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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 1/5/2026 has been entered.
Claim 10 remains withdrawn. Claims 1-9 are pending and are examined on the merits herein.
Response to Applicant’s Amendments
Claim Objections
Claims 1 and 6 were objected to due to minor informalities. In light of Applicant’s amendments to the claims submitted 1/5/2026, these objections have been withdrawn, but see new grounds of objection below.
35 USC 112(b) Rejections
Claims 1-9 were objected to due to various indefiniteness issues. In light of Applicant’s amendments to the claims submitted 1/5/2026, these rejections have been withdrawn, but see new grounds of rejection below.
35 USC 103 Rejections
Claims 1-9 are rejected under 35 U.S.C. 103 as being unpatentable over Bau et al. (WO 2019/178346 A1). These rejections have been withdrawn, but see new grounds of rejection and “Response to Applicant’s Arguments” below.
Response to Applicant’s Arguments
Regarding the 35 USC 103 Rejections presented in the Final Rejection mailed 9/5/2025, Applicant argues that Bau does not teach performing a PCR and a cleavage reaction simultaneously, and cites the examples of Bau as evidence of this (Remarks, page 7). Applicant also notes that the polymerase used by Bau has a working temperature that is lower than PCR denaturation temperature, and so the reactions could not be performed simultaneously (Remarks, page 8). Applicant also argues that though Bau teaches that conditions may be developed to perform amplification and cleavage simultaneously in their methods, the reference does not provide any optimized conditions for such simultaneous reactions, and these specifics are alleged unknown in the art (Remarks, page 9). Finally, Applicant argues that the instant invention produces superior and unexpected results compared to those of Bau.
In the Final Rejection, the teachings of Bau used to read on simultaneous cleavage and amplification reactions were paras. 53, 57, and 64. However, the Examiner notes that the embodiments encompassing simultaneous reactions in this reference appear to mainly be cleavage reactions in conjunction with isothermal amplifications (e.g. “While isothermal amplification can allow the simultaneous cleavage and amplification of non-target and target nucleic acids, respectively, thermocycling methods can also be used when the amplification process is subsequent to the cleavage assay,” in para. 60). Thus, Applicant’s arguments that Bau does not provide enough information for creating optimized simultaneous PCR and cleavage reaction conditions is persuasive. This is the basis for the new grounds of rejection presented below. It is noted in these rejections that at least one of the new references cited does teach simultaneous PCR and cleavage reactions, making Applicant’s assertion that this has not been done before in the prior art not persuasive.
Regarding Applicant’s other arguments, firstly, Applicant pointed to the temperatures of TtAgo used in Table 4 to state why this enzyme would not work under PCR conditions. In the Final Rejection, PfAgo is cited for use, not TtAgo (see para. 24 of the Final Rejection). Furthermore, Applicant states that denaturation for PCR must be 95°C, when this is not in fact always required – in fact, in newly amended claim 1, denaturation temperatures from 85-95°C are recited. This argument is therefore not persuasive. Finally, regarding Applicant’s allegedly superior and unexpected results, firstly, these are compared to those of Bau, which, by Applicant’s own admission, does not perform the same method. Applicant’s results from the instant invention are also based on Example 5 and Figure 6A, which are currently not commensurate in scope with the claimed invention. For example, these teachings focus on a specific mutant gene and contain a pre-amplification step that are not claimed. See MPEP 716.02 for a list of the requirements needed to prove unexpected results. Therefore, this argument is currently not persuasive.
Claim Objections
Claim 1 is objected to because of the following informalities: in lines 3-4 of step (b), the newly amended portion should read “for amplification, wherein C cycles of PCR are performed, wherein C is 10-30, and a denaturation temperature…” Additionally, in the third to last line on the first page of the claims, “PCR reaction” should just read “PCR.” In the newly amended wherein clause describing the ratio of F1b/F1a, the word “and” should be inserted before “when F1a≤0.1%.” Finally, the third to last line of the claim should read “from the following Argonaute proteins and Appropriate correction is required.
Claim 6 is objected to because of the following informalities: in line 2, “for PCR reaction” should read “for the PCR.” Additionally, in line 3, “1-5 Mm” should read “1-5 mM.” Appropriate correction is required.
Claim 9 is objected to because of the following informality: for clarity and to better match the language used in claim 1, it is recommended that “the amount of nucleic acid used as a template” should read “the amount of the target nucleic acid in the nucleic acid sample used as a template.” Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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 1-9 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.
Claim 1 recites the limitation "the gDNA" in the first newly added wherein clause after step (b). “A gDNA” does not appear earlier in the claim, and so there is insufficient antecedent basis for this limitation in the claim. Additionally, this same clause of the claim recites “the nucleic acid sequence of the target region” for both the target and non-target nucleic acid without first reciting “a nucleic acid sequence of a target region” for both of these sequences. Therefore, these also have insufficient antecedent basis in the claim.
Additionally in claim 1, in the second newly added wherein clause after step (b), the phrase “the nucleic acid cleavage tool enzyme” is recited without first reciting “a nucleic acid cleavage tool enzyme” earlier in the claim. This phrase therefore also lacks antecedent basis. Furthermore, later in this wherein clause, the “concentration of initial cleavage tool enzyme” (emphasis added) is recited. The use of the word “initial” makes it unclear if more enzyme is to be provided later in the method, particularly as no step for such addition is recited in the claim. It is recommended to have this phrase read “concentration of the nucleic acid cleavage tool enzyme” if this is Applicant’s intention.
Finally in claim 1, the final wherein clause recites “a nucleic acid cleavage tool enzyme.” It is unclear if this Is the same enzyme as described earlier in the claim, or if this clause is introducing a new cleavage tool enzyme. It will be interpreted as though the former is the case.
Claims 2-9 are rejected due to their dependence on rejected claim 1.
Claim 5 is also rejected because it uses the phrase “the complex” throughout. “A complex” is not recited earlier in the claim or in instant claim 1, from which this claim depends. This phrase therefore lacks antecedent basis. It is unknown what components comprise this complex, nor if “the complex” is intended to include the same components as it relates to the target and non-target nucleic acids. Based on the general method of claim 1 and the fact that the complex must be capable of cleaving, “the complex” will be interpreted to at least include the nucleic acid cleavage tool enzyme.
Claim 6 is rejected based on its dependence on rejected claim 5.
Claim Interpretation
In instant claim 1, Applicant has added a limitation requiring particular ratios of the abundance of the target nucleic acid before and after amplification/cleavage given a particular abundance of the target in the initial sample. It is first noted that these ratios do not cover all possible abundances of the target in the initial sample (e.g. when the target comprises more than 10% of the nucleic acids in the sample or when the target is above 0.5% but less than 1% of the sample). Overall, these are considered contingent limitations (see MPEP 2111.04 II), and as the claim is a method claim, these limitations will only be considered as they are relevant to the embodiments described by the prior art. Therefore, if cited prior art teaches an embodiment including abundances in one or more of the specifically claimed ranges, than the particular ratio associated with said range(s) will apply. If the cited prior art teaches an embodiment including abundances outside of the claimed ranges, than no specific ratio will be required. For example, if the prior art teaches an initial abundance of 3% for a target sequence, then the ratio of the abundance of the target before and after amplification/cleavage must be greater than or equal to 10 in order to read on the instant claim. However, if the prior art teaches an initial abundance of 12% for a target sequence, than no specific ratio of the abundance of the target before and after amplification/cleavage would be required in order to read on the instant claim.
In instant claim 5, particular levels of complementarity in the binding regions established in claim 1 are recited. For each particular level of complementarity/mismatch, the claim states “thereby causing…” With this wording, it will be interpreted that the inherent structure of the binding regions leads to the functional limitation described by the “thereby” statements.
Claim Rejections - 35 USC § 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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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 and 7-9 are rejected under 35 U.S.C. 103 as being unpatentable over Bau et al. (WO 2019/178346 A1), in view of Sorge et al. (US 2007/0292865 A1), and in view of Swarts et al. (Nucleic Acids Research, 2015).
Bau teaches enrichment methods for biological samples, particularly for low-copy nucleic acids relative to high-copy nucleic acids and mutant alleles (Abstract). Generally, this method involves the use of guide nucleic acids, endonucleases, and amplification to enrich a target nucleic acid (para. 9). The cleavage assay and amplification reaction may be combined and run simultaneously in a single reaction vessel (paras. 53, 57, and 64). Mutations can be examined that are single nucleotide polymorphisms (see Table 1 for example; instant claims 2-3). Bau teaches that an Argonaute protein can be used as the endonuclease, and specifically Pyrococcus furiosus Argonaute (PfAgo; para. 54; instant claim 4). In their working examples, Bau shows that the Ago protein can be used to cleave non-target wild-type sequences while leaving mutant sequences untouched (paras. 79 and 85). Bau teaches that the target may be present at concentrations as low as 0.1% of that of the wild-type sequences (see claims 1 and 6 and para. 52).
Bau does not specifically teach a ratio of the abundance of the target nucleic acid before and after the cleavage and amplification reactions. However, Bau does teach PCR using primer specific for the target nucleic acid with a number of cycles ranging from 30-45 (paras. 59, 61, 125 and 127), and general temperatures for the different portions of the reaction ranging from 60°C to 95°C (paras. 125, 127-129, 131, and 133). As only the target nucleic acid is being amplified, this would result in an abundance of the target in the cleavage-amplification product that is well over 500 times the abundance present in the original sample. Additionally, though Bau teaches that cleavage assay and amplification reactions may be run simultaneously, this appears to mainly involve cleavage in accordance with isothermal amplification reactions (e.g. “While isothermal amplification can allow the simultaneous cleavage and amplification of non-target and target nucleic acids, respectively, thermocycling methods can also be used when the amplification process is subsequent to the cleavage assay” in para. 60), and not PCR.
It is also noted that the specific cleavage methods described by Bau generally involve the use of a guide DNA that binds to the non-target sequence, and then providing an endonuclease that has affinity for the hybridized complex, and notes that these cleaving methods can produce fluorescent signals or detectable by-products (e.g. paras. 63, 67, and 70). The enzyme PfAgo is also noted to operate at 95°C (paras. 87 and 114).
Sorge teaches methods for generating a signal indicative of a target nucleic acid in a sample utilizing cleavage methods (para. 16). Specifically, these methods involve conditions under which a polymerase extends a target sequence and a nuclease performs cleavage (para. 18). Both the polymerase and the nuclease may be thermostable (paras. 50-51), and the reference teaches that thermostable enzymes may be taken from Pyrococcus furiosus (e.g. paras. 33 and 42). Specifically, the methods of Sorge involve performing PCR simultaneously with a cleavage reaction (para. 213). Para. 675 uses such a method in Example 11, where thermocycling parameters are recited, with a specific denaturation temperature of 95°C, and a general range of temperatures from 60°C to 95°C.
Swarts demonstrates that PfAgo is capable of high levels of nuclease cleavage activity at 87°C to 99°C, though it generally exhibits activity at 37°C and above (page 5124, columns 1 and 2 joining para. and Figure 3).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Sorge and Swarts to optimize the methods of Bau to be capable of performing PCR amplification and cleavage simultaneously. Specifically, Sorge provides evidence that cleavage reactions and PCR can be performed simultaneously under typical PCR thermocycling temperature conditions, provided that the enzymes used are thermostable, and Swarts and Bau both teach that PfAgo is capable of acting at a wide range of temperatures, and particularly those temperatures that would be encompassed by a PCR reaction. MPEP 2143.01 V states, “‘[a] given course of action often has simultaneous advantages and disadvantages, and this does not necessarily obviate motivation to combine’ (quoting Medichem, S.A. v. Rolabo, S.L., 437 F.3d 1157, 1165, 77 USPQ2d 1865, 1870 (Fed. Cir. 2006),” and thus, though isothermal methods may also present some advantages over PCR (e.g. the ability to amplify without a thermocycler), this does not inherently obviate the use of PCR in the methods of Bau, provided such methods have support in the prior art. It is noted that the ordinary artisan would similarly recognize advantages of PCR over isothermal methods, such as the need for fewer primers when performing PCR (see the LAMP primers used by Bau for example, para. 130 and Table 5). Additionally, this change of isothermal amplification for PCR would amount to a simple substitution of the amplification method used in Bau. MPEP 2143 I (B) states, “The rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.” This change would still result in the amplification of a target sequence and the cleavage of a non-target sequence, and there would be a reasonable expectation of success given the teachings of Sorge and Swarts. This, the results of this substitution would be predictable.
Regarding the PCR parameters described by claim 1, the PCR amplification teachings of Bau described above overlap with the number of cycles and denaturation temperature recited. It would be prima facie obvious to use the PCR parameters already established by Bau in the method of Bau in view of Sorge, and in view of Swarts, as these parameters have already been shown to work on the target sequences of Bau.
Regarding the target regions described by claim 1, in the invention of Bau, in view of Sorge, and in view of Swarts described above, the target and non-target sequence may only differ by a single nucleotide. Bau also teaches that their cleavage methods work when in the presence of phosphorylated gDNAs specifically that are sufficiently complementary to the non-target nucleic acid (para. 49). The reference also notes that a single base-pair mismatch between the gDNA and the wild-type sequence increases specificity (para. 79). Figure 1 shows that the guide DNA can attach to both a wild-type and a mutant sequence that differ by a single nucleotide, and will still result in only the wild-type sequence being cleaved. In this figure, there is a single mismatch between the gDNA and the wild-type sequence (at the 5’ phosphorylated end), and two mismatches between the gDNA and the mutant sequence (at the 5’ phosphorylated end and at the location of the mismatch). Similar scenarios are shown in Figures 10-13. Thus, Bau teaches many scenarios in which the mutant sequence has two mismatches with the gDNA and the wild-type sequence has one. In the method of Bau, in view of Sorge, and in view of Swarts, the cleavage methods described by Bau are used, and so it would be prima facie obvious to keep the gDNA design of Bau in relation to target and non-target sequence (instant claim 5).
Regarding the claimed molar ratio of the cleavage enzyme to the gDNA, Bau teaches utilizing a 1:10-1:20 total ratio in their cleavage assays (paras. 30, 104, 126 and 131-132). Para. 114 shows an embodiment with this ratio concentration range specifically using PfAgo. It would thus be prima facie obvious to use this ratio concentration range in the teachings of Bau, in view of Sorge, and in view of Swarts, which utilizes the cleavage enzymes and methods of Bau, as such a molar concentration ratio has been shown to result in successful cleavage.
Regarding the claimed concentration of the gDNA, Bau teaches concentrations ranging from 250 nM to 2,500 nM (paras. 80 and 114). MPEP 2144.05 I states, “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990).” As the ranges of gDNA in Bau overlap with those described by the instant invention, and as noted above in the “Response to Applicant’s Arguments” section, Applicant has not shown any unexpected or critical results commensurate in scope with the claimed invention and associated with this range, this range of gDNA is rendered obvious by Bau. Additionally, it would be prima facie obvious to use the concentration range taught by Bau in Bau, in view of Sorge, and in view of Swarts, which utilizes the cleavage enzymes and methods of Bau, as such concentrations have been shown to result in successful cleavage.
Regarding the claimed concentration of the cleavage enzyme, it is noted that Bau, in view of Sorge, and in view of Swarts renders obvious the claimed gDNA concentration and the claimed molar ratio of enzyme to gDNA. Thus, for the given concentrations of gDNA described above, the ordinary artisan would recognize that the corresponding amount of enzyme needed to obtain the given molar ratio range would be 12.5-250 nM. This range overlaps with the claimed range, and as noted in the paragraph above, in light of a lack of unexpected or critical results commensurate in scope with the claimed invention and associated with the claimed range, this overlapping range renders the claimed range obvious.
Thus, claims 1-5 are prima facie obvious over Bau, in view of Sorge, and in view of Swarts.
Regarding claim 7, Bau teaches various detection methods for the cleavage-amplification products, such as gel electrophoresis (e.g. para. 16 and Figure 2) and sequencing (e.g. para. 33 and Figure 19). Bau also generally mentions detection methods as part of their invention (para. 53 and 57). It would be prima facie obvious to use such methods in Bau, in view of Sorge, and in view of Swarts for detection, as they are already taught to work successfully with amplification/cleavage reactions, and are generally methods that would be known to the ordinary artisan.
Thus, claim 7 is prima facie obvious over Bau, in view of Sorge, and in view of Swarts.
Regarding claim 8, Bau teaches that their assay can analyze multiple mutations concurrently in a multiplex reaction, where the multiple mutations are on different genes (para. 82). Paras. 113 and 116 also provide evidence of multiple targets being examined in the working examples. Therefore, the ordinary artisan would be motivated to include multiple target sequences when using the method of Bau, in view of Sorge, and in view of Swarts described above in the rejection of claim 1. This would allow the detection of multiple mutations that may be associated with a disease such as cancer, as described in Table 1 of Bau. Detecting multiple mutations in a single reaction would eliminate the need for running multiple reactions, increasing efficiency and decreasing the time required to perform diagnostics, and may provide additional helpful clinical information for practitioners with regard to patient diagnostics and risk assessments.
Thus, claim 8 is prima facie obvious over Bau, in view of Sorge, and in view of Swarts.
Regarding claim 9, a specific amount of the target nucleic acid is required. Para. 52 of Bau notes that the target can be less than 10%, or even less than 1% of the amount of the non-target nucleic acid. Though Bau does not explicitly recite an embodiment in which the amount of target nucleic acid is in the range presented in the claim, the reference does recite an embodiment in which 250 nM non-target (i.e. wild-type) nucleic acid is used (para. 80). Thus, the ordinary artisan would be able to use ordinary logic and creativity to combine the teachings of Bau, and so would be capable of developing a sample in which the non-target sequence is present in 250 nM, the target could be anywhere from about 2.5-25 nM. The ordinary artisan would motivated to use such a sample in the methods of Bau, in view of Sorge, and in view of Swarts because the successful use of such a sample would be a promising proof-of-concept for detecting rare alleles, which are frequently disease markers, as taught by Bau (paras. 7, 62, 78). Being able to detect a target present in such small amounts of a sample would allow for earlier diagnosis of diseases associated with rare alleles and would likely improve patient outcomes. There would be a reasonable expectation of success as Bau teaches that their wild-type cleavage methods can be used when there are rare alleles in the presence of wild-type alleles (para. 50).
Thus, claim 9 is prima facie obvious over Bau, in view of Sorge, and in view of Swarts.
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Bau et al. (WO 2019/178346 A1), in view of Sorge et al. (US 2007/0292865 A1), in view of Swarts et al. (Nucleic Acids Research, 2015), and further in view of Fisher Scientific (“New England Biolabs, Inc Q5 Hot Start High-Fidelity 2X Master Mix,” 20151).
Regarding claim 6, Bau, in view of Sorge, and in view of Swarts teaches the method of claim 1, as described above. These references also teach the use of PCR, and Bau notes that in their PCR methods, the Q5 Hot Start High-Fidelity Master Mix is used (see paras. 124-125 and 128).
Fisher Scientific notes that this master mix includes a DNA polymerase, dNTPs, a buffer, and Mg++. When used at the recommended concentration, the master mix contains 2 mM of Mg++ (see product description).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the specific master mix stated by Bau for PCR in the method of Bau, in view of Sorge, and in view of Swarts, which utilizes PCR. This master mix has already been shown by Bau to successfully amplify their samples, and the master mix is commercially available, and thus would be easily accessible to the ordinary artisan. Fisher Scientific notes that this particular master mix allows for “robust DNA amplification,” allows for room temperature setup, works with difficult amplicons, operates regardless of the GC content of sequences, and has a polymerase with a lower error rate than Taq polymerase. These advantages would also motivate the ordinary artisan to use this master mix.
Thus, claim 6 is prima facie obvious over Bau, in view of Sorge, in view of Swarts, and in view of Fisher Scientific.
Conclusion
No claims are currently allowable.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to FRANCESCA F GIAMMONA whose telephone number is (571)270-0595. The examiner can normally be reached M-Th, 7-5pm.
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/F.F.G./Examiner, Art Unit 1681
/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681
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