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 .
Status of the Claims
It is acknowledged that the Applicant amended claims 1 and 7 and cancelled claims 4-6 and 8-10 in their response filed April 22, 2026.
Claims 1-3 and 7 are currently pending and have herein been examined.
Summary of Response to Applicant’s Arguments
This action is in response to the papers filed April 22, 2026. Applicant’s remarks and amendments have been fully and carefully considered but are not found to be persuasive. Detailed responses are documented on pages 11-15 of this office action. Any new grounds of rejection presented in this Office Action are necessitated by Applicant’s amendments. Any rejections or objections not reiterated herein have been withdrawn. This action is made FINAL.
As a result of the amendments to the claims filed on April 22, 2026, prior rejections under 35 U.S.C. 112(b) were withdrawn. However, amendments raised issues of indefiniteness, and new 35 U.S.C. 112(b) rejections are set forth below.
As a result of the declaration under 37 CFR 1.130(b) filed on April 22, 2026, the 35 U.S.C. 102(a)(1) rejection and the 35 U.S.C. 103 rejections over Lai et al. have been withdrawn. As a result of the amendments to the claims filed on April 22, 2026, the remaining 35 U.S.C. 103 rejections have been modified and are set forth below.
Priority
Claims to the priority of PCT/US/2022/030370 and to the benefit of the provisional application 63/191,455 are acknowledged. The effective filing date of the claims under examination is considered to be May 21, 2021.
Information Disclosure Statement
No information disclosure statement was filed at the time of this action.
Claim Objections
Claim 1 is objected to because of the following informalities: unclear sentence construction/minor grammatical error.
In claim 1, line 17-18, the phrase “for synthesizing copper nanoclusters” follows the term “target gene sequence,” instead of “the AAT repeat sequence.” The claim should instead read “…, wherein the amplified product comprises the target gene sequence with the AAT repeat sequence for synthesizing copper nanoclusters at a first end of the target gene sequence…”
Appropriate correction is required.
Claim Rejections - 35 USC § 112
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.
Claims 1-3 and 7 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 1-3 and 7 are rejected as indefinite for the recitation of “the digested amplified product is digested by the restriction enzyme” in step (c) of claim 1. It is unclear whether the claim is merely describing the products of digesting the amplified product, or if it’s requiring that the already-digested amplified product be again digested. As a result, one of skill in the art would not be able to determine the metes and bounds of the claimed subject matter so as to avoid infringement. If what is meant is “wherein the amplified product is digested by the restriction enzyme…”, the claim must be amended to reflect that meaning.
Claims 1-3 and 7 are rejected as indefinite for the recitation of “adding streptavidin-coated magnetic beads to bind with the biotin-modified double-stranded DNA fragment and the undigested amplified product…” in step (d) of claim 1. In step (c), the claim allows that there may be a digested amplified product (in the forms of a fragment containing a biotin modification and a fragment containing an AAT repeat sequence) and/or an undigested amplified product. The ‘and’ in step (d) therefore causes uncertainty with regard to what the claim requires when one or the other is not present. As a result, one of skill in the art would not be able to determine the metes and bounds of the claimed subject matter so as to avoid infringement. If what is meant is that beads are added to bind with the “biotin-modified double-stranded DNA fragment and/or the undigested amplified product”, the claim must be amended to reflect that meaning.
Claims 1-3 and 7 are rejected as indefinite for the recitation of “double-stranded DNA template copper nanoclusters” in step (e) of claim 1 but “the double-stranded DNA-templated copper nanoclusters” in step (d) of claim 1. It is unclear if these nanoclusters are meant to be the same. If they are not the same, it is not clear what is meant by “double-stranded DNA template copper nanoclusters” in step (e) – does the limitation require that the DNA remain in complex with the nanoclusters, or just have been produced by the nanoclusters? If the nanoclusters of step (d) are meant to serve as antecedent basis for the nanoclusters of step (e), the claim must be amended so that the two are the same (i.e. both should read “double-stranded DNA-templated copper nanoclusters”).
Response to Arguments Regarding 35 U.S.C. 112(b)
In the reply filed April 22, 2026, Applicant provided arguments and amendments to the claims. As a result of the amendments to the claims filed on April 22, 2026, prior rejections under 35 U.S.C. 112(b) were withdrawn. New rejections were applied due to language introduced by amendment which is considered indefinite (as discussed above). Relevant to the new rejections, Applicant provided clarification for the logic of amended claim 1:
“In a heterozygous sample, step (a) establishes that the sample contains both a wild-type nucleotide sequence (first allele) and a single- nucleotide variant sequence (second allele). Step (b) provides that the amplified product comprises the target gene sequence derived from both alleles. In step (c), the restriction enzyme selectively digests the wild-type sequence (first allele) but not the single-nucleotide variant sequence (second allele). Accordingly, a heterozygous sample will necessarily and simultaneously yield both a digested product (from the wild-type sequence) and an undigested amplified product (from the single-nucleotide variant sequence) within a single reaction. This is not a logical contradiction-it is the very mechanism by which the claimed method distinguishes heterozygous from homozygous samples.
The Examiner's concern that "step c would render the claim contradictory" appears to have assumed a homozygous context. In a heterozygous sample, as explicitly contemplated by the claims, both products are expected and necessary outcomes of the restriction digestion step. Amended claim 1 therefore presents no internal inconsistency.”
The applicant further states that steps (a) through (f) of claim 1 were rewritten in accordance with embodiment 1 (par. 35) of the original specification and argues that the presently amended claims comply with 35 U.S.C. 112(b).
These arguments have been fully considered and not found fully persuasive. MPEP 2173.01(I) states:
“But c.f. In re Am. Acad. of Sci. Tech. Ctr., 367 F.3d 1359, 1369, 70 USPQ2d 1827, 1834 (Fed. Cir. 2004) ("We have cautioned against reading limitations into a claim from the preferred embodiment described in the specification, even if it is the only embodiment described, absent clear disclaimer in the specification.””
Therefore, although the logic of the embodiment represented in Embodiment 1 of the specification may be clear, the amended claims still contain language which renders the claims indefinite. It is true that in the case that the test sample was heterozygous (i.e. the target gene sequence is a combination of a wild-type and a single nucleotide variant sequence), both biotin-modified double-stranded DNA fragments and undigested amplified product containing the biotin-modified end should be present in the reaction as of step (d). However, the claim is not directly solely to a heterozygous context. Instead, the claim allows in step (a) for three situations: 1) only the wild-type sequence is present (homozygous wild-type), 2) only the single nucleotide variant sequence is present (homozygous variant), and 3) both the wild-type and the single nucleotide variant sequence are present (heterozygous). A restriction enzyme specific for the wild-type sequence is added to the amplified sequence(s), and in newly amended step (d), streptavidin-coated magnetic beads are added “to bind with the biotin-modified double-stranded DNA fragment and the undigested amplified product.” The word “and” requires that both elements be present, but only in situation (3) would this be the case. In the other situations (homozygous wild-type or homozygous variant), both would not be present, and it is unclear how step (d) should be applied. Although amendments to the claims have overcome the previous rejections (which were based on similar logical inconsistencies), the newly amended limitations are subject to new rejections, as discussed here and above in the rejections 35 U.S.C. 112(b).
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.
Claims 1, 3, and 7 are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (published September 11, 2018; Analytical Chemistry 2018 90 (19), 11599-11606), in view of Jia et al. (published March 14, 2012; ACS Nano 2012 6 (4), 3311-3317), and Song et al. (published February 2, 2015; Song et al. Chem Eur J 21(4), 2417-2422).
Wang et al. teaches a method of detecting gene variants using streptavidin magnetic beads combined with PCR and restriction fragment release (Abstract).
Regarding claim 1, Wang teaches a test sample containing a wild-type target gene sequence and/or a variant; adding a specific primer pair to perform an amplification via PCR, wherein the reverse primer has a biotin modification on the 5’ end, the forward primer has a detectable element on the 5’ end, and the amplification product includes the biotin modification on one end and the detectable element on the opposite end (Figure 1); digesting the product with a restriction enzyme that only digests the wildtype sequence (pg. 11600, col 2, last paragraph), and interpreting the quantitative results of a fluorescence spectrometry analysis to determine the condition of the target gene (pg. 11600, col 2, first and last paragraphs). Wang teaches that the amplified product is digested by the restriction enzyme into a biotin-modified double-stranded DNA fragment and a DNA fragment having a detectable element, adding streptavidin-coated magnetic beads to bind with the biotin-modified double-stranded DNA fragment and/or undigested amplified product (Fig. 1), and separating, collecting, and analyzing the supernatant containing the double-stranded DNA fragment having the detectable element (pg. 11600, col. 2, last par. – pg. 11601, 1st par.). Wang teaches analyzing the fluorescence intensity of the detectable double-stranded DNA fragments, where a higher fluorescence intensity indicates a greater proportion of wild-type nucleotide sequence, while a lower fluorescence intensity indicates a greater proportion of single-nucleotide variant sequence (pg. 11601, col. 1, 1st par.).
Regarding claim 3, Wang teaches that the gene amplification reaction is polymerase chain reaction (Fig. 1).
Regarding claim 1, Wang does not teach any sequence for copper nanocluster synthesis on the non-biotin-modified end of the amplified product. Wang does not teach conducting copper nanocluster synthesis in the supernatant by adding copper ions and a reducing agent, wherein the repeat sequence serves as a template for the reduction of copper ions during the copper nanocluster synthesis reaction to form fluorescent copper nanoclusters specifically on the double-stranded DNA structure, thereby producing double-stranded DNA-templated copper nanoclusters. However, Wang does teach a fluorescent chromophore on the non-biotin-modified end of the amplified product (Figure 1).
Jia teaches synthesis of copper nanoclusters using AT repeat sequences (pg. 3315, col 1, first paragraph) where the amount of copper nanoclusters is used to determine the condition of the target gene sequences (pg. 3312, col 1, first paragraph). Jia teaches adding copper ions and a reducing agent to the supernatant containing the double-stranded DNA fragment with a repeat sequence for conducting a copper nanocluster synthesis reaction, wherein the repeat sequence serves as a template for the reduction of copper ions during the copper nanocluster synthesis reaction to form fluorescent copper nanoclusters specifically on the double-stranded DNA structure, thereby producing double-stranded DNA-templated copper nanoclusters (pg. 3312, col. 1, 2nd par.)
As the copper nanoclusters and the fluorescent chromophore are used for the same purpose (generating differential levels of fluorescence in a sequence-specific manner), it would be obvious to a person with ordinary skill in the art before the effective filing date of the invention to substitute the chromophore modification of Wang for a repeat sequence for conducting a copper nanocluster synthesis reaction to arrive at the instant invention. One would be motivated to do so in order to achieve high selectivity in detection of DNA mismatches (Jia: pg. 3311, col 2).
Regarding claims 1 and 7, neither Wang nor Jia teach that the repeat sequence for conducting a copper nanocluster synthesis reaction is an AAT sequence. For clarity of the record, the Examiner is interpreting that the ‘AAT repeat sequence’ of claim 1 may be ‘AAT, ATA, TAA, TTA, TAT, and ATT repeat sequences,’ consistent with claim 7 and in light of the specification (par. 18-20 in the originally-filed specification).
Song et al. teaches AAT repeat sequences including AAT and ATA repeat sequences in duplexed DNA as sequences capable of synthesizing copper nanoclusters sufficient to generate a qualitative result by direct observation (Figure S7, and pg. 2419 col 1, last paragraph – col 2, first paragraph).
It would have been obvious to a person with ordinary skill in the art before the effective filing date of the invention to combine the use of AAT and ATA sequences for copper nanocluster synthesis with the teachings of Wang and Jia to arrive at the instant invention. One would have been motivated to do so to control the formation and distribution of copper nanoclusters onto DNA templates (Song: pg. 2421, col 2) and in order to screen for sequence variation in specific segments of genomic DNA using differences in fluorescence intensity (Jia: pg. 3312, col 1, first paragraph).
Claim 2 is rejected under 35 U.S.C. 103 as unpatentable over Wang et al. (published September 11, 2018; Analytical Chemistry 2018 90 (19), 11599-11606), in view of Jia et al. (published March 14, 2012; ACS Nano 2012 6 (4), 3311-3317) and Song et al. (published February 2, 2015; Song et al. Chem Eur J 21(4), 2417-2422), as applied to claim 1 above, and further in light of Escosura-Muñiz et al. (published January 13, 2016; Escosura-Muñiz et al. Small. 2016 Jan 13;12(2):205-13).
The limitations of claim 1 are taught by the combination of Wang, Jia, and Song, as discussed above.
Regarding claim 2, the combination of Wang, Jia, and Song does not teach the use of recombinase polymerase amplification for gene amplification.
Escosura-Muñiz teaches the use of recombinase polymerase amplification in the context of assay that uses optical detection of metal nanoparticles to detect the presence of DNA (pg. 206, col. 1, 2nd par.).
It would have been obvious to a person with ordinary skill in the art before the effective filing date of the invention to combine the teachings of Wang, Jia, and Song with the teachings of Escosura-Muñiz, in order to overcome the complications of traditional PCR (Escosura-Muñiz: pg. 206, col 1, 2nd par.).
Response to Arguments Concerning 35 U.S.C. 103 Rejections
In the reply filed April 22, 2026, Applicant provided arguments and amendments to the claims. In the response, Applicant stated the inventive core of the present invention lies with the particular design and placement of an AAT repeat sequence for copper nanocluster synthesis at one end of an amplified product which exploits a mismatching self-pairing structure in the absence of a proper-double-stranded amplicon (embodiment 5, Fig. 7-9) which suppresses background fluorescence (Fig. 8-10).
In pg. 9 of the response, Applicant recited specific deficiencies of each reference, treated individually:
Wang et al. does not disclose copper nanoclusters, AT/AAT repeat
motifs, or any specific primer sequence design aimed at lowering background fluorescence under no-template conditions. Wang et al. focuses on genotyping via labeled fragments and quantitative fluorescence at 520 nm (see Figures 2-5 of Wang et at.).
Jia et al. and Song et al. emphasize enhancing fluorescence signal intensity, but neither discloses a low-background, mismatch-based suppression strategy applicable to blank samples (see Abstract and Figure 4 of Jia et al.; Figure 1 of Song et al.).
Zhou et al. employs standard fluorescent probes rather than copper nanoclusters or label-free detection, and does not suggest replacing fluorescent labels with an AAT-repeat copper nanocluster sequence in a magnetic bead/restriction enzyme platform, nor does it suggest using mismatched self-pairing for background suppression (see Abstract of Zhou et al.).
Escosura-Muniz et al. discloses recombinase polymerase amplification (RPA) as a general isothermal amplification technique, but does not teach its integration with copper nanocluster synthesis, restriction digestion, and magnetic bead capture in a single tube under conditions specifically enabled by RPA buffer components.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Also, the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). As discussed in the rejections under 35 U.S.C. 103 above, the combined references teach all elements of the claimed invention. Any deficiencies of the individual reference are cured by additional references, as discussed in the rejections above.
Applicant argued that none of the cited references, alone or in combination, teach to select an AAT repeat sequence specifically to reduce background fluorescence in a PCR-based or RPA-based diagnostic assay when only primers are present, nor to exploit the mismatching self-pairing of such a sequence for that purpose. In pg. 11-12 of the response, Applicant argued that the combination of claimed features provides technical advantages which are neither taught nor suggested by the cited prior art, either individually or in the proposed combinations. Applicant stated that therefore, the rejections under 35 U.S.C. 103 represent impermissible hindsight based on the knowledge gleaned only from Applicant’s disclosure.
Regarding arguments that the combined references do not teach the elements of the claimed invention for the purposes described, it is not required that the motivations to combine reference be exactly the same as those disclosed by the Applicant. MPEP 2144(IV) states that “The reason or motivation to modify the reference may often suggest what the inventor has done, but for a different purpose or to solve a different problem. It is not necessary that the prior art suggest the combination to achieve the same advantage or result discovered by applicant.” As the applicant has not argued that the motivations used by the Examiner are invalid, the rejections are not overcome. Further, regarding arguments that the combined references do not teach the technical advantages of background suppression, naked-eye readability, or low cost and rapid single tube operation, MPEP 2145(II) states that “Mere recognition of latent properties in the prior art does not render nonobvious an otherwise known invention” and "The recitation of an additional advantage associated with doing what the prior art suggests does not lend patentability to an otherwise unpatentable invention." The recognition by the applicant of advantages which are not explicitly provided by the prior art references therefore does not invalidate their use in combination to reject the claims.
Regarding the argument that the rejections represent impermissible hindsight, MPEP 2145(X)(A) states: ‘"[a]ny judgment on obviousness is in a sense necessarily a reconstruction based on hindsight reasoning, but so long as it takes into account only knowledge which was within the level of ordinary skill in the art at the time the claimed invention was made and does not include knowledge gleaned only from applicant’s disclosure, such a reconstruction is proper." In re McLaughlin, 443 F.2d 1392, 1395, 170 USPQ 209, 212 (CCPA 1971).’ The rejections under 35 U.S.C. 103 relied on knowledge that a person with ordinary skill in the art would have had before the filing date of the instant invention, as discussed above.
In pg. 11, Applicant argued that the substitution of RPA for PCR in the previous office action was invalid because the present invention does more than simply substitute one amplification method for another. Applicant argued that the integrated arrangement disclosed in par. 41 (using RPA to conduct the entire reaction in a single tube at 37ºC to achieve the desired detection effect) is neither taught nor suggested by Escosura-Muniz or any other cited reference in the context of an AAT-based CuNC platform.
Regarding the argument that the substitution of RPA for PCR is invalid, the Applicant has not provided evidence to support the invalidity of such a substitution. The claims themselves present RPA and PCR as alternatives (claims 2 and 3 depend on claim 1). MPEP 2145 states that: “If a prima facie case of obviousness is established, the burden shifts to the applicant to come forward with arguments and/or evidence to rebut the prima facie case.” No specific arguments nor evidence were presented which disqualify the substitution of RPA for PCR in the claimed invention.
Regarding the argument that the applied combinations of references do not disclose the integrated arrangement of par. 41, the claims of the instant application do not require said arrangement. For example, the claims do not require any particular temperature and do not require that the reaction occur in a single tube. MPEP 2145(VI) states: “Although the claims are interpreted in the light of the specification, limitations from the specification are not read into the claims.” As the arrangement of par. 41 may not be imported into the claims, it is not required that rejections address those specific limitations.
Applicant argued the inventive concept/core of the invention was reinforced by the incorporation of claims 5 and 6 into claim 1, and that the claimed method embodies a particular an non-obvious combination of features (pg. 11 of the remarks):
A restriction enzyme/magnetic bead capture architecture adaptable to diverse single nucleotide variants;
A specially designed AAT repeat copper nanocluster synthesis sequence at one end of the amplicon, which exploits mismatched self- pairing under no-template conditions to suppress background fluorescence while maintaining adequate signal in true positive samples;
A single-tube integration with RPA that makes use of RPA buffer conditions to facilitate restriction digestion and copper nanocluster synthesis at 37 °C within approximately 30 minutes
Feature (a) is fully addressed by the rejections set forth above, as well as the AAT repeat sequence of feature (b). As discussed in the response above, it is not required that the rejections address unclaimed features or properties such as the purposes of exploiting mismatches to suppress background, and single-tube integration with RPA.
In summary, the arguments provided in the remarks filed April 22, 2026 have not been found persuasive. However, due to amendments filed April 22, 2026, the rejections under 35 U.S.C. 103 have been modified and are set forth above.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Christine M Jones whose telephone number is (571)272-2585. The examiner can normally be reached Monday - Friday, 8AM - 4PM.
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/C.M.J./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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