, such as a 2'-fluoronucleoside may be substituted”; [0293] “Thus, Pyrococcus abyssi RNase H2 can be used to cleave substrates which do not contain any RNA bases but instead contain 2′-modified bases.”; see also [0131]-[0132], 2'-fluoro modified RNA residue is used with a 2' LNA modified RNA residue, both 2′-F RNA modification and LNA are non-natural 2’ modifications, see Collingwood, p. 196, right-hand col, lines 5-14). Walder teaches that incorporating modified residues within an RNase H cleavage domain can suppress background cleavage of a primer or probe due to water catalyzed hydrolysis or cleavage by single stranded ribonucleases ([0134]). Accordingly, a person of ordinary skill in the art before the effective filing date of the claimed invention would have found it prima facie obvious to modify Jung’s method by replacing the RNA nucleobase in the inner primers with a non-natural nucleobase that is also cleavable by RNase H, specifically RNase H2, as disclosed by Walder. Such a modification represents a simple substitution of one known cleavable linker for another to obtain predictable results, see MPEP 2141. Both the RNA nucleobase in Jung and the non-natural nucleobase taught by Walder serve the same function as cleavable regions in a primer for RNase H cleavage. Therefore, a skilled artisan would have reasonably expected RNase H2 to cleave the primer at the site comprising the non-natural modified base. A skilled artisan would have been particularly motivated to make this modification because Walder suggests that the modified bases are resistant to background cleavage by hydrolysis and cleavage by other RNase enzymes ([0134]; [0294]). Thus, a skilled artisan would have understood that the modification would improve primer stability and prevent non-specific degradation of primers. B) Regarding claim 2, Walder teaches 3’ blocked cleavable primers for improving specificity during amplification (see Abstract; Fig. 18; [0307] for examples). Accordingly, a skilled artisan would have found it prima facie obvious to further modify the inner primers of Jung to include a 3’ block (corresponding to the recited terminal 3' cap), as taught by Walder, in order to improve amplification specificity. There would have been a reasonable expectation of success because the references disclose technically compatible teachings. Jung teaches primers for amplification that are not 3’ blocked, and Walder teaches an improved method of including a 3’ block in amplification primers for more specific amplification. Walder also provides detailed guidance on primer design (Fig. 18; [0307]). Regarding claim 3, Walder teaches target- specific region comprises a target-specific cleavage domain (FIG. 18). Regarding claim 4, Walder teaches the target-specific cleavage domain comprises a ribonucleotide (FIG. 18, cleavable domain being rN). Regarding claim 5, Walder teaches wherein the non- natural nucleotide base is a ribonucleotide ([0131]-[0132], 2'-fluoro modified RNA residue with a 2' LNA modified RNA residue, for example). Regarding claim 16, as discussed for claim 1, the combined teachings of Jung and Walder teaches a forward and reverse primer comprising the nucleic construct of claim 1. Regarding claim 17, Walder teaches forward primer comprise a terminal 3' cap (FIG. 18). Regarding claim 19, it recites “wherein the non-natural nucleotide base of forward primer is the same as the non-natural nucleotide base of reverse primer.” This limitation would have been obvious in view of the combined teachings of Jung and Walder. As discussed for claim 1, a person of ordinary skill in the art before the effective filing date of the claimed invention would have found it prima facie obvious to modify Jung’s method by replacing the RNA nucleobase in the inner primers with a non-natural nucleobase that is also cleavable by RNase H, specifically RNase H2, as disclosed by Walder. Jung teaches both forward and reverse inner primers comprising the same RNA bases suitable for RNase H cleavage (Figure 1; Table 1). Accordingly, with the proposed modification discussed for claim 1, a skilled artisan would have found it obvious to apply the same non-natural nucleotide base cleavable by RNase H2, taught by Walder, in both the forward and reverse primers. Regarding claim 21, Walder teaches wherein the non- natural nucleotide base is a ribonucleotide ([0131]-[0132], 2'-fluoro modified RNA residue with a 2' LNA modified RNA residue, for example). Claims 7-9 are rejected under 35 U.S.C. 103 as being unpatentable over Jung, in view of Walder, as applied to claim 1 above and further in view of Yang (Yang et al. , Expanded genetic alphabets in the polymerase chain reaction. Angew Chem Int Ed Engl. 2010;49(1):177-80. doi: 10.1002/anie.200905173. PMID: 19946925; PMCID: PMC3155763.). The teachings of Jung and Walder are recited above and applied as for claim 1. Regarding claims 7-9, although the combined teachings of Jung and Walder does not explicitly teach a specifically recited non-natural nucleotide base such as 2-amino-8-(1'-beta-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, this feature is obvious in view of Yang. Yang teaches an artificially expanded genetic information system (AEGIS), which adds extra nucleotide "letters" and additional base-parings to DNA alphabets (Abstract). The recited “2-amino-8-(1'-beta-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one” is one of the AEGIS nucleotides, represented by the letter “P,” which base-pairs with the AEGIS nucleotide “Z”(Figure 1; p.2, para 3). Yang teaches including AEGIS nucleotides, such as P, in the external tag (a region 5’ to target-binding region, see Figure 2) of a primer could reduce off-target priming. Because AEGIS-containing tags do not pair with DNA in any natural genomic background, they cannot create amplicons through off-target priming regardless of how complex the assay environment becomes (Abstract; p. 2, para 2). Accordingly, a person of ordinary skill in the art before the effective filing date of the claimed invention would have found it prima facie obvious to further modify the 5’ regions (corresponding to the “external tag” in Yang; also corresponding to the claimed “universal adapter sequence”) of the inner primers in the combined teachings of Jung and Walder, to include additional AEGIS nucleotides, such as P (2-amino-8-(1'-beta-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one), as taught by Yang, in order to reduce off-target priming and thereby improve amplification specificity. There would have been a reasonable expectation of success because the references provide overlapping and technically compatible teachings. All three references teach performing amplification using primers. Jung already teaches primers comprising an external tag, and Yang provides a particular improvement for reducing off-target priming by including the AEGIS nucleotides P, in the primer’s external tag. Prior Art Below are relevant prior art not used in rejection but pertinent to the claims or disclosure. Steelman (US20170218455A1 -- Methods, compositions, and devices for rapid analysis of biological markers; published 2017-08-03) also teaches primer pairs comprising 3' blocker, cleavable by RNase H, and an additional cleavage site in the 5' region (FIG. 14). Conclusion No claims are allowed. Any inquiry concerning this communication or earlier communications from the examiner should be directed to TIAN NMN YU whose telephone number is (703)756-4694. The examiner can normally be reached Monday - Friday 8:30 am - 5:30 pm. Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. 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If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000. /TIAN NMN YU/Examiner , Art Unit 1681 1 Claims 10-15 and 23 are withdrawn as being drawn to non-elected groups II-III. 2 Claim 20 is withdrawn as being drawn to non-elected species b.