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 Claims / Response to Amendment
This office action is in response to an amendment filed on May 14, 2026.
Claims 1-20 were previously pending. Applicant amended claims 1, 6-7, 9 and 11-12.
Claims 1-20 are currently pending, with claims 14-16 and 20 withdrawn.
Claims 1-13 and 17-19 are under consideration.
All of the amendment and arguments have been thoroughly reviewed and considered.
Applicant's submission of the specification amendment have addressed the previously presented objection to specification.
The objection to claim 11 has been withdrawn in view of amendment to the claim.
All of the previously presented objection and rejections have been withdrawn as being addressed or obviated by the amendment of the claims, which added new limitations to the claims, that were not considered in the previous rejections.
The previously set forth prior art rejections have been withdrawn in view of the recent claim amendment filed on May 14, 2026, which added new limitations to the claims (e.g., the newly amended method in claim 1 now recites "an autocatalytic template comprising a partial repeat structure including a nicking enzyme recognition site" and "a conversion template comprising a target-binding region"). Thus, the scope of the claims has been changed in a manner that were not considered in the previous rejections.
Applicant's amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
This office action contains new grounds for rejection necessitated by amendment.
Priority
The priority date of the instant claims 1-6, 8-13 and 17-19 is May 27, 2019, filling date of the EP Patent Application Number 19305669.4, to which the present application claims priority.
Regarding claim 7, the earliest priority is 05/27/2020 because the priority document (PCT/EP2020/064771) filed that date is the first to disclose a "fifth oligonucleotide".
Claim Interpretation -- Updated
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, amended claim 1 recites "an autocatalytic template comprising a partial repeat structure." The application's disclosure does not define the term "partial repeat structure."
The specification describes "partial repeat structure" as containing a nicking enzyme recognition site (page 11, line 25). Accordingly, under BRI and in light of the specification, "partial repeat structure" is interpreted to encompass any nucleotide sequence structure containing a nicking enzyme recognition site.
For the purpose of applying prior art, amended claim 1 recites a "bistable amplification system," which is not defined in the application's disclosure.
The specification describes the relevant term "bistable module" as "autocatalytic template + pseudotemplate"(page 13, line 22)
Accordingly, under BRI and in light of the specification, "bistable amplification system" is interpreted as a system comprising an autocatalytic template and a pseudotemplate.
For the purpose of applying prior art, claim 1 recites "converting the target biomolecule into a signal." Under BRI, the term "signal" could encompass any observable or measurable signal. However, in this present application, Applicant has acted as their own lexicographer by expressly assigning a narrower meaning to the term, specifically defining it as a nucleic acid sequence as follows:
"As used herein, the term “signal” or “signal sequence” relates to nucleic acid sequence, preferably a single strand DNA which is obtained by converting the sequence of the target biomolecule, preferably nucleic acid molecule, more preferably, a microRNA into a sequence which may be amplified." (specification, page 20, lines 11-14)
Accordingly, within the context of the claim, the term "signal" is interpreted as encompassing nucleic acid sequences.
For the purpose of applying prior art, claim 5 recites "the second oligonucleotide is able to bind, extend, deactivate, and slowly release the products of polymerization along the first oligonucleotide, thereby inducing a threshold effect."
The phrase "able to bind, extend, deactivate, and slowly release the products of polymerization along the first oligonucleotide, thereby inducing a threshold effect" does not recite any specific structures in the oligonucleotide that directly support or relate to this functional language. Therefore, it is interpreted as intended use that does not distinguish the claimed oligonucleotide over the prior art oligonucleotide with the same physical components. See MPEP § 2111.05.
New Grounds of 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.
Claims 1-13 and 17-19 are rejected under 35 U.S.C. 112(b), 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.
A) Regarding claim 1, it recites "said sequence being able to activate the first oligonucleotide above a threshold adjusted by controlling concentration of the second oligonucleotide," which is indefinite because the terms "said sequence," "the first oligonucleotide," and "the second oligonucleotide" lack antecedent basis.
It is unclear whether "said sequence" refers to the previously recited "signal sequence," or something else.
Regarding the terms "the first oligonucleotide," and "the second oligonucleotide," they are not previously recited in the claim.
For the purpose of compact prosecution and applying prior art under 35 USC§ 102 and 103, this phrase "said sequence being able to activate the first oligonucleotide above a threshold adjusted by controlling concentration of the second oligonucleotide" is interpreted as not further limit the claimed method, as the method does not require a "first oligonucleotide" nor a "second oligonucleotide" in any of its steps.
Claims 2-13 and 17-19 are rejected for depending from claim 1 and not remedying the indefiniteness.
B) Regarding claim 12, it recites "the compartment(s) that receive(s) a fluorescent signal" and "the compartment(s) that do(es) not receive a fluorescent signal," which are indefinite for lacking antecedent basis.
Specifically, the base claim 11 recites "compartments emitting a fluorescence," NOT compartments that receive a fluorescent signal. Thus, it is unclear to what these compartments in claim 12 refers.
New Grounds of 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.
The following are new grounds of rejections necessitated by Applicant's amendments. Although the claims were previously rejected as being unpatentable over the same reference(s), Applicant's amendments have necessitated the inclusion of new grounds of rejections in this Office action. It is noted that, to the extent that they apply to
the present rejection; Applicant's arguments are addressed following the rejection.
Claims 1-6, 8-13 and 17-19 are rejected under 35 U.S.C. 103 as being unpatentable over
Zhang (Zhang et al. Digital quantification of miRNA directly in plasma using integrated comprehensive droplet digital detection. Lab Chip. 2015 Nov 7;15(21):4217-26. doi: 10.1039/c5lc00650c. Epub 2015 Sep 21. PMID: 26387763; PMCID: PMC4631652) , in view of
Rondelez (WO2017141067A1 - Method of eliminating background amplification of nucleic acid targets; 2017-08-24) as evidenced by
Choudhary (Choudhary et al. ; Digital PCR: Helpful Tips When Using Droplet Partitioning Technology; November 9, 2016);
YUAN (Lin Yuan; Introduction of Droplet Digital PCR ; 2016.11.30) ;
Chang (WO2017209906A1 - Ac electrosprayed droplets for digital and emulsion pcr ; Published 2017-12-07); and
Kaushik (Kaushik et al. Droplet microfluidics for high-sensitivity and high-throughput detection and screening of disease biomarkers. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2018 Nov;10(6):e1522. doi: 10.1002/wnan.1522. Epub 2018 May 24. PMID: 29797414; PMCID: PMC6185786.).
A) Zhang teaches performing isothermal exponential amplification for detection of target nucleic acids in monodispersed droplets for digital quantification (Abstract; Scheme 1).
Regarding claim 1, Zhang teaches a digital method for detecting and/or quantifying at least one target biomolecule in a sample(Integrated Comprehensive Droplet Digital Detection; Scheme 1) comprising:
mixing said sample with a mixture including a buffer (pages 5-6, “Droplet generation of synthetic miRNA Let-7a spiked plasma,” lines 6-7), enzymes selected from the group consisting of polymerase, nicking enzyme or restriction enzyme, and exonuclease (pages 5-6, “Droplet generation of synthetic miRNA Let-7a spiked plasma,” lines 6-7), a first oligonucleotide (Fig. 1, template oligo for Exponential amplification reaction (EXPAR)) ;
partitioning the mixture obtained in step a) into several monodisperse compartments (Scheme 1; Fig. 2; ) so that a fraction higher than 1 % of the compartments does not contain the target biomolecule (Fig. 2; most droplets are not positive for containing target), and that the target biomolecules are randomly distributed following a Poisson law, in such a way that from zero to ten biomolecules are present in the compartments (page 8, lines 28-30, “8.85% of droplets with 1 miRNA per droplet on average after encapsulation”),
converting the target biomolecule into a signal (Fig. 1);
isothermally amplifying the signal (Fig. 1; Scheme 1), and
detecting and/or measuring said signal in each said monodisperse compartment, thereby allowing a direct counting of discrete events for each target biomolecule population and therefore the absolute quantification of the target biomolecules in the initial sample. (Page 8, “Droplet digital (dd) EXPAR for miRNA detection”; Figure 2; Scheme 1).
Zhang teaches the encapsulation of target molecule in droplets follows Poisson model by teaching droplet digital detection assay (Abstract). This is supported by Choudhary (pages 1-2) and YUAN (page 12), which describe that in digital assays ꟷ target molecules are randomly disturbed accordingly to a Poisson model.
With respect to compartment size, Zhang teaches droplet digital assays using a Y-junction for droplet generation (see Scheme 1), which are known in the art to generate droplets ranging from 10-50 microns in diameter, corresponding to about 1 pL per droplet, as evidenced by Chang (page 18, lines 24-28):
"With currently available technology, almost all droplets have been generated sequentially by flow focusing using Y junction microfluidic chips. The fastest droplet generation rate by this technique is about 30,000 droplets/second. The droplets generated are about 10-50 μm in diameter (about 1 picoliter per drop)."
A skilled artisan would have found it obvious to generate droplets within the claimed size range (e.g., 1 picoliter per drop), because Zhang already teaches a microfluidic system capable of generating 1 picoliter droplets, and droplet size is a well-known variable that need to be optimized in droplet microfluidic assays, as evidenced by Kaushik (page 22, lines 3-7):
“Optimization between droplet volume and sample volume for each assay may also be important. This is because although smaller droplet volumes can accelerate time-to-detection (Boedicker et al., 2008; Kaushik et al., 2017), they also reduce the volumetric throughput for performing the assay and can consequently lengthen the overall turn-around time if the target biomarker is rare (Rosenfeld, Lin, Derda, & Tang, 2014).”
Zhang teaches most of the claimed limitations, including an assay reaction mixture comprising buffer, enzymes, and oligonucleotides (pages 5-6; Fig. 1). Although Zhang does not explicitly disclose the use of a combination of oligonucleotides comprising an autocatalytic template; a pseudotemplate; and a conversion template, this feature is obvious in view of the knowledge in the art, as supported by Rondelez.
Rondelez specifically teaches an improved isothermal assays with reduced background amplification (Abstract; Fig. 1; Fig. 2). Rondelez teaches that its improved assay is “a move from an amplify-only exponential amplification system, which is bound to initiate amplification, toward a bistable system, which can stay indefinitely in any one of two alternative states, but also switch from one to the other.” Based on this new and general principle, background amplification can be delayed and even completely removed ([0071]).
Rondelez teaches an assay reaction mixture including buffer, enzymes, (Fig.1, 2 ; claim 1);
an autocatalytic template comprising a partial repeat structure including a nicking enzyme recognition site and capable of exponential amplification of a signal sequence (Fig.1, 2, amplification template ; claim 2, first oligonucleotide; page 19, lines 1-5 “amplification reaction was driven by a single amplification template, which was either a perfect dual repeat of an 11-mer containing the nicking enzyme recognition site, or had been shortened by one or two bases on the input (3') side”),
a pseudotemplate at a concentration of 5 nM to 15 nM (Fig.1, 2 ; claim 2; claim 6, “ threshold is adjusted by controlling a concentration of the second oligonucleotide”; [0044] lines 21-22, pseudo-template pTBe12T5SP is at concentration 6nM) comprising:
a 3' end sequence complementary to the signal sequence amplified by the autocatalytic template (Fig.1, 2), and
a 5' end sequence configured to be a template to add a deactivating tail to the signal sequence, preventing further priming on the autocatalytic template(Fig.1, 2), and
a conversion template comprising a target-binding region configured to generate the signal sequence upon polymerization and nicking (FIG. 1; 17); wherein the autocatalytic template and the pseudo template form a bistable amplification system having a threshold controlled by the concentration of the pseudotemplate ([0057]; claim 6, “ threshold is adjusted by controlling a concentration of the second oligonucleotide”; [0094]).
Rondelez directly cites Zhang (referred to as NPL 3) and identifies a limitation in Zhang's autocatalytic assaying approach ꟷ namely, high background amplification resulting in false positive signals:
"[0026] The problem is that template/polymerase and template/polymerase/nickase mixtures are prone to leaking reactions, and/or that the starting mixture may contain some analogs of the target that may induce some leaky production, albeit at a lower rate than the true target (see NPL 5). Because the detection is based on a positive feedback, even a minute production of trigger will inevitably lead to the start of the amplification loop, which will ultimately reach the high level, leading to a false positive detection in an end-point assay, or background amplification in the case of real-time monitoring. This weakness has been reported many times (see, for example, NPL 1-4), and the initiation by spurious polymerization or impurities has been investigated (see, for example, NPL 5). This "self- start" phenomenon is usually observed within the first hour, sometimes within a few minutes only. Such non-specific leak reaction significantly reduces the limit of detection, because self-triggering becomes in fact faster than triggering by minute amounts of targets. Moreover, it makes the analysis delicate, because, since the amplification will eventually happen anyway, one needs to monitor the time of apparition of the signal (and not only the signal itself) in order to infer the initial presence or absence of the target. However, "self- start" is a fundamental property of the design with positive feedback: it is well known from theory of dynamical system that autocatalytic loop produces first-order amplification with an intrinsically unstable 0 state. Whatever happens, they will eventually start to amplify, leading to a false positive." [emphasis added]
Rondelez provides an improvement to the method of Zhang by introducing additional oligos designed to "absorb" amplification signals (Fig. 1; Fig 2B; [0027]). Rondelez states that these improvements only "requires a few modifications of the protocol," which can act to reduce and even suppress completely the background amplification ([0027]).
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 droplet digital assay method taught in Zhang by incorporating the improved isothermal amplification strategy using the oligonucleotides combination disclosed in Rondelez, to reduce background amplification and mitigate false positives in Zhang's assay. A skilled artisan would have been motivated to make this modification because background signal is a known issue in autocatalytic isothermal amplification methods such as those in teachings of Zhang, as acknowledged in Rondelez. And as suggested by Rondelez, its assay utilizing additional oligonucleotides directly addresses this issue and provides a solution to improve assay performance by reducing background noise.
The person of ordinary skill would have had a reasonable expectation of success in combining these teachings, as these references are technically related and fall within the same field of nucleic acid detection using isothermal, autocatalytic amplification. The teachings of Rondelez are complementary to those of Zhang, and Rondelez specifically notes that the improvements as requiring minimal protocol changes. Therefore, a skilled artisan would have reasonably expected that applying the improved assay design with additional oligonucleotides and reaction conditions of Rondelez to the droplet-based digital quantification system of Zhang would yield the predictable result of reduced background.
B) Regarding claim 2, Zhang teaches the target biomolecule is a nucleic acid (Abstract, miRNA).
Regarding claim 3, Zhang teaches micro RNA (Abstract, miRNA).
Regarding claim 4, Rondelez teaches Bst 2.0 DNA polymerase (FIG.5).
Regarding claim 5, Rondelez teaches the first oligonucleotide includes a partial repeat structure containing a nicking enzyme recognition site ([0009]).
Regarding claim 6, Rondelez teaches a reporting probe (Fig. 1, reporter module; Fig. 25-26).
Regarding claim 8, Zhang teaches partitioning mixture into droplets (Scheme 1).
Regarding claim 9, as discussed above for claim 1, Zhang evidenced by Chang teaches 1 pL droplet size.
Regarding claim 10, Zhang teaches signal is labelled (Fig. 1, EvaGreen labeling).
Regarding claim 11, Zhang teaches detecting and/or counting the compartments emitted a fluorescence (Scheme 1).
Regarding claim 12, it recites
"wherein, for measuring the absolute concentration of the target biomolecule in the tested biological sample, the compartment(s) that receive(s) a fluorescent signal and the compartment(s) that do(es) not receive a fluorescent signal are counted and the ratio of compartment(s) that receive a fluorescent signal to compartment(s) that do not receive a fluorescent signal is calculated."
This limitation is obvious in view of the combined teachings of Zhang and Rondelez because it does not further limit the claimed method.
Per MPEP 2111.04, a wherein clause can limit a method claim if it contributes meaning and purpose to the manipulative steps. In this instant case, the "wherein" clause does not introduce any required additional steps or modify any existing step. The base claims 11 and 1 do not require any step of "measuring the absolute concentration of the target biomolecule in the tested biological sample."
Therefore, this claim language is interpreted as descriptive statement without any associated active steps and do not distinguish the claims from the prior art.
Regarding claim 13, Zhang teaches using microRNAs as biomarkers (page 2, introduction, para 1).
Regarding claim 17, Rondelez teaches converted signal is a DNA single strand (Fig. 17, trigger is ssDNA).
Regarding claim 18, Rondelez teaches fluorescent probe (Fig. 1, reporter module; Fig. 25-26).
Regarding claim 19, Zhang teaches water-in-oil emulsion droplets (scheme 1).
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang, in view of Rondelez, as applied to claim 1 above and further in view of Padirac (Padirac et al., Bottom-up construction of in vitro switchable memories. Proc Natl Acad Sci U S A. 2012 Nov 20;109(47):E3212-20. doi: 10.1073/pnas.1212069109. Epub 2012 Oct 29. PMID: 23112180; PMCID: PMC3511151)
The teachings of Zhang and Rondelez are recited above and applied as for base claim 1.
Regarding claim 7, Rondelez teaches a reporter oligonucleotide that emits fluorescence upon hybridizing to the amplified trigger sequence (Fig 1). Although Rondelez does not specifically teach using this reporter oligonucleotide for detecting two or more targets, wherein the oligonucleotide comprises the claimed 3’ and 5’ structures, these features are obvious in view of the knowledge in the art.
Padirac specifically teaches a nucleic acid circuit system that allows for specific and simultaneous multiplex detection of two templates (see Fig. 3A and legends; page E3215, left-hand col, lines 8-9), wherein the reporter oligonucleotide, by binding to its corresponding target produces the inhibitor sequence (referred to as “leak-absorption oligonucleotide” in Rondelez) for the other target.
Padirac teaches that the reporter oligonucleotide is composed of a 3' end at least partially complementary to the signal strand amplified by a first bistable system, a 5' end complementary to a leak absorption oligonucleotide of a second bistable system, and a nicking recognition in between (FIG. 3A; FIG. 6A, αtoiβ for example).
Padirac further teaches advantages of its system:
“The advantages of the present design are twofold: (i) Both stable states correspond to a high concentration of one of two species (and not to the presence or absence of a single species), making the reading and interfacing easier, and (ii) the symmetry facilitates the identification of the control parameters for the network behavior. In particular, even for sequences that are not symmetrical, one can theoretically tune the concentrations of templates to obtain and balance the bistable domain. In practice, this proved to be a useful feature for the construction of the more complex target behaviors.”(page E3217, right-hand col, para2, lines 9-19).
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 combined teachings of Zhang and Rondelez to employ a nucleic acid circuit system in droplet digital detection, in view of the specific teachings of Padirac regarding reporter oligonucleotide design that permits production of an inhibitor sequence for another target, thereby enabling simultaneous multiplex detection.
The skilled artisan would have been motivated to make this modification in order to leverage the additional advantages suggested by Padirac, including simultaneous, real-time multiplex detection and easier reading and interfacing of results from multiple targets.
The person of ordinary skill would have had a reasonable expectation of success because the references are in the same or closely related field of nucleic acid target detection using nucleic acid circuits, and their teachings are technically compatible.
Response to Arguments: Claim Rejections - 35 USC § 103
The previously set forth 103 rejections of claims 1-13 and 17-19 have been withdrawn in view of the recent claim amendment filed on May 14, 2026, which added new limitations to the claims, that are not addressed in the previous rejections.
Applicant's arguments filed on May 14, 2026 have been fully considered but are not found persuasive.
First, with respect to Zhang, Applicant asserts that the droplet volume is 15 pL (remarks, page 19), “which is incompatible with the problem Applicants' invention solves/would not work for Applicants' claimed invention.” And that Zhang’s method improperly dilutes the target template.
This argument is not persuasive as it fails to consider the rejection as a whole based on combinations of references. One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. In reKeller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In reMerck & Co., Inc., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Here, Zhang does not teach away from a smaller droplet size, and, as discussed in the rejection, Zhang teaches droplet digital assays using a Y-junction for droplet generation (see Scheme 1), which are known in the art to generate droplets ranging from 10-50 microns in diameter, corresponding to about 1 pL per droplet, as evidenced by Chang (page 18, lines 24-28).
Applicant further asserts that a person of ordinary skill in the art would not have had a reasonable expectation of success that the method of Rondelez would function reliably in picoliter digital compartments containing zero or ten target molecules, because Rondelez teaches reactions in bulk, and that "one of ordinary skill in the art would not have known what is required to transfer an amplification method to a digital format." (Remarks, page 23-25).
Although this argument appears detailed, it does not provide any objective evidence sufficient to support Applicant's assertions regarding the knowledge in the art or a skilled artisan's reasonable expectation of success. For example, does the prior art consistently teach failures in transferring bulk amplification assays to digital counterparts?
No objective evidence has been provided to support these assertations or to establish what would be considered general knowledge in the art. As the arguments of counsel cannot take the place of evidence in the record, assertions made without objective evidence from relevant references are insufficient to overcome the prior art rejections under 35 USC 103.
Applicant also argues that because the teachings of Rondelez differ from those of digital assay with respect to kinetic behavior and low target-copy input, one of ordinary skill in the art would not have had a reasonable expectation of success (Remarks, page 23-25).
These points are likewise unpersuasive for the same reason as above.
Moreover, Rondelez does not teach away from digital assays or low-target-copy inputs. To the contrary, it teaches ultrasensitive detection of low-copy-number targets, including detection of microRNA targets at 1 fM ([0073]-[0074]), which is a far lower concentration of a single molecule in a 1 picoliter droplet (approximately 1.66 pM).
Rondelez further teaches that its background-removal mechanism is rooted in a bistable dynamical system, rather than changes in kinetic rates ([0057]).
"[0057] FIG. 8A shows an evaluation of the evolution of the background amplification time as a function of the concentration of pseudo-template in the mixture. When the inverse of the background amplification time is plotted as a function of pseudo-template, a line intersects the x-axis. This confirms that the background amplification time is delayed to hyperbolically increasing time, using small and finite concentrations of pseudo-templates. This is a signature of the bifurcation to the bistable regime. This proves that the working principle of background removal as presented here is rooted in out-of- equilibrium dynamical systems, and not in improvement of the specific kinetic rates versus the non-specific kinetic rates. "
Accordingly, a person of ordinary skill in the art would have had a reasonable expectation of success to modify the droplet digital assay method taught in Zhang by incorporating the improved isothermal amplification strategy using the oligonucleotides combination disclosed in Rondelez, to reduce background amplification and mitigate false positives in Zhang's assay
Double Patenting- Obvious Type -- New Grounds
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-3, 5-6, 9-11, 13 and 17-18 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over claims 1, 9-10, 12-15 of U.S. Patent No.11111529B2 in view of Zhang (Zhang et al. Digital quantification of miRNA directly in plasma using integrated comprehensive droplet digital detection. Lab Chip. 2015 Nov 7;15(21):4217-26. doi: 10.1039/c5lc00650c. Epub 2015 Sep 21. PMID: 26387763; PMCID: PMC4631652).
Instant claim 1 recites:
A digital method for detecting and/or quantifying at least one target biomolecule in a sample comprising the following steps:
a) mixing said sample with a mixture including
a buffer(‘529 Patent, claim 1),
enzymes (‘529 Patent, claim 1) selected from the group consisting of polymerase, nicking enzyme or restriction enzyme, and exonuclease,
an autocatalytic template (‘529 Patent, claim 1)comprising a partial repeat structure including a nicking enzyme recognition site and capable of exponential amplification of a signal sequence,
a pseudotemplate at a concentration of 5 nM to 15 nM (‘529 Patent, claim 1) comprising:
a 3' end sequence complementary to the signal sequence amplified by the autocatalytic template, and
a 5' end sequence configured to be a template to add a deactivating tail to the signal sequence, preventing further priming on the autocatalytic template, and
a conversion template (‘529 Patent, claim 1) comprising a target-binding region configured to generate the signal sequence upon polymerization and nicking,
said sequence being able to activate the first oligonucleotide above a threshold adjusted by controlling concentration of the second oligonucleotide
;
wherein the autocatalytic template and the pseudo template form a bistable amplification system having a threshold controlled by the concentration of the pseudotemplate (‘529 Patent, claim 6);
b) partitioning the mixture obtained in step a) into several monodisperse compartments so that a fraction higher than 1 % of the compartments does not contain the target biomolecule, and that the target biomolecules are randomly distributed following a Poisson law, in such a way that from zero to ten biomolecules are present in the compartments, the size of said compartments being 0.01 to 10 pL;
c) converting the target biomolecule into a signal (529 Patent, claim 14);
d) isothermally amplifying the signal (‘529 Patent, claim 1), and
e) detecting and/or measuring said signal in each said monodisperse compartment, thereby allowing a direct counting of discrete events for each target biomolecule population and therefore the absolute quantification of the target biomolecules in the initial sample.
The ‘529 Patent claims most of the limitations recited in the instant claim 1, including performing an isothermal amplification assay by adding a reaction mixture to a sample, wherein the mixture comprises buffer, enzymes, and a combination of three oligonucleotide as claimed.
While the ‘529 Patent's claims do not cover the digital assay aspect ꟷ specifically, partitioning the mixture into monodisperse compartments and detecting signal within each compartment for counting discrete amplification events ꟷ these features are obvious modifications in view of Zhang.
Zhang teaches methods of performing droplet digital amplification assay, which adapts conventional isothermal amplification reactions to a microfluidic droplet format, thereby enabling digital quantification. Zhang discloses partitioning the reaction mixture into monodisperse compartments and detecting amplification signals within individual compartments to count discrete positive events (see. e.g., Scheme 1)
Zhang also highlights the advantages of this approach, including the ability to perform digital quantification, specific target detection, and rapid, high-throughput droplet counting (page 3, lines 5-8).
Accordingly, it would have been prima facie obvious to a person of ordinary skill in the art to modify the isothermal amplification method of the ‘529 Patent by incorporating the droplet digital amplification format taught by Zhang, to leverage the benefit of digital quantification and improved analytical performance, as suggested by Zhang.
Therefore, the instant claim 1 lacks patentable distinction over the ‘529 Patent in view of Zhang.
Therefore, instant claims 1, 5, 9 are obvious over claim 1, 9, 14 of the '529 patent, in view of Zhang. Instant claims 2,3,13; 6; 10, 17; 11,18; are obvious over claims 10;12; 15; 13 of the '529 patent, in view of Zhang.
Claims 1-3, 6, 8-11, 13 and 19 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 5-6, 11-12, 17-18 of copending Application No. 17/614,023 (reference application, amended claims filed on 05/28/2026), in view of Rondelez (WO2017141067A1 - Method of eliminating background amplification of nucleic acid targets; 2017-08-24).
Instant claim 1 recites:
A digital method for detecting and/or quantifying at least one target biomolecule in a sample comprising the following steps:
a) mixing said sample with a mixture including
a buffer (‘023 Application, claim 1),
enzymes (‘023 Application, claim 1)selected from the group consisting of polymerase, nicking enzyme or restriction enzyme, and exonuclease,
an autocatalytic template (‘023 Application, claim 1)comprising a partial repeat structure including a nicking enzyme recognition site and capable of exponential amplification of a signal sequence,
a pseudotemplate (‘023 Application, claim 1) at a concentration of 5 nM to 15 nM comprising:
a 3' end sequence complementary to the signal sequence amplified by the autocatalytic template, and
a 5' end sequence configured to be a template to add a deactivating tail to the signal sequence, preventing further priming on the autocatalytic template, and
a conversion template (‘023 Application, claim 1)comprising a target-binding region configured to generate the signal sequence upon polymerization and nicking,
said sequence being able to activate the first oligonucleotide above a threshold adjusted by controlling concentration of the second oligonucleotide
;
wherein the autocatalytic template and the pseudo template form a bistable amplification system having a threshold controlled by the concentration of the pseudotemplate (‘023 Application, claim 22);
b) partitioning the mixture obtained in step a) into several monodisperse compartments so that a fraction higher than 1 % of the compartments does not contain the target biomolecule, and that the target biomolecules are randomly distributed following a Poisson law, in such a way that from zero to ten biomolecules are present in the compartments, the size of said compartments being 0.01 to 10 pL (‘023 Application, claim 1, 17);
c) converting the target biomolecule into a signal (‘023 Application, claim 1);
d) isothermally amplifying the signal (‘023 Application, claim 1), and
e) detecting and/or measuring said signal in each said monodisperse compartment, thereby allowing a direct counting of discrete events for each target biomolecule population and therefore the absolute quantification of the target biomolecules in the initial sample (‘023 Application, claim 1, microchamber arrays comprise monodisperse compartments).
The claims of the '023 Application largely overlap with the instant claim 1. While the '023 Application claims a set of oligonucleotides including a conversion template (‘023 Application, claim 1, conversion oligonucleotide), a pseudotemplate (‘023 Application, claim 1, leak absorption oligonucleotide), and autocatalytic template (‘023 Application, claim 1, amplification oligonucleotide), it does not specifically claim structural features for these oligonucleotides. However, these are obvious, known structure features for these oligonucleotides in a nucleic acid circuit system, this is supported by Rondelez.
Rondelez teaches:
an autocatalytic template comprising a partial repeat structure including a nicking enzyme recognition site and capable of exponential amplification of a signal sequence (Fig.1, 2, amplification template ; claim 2, first oligonucleotide; page 19, lines 1-5 “amplification reaction was driven by a single amplification template, which was either a perfect dual repeat of an 11-mer containing the nicking enzyme recognition site, or had been shortened by one or two bases on the input (3') side”),
a pseudotemplate at a concentration of 5 nM to 15 nM (Fig.1, 2 ; claim 2; claim 6, “ threshold is adjusted by controlling a concentration of the second oligonucleotide”; [0044] lines 21-22, pseudo-template pTBe12T5SP is at concentration 6nM) comprising:
a 3' end sequence complementary to the signal sequence amplified by the autocatalytic template (Fig.1, 2), and
a 5' end sequence configured to be a template to add a deactivating tail to the signal sequence, preventing further priming on the autocatalytic template(Fig.1, 2), and
a conversion template comprising a target-binding region configured to generate the signal sequence upon polymerization and nicking (FIG. 1; 17),
Given these teachings, it would have been prima facie obvious for one of ordinary skill in the art to use a combination of nucleic acid circuit oligonucleotides having specific structural features as taught by Rondelez in the method in the ‘023 Application, because both references are in the overlapping field of detecting target molecules using nucleic acid circuits. The use of a combination of oligonucleotides with specific structures, as disclosed in Rondelez, to achieve the same function of generating detectable amplification signal, as claimed in ‘023 Application, represents a predictable use of prior art elements according to known methods to yield predictable results (see MPEP §2143).
Therefore, instant claims 1, 6, 8-10 are obvious over claim 1, 5, 17 of the '023 Application , in view of Rondelez.
Instant claims 2-3; 11; 13; 19 are obvious over claims 11;18; 12; 6 of the '023 Application , in view of Rondelez.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Prior Art
Below are relevant prior art not used in rejection but pertinent to the claims or disclosure.
Digital detection of Target nucleic acid using Autocatalytic Nucleic Acid Circuits with inhibitor sequence, in monodisperse compartments is known in the art:
See Desbois et al., A microfluidic device for on-chip agarose microbead generation with ultralow reagent consumption. Biomicrofluidics. 2012 Oct 9;6(4):44101. doi: 10.1063/1.4758460. PMID: 24106525; PMCID: PMC3482248.
The use of a bistable autocatalytic loop with tunable threshold by varying concentration of a drain template is known in the art:
See Montagne, K., Gines, G., Fujii, T. et al. Boosting functionality of synthetic DNA circuits with tailored deactivation. Nat Commun 7, 13474 (2016). https://doi.org/10.1038/ncomms13474;
See GINES et al. ; WO2017141068A1 - Molecular computing component and method of molecular computing; Published on 2017-08-24.
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.
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/TIAN NMN YU/Examiner , Art Unit 1681 /AARON A PRIEST/Primary Examiner, Art Unit 1681
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