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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 September 05, 2025 has been entered.
Examiner Assignment
This application has been transferred from Examiner Suryaprabha Chunduru to Examiner Tian Yu. All future correspondence should be directed to Examiner Tian Yu whose contact information appears at the end of this Office Action.
Objection to Specification
The disclosure is objected to because page 55, line 3 of the specification contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Status of Claims / Response to Amendment
This office action is in response to an amendment filed on September 05, 2025.
Claims 1-20 were previously pending. Applicant amended claims 1 and 4.
Claims 1-20 are currently pending, with claims 14-16 and 20 withdrawn.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. The objection to claim 4 has been withdrawn in view of amendment to the claim. All of the previously presented rejections have been withdrawn as being obviated by the amendment of the claims.
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.
Claim Objections
Claim 11 is objected to because of the following informalities:
In claim 11, line 1, "step d)" should read "step e)."
In claim 11, line 3, "emitted a fluorescence" should read "emitting a fluorescence."
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
In evaluating the patentability of the claims presented in this application, claim terms have been given their broadest reasonable interpretation (BRI) consistent with the specification, as understood by one of ordinary skill in the art, as outlined in MPEP§ 2111.
Regarding claim 1, it recites oligonucleotides in a mixture, described in functional languages.
MPEP§ 2111.04 states: "Claim scope is not limited by claim language that suggests or makes optional but does not require steps to be performed, or by claim language that does not limit a claim to a particular structure."
Claim 1 recites:
"a first oligonucleotide which is an amplification oligonucleotide exponentially amplifying a signal sequence,
a second oligonucleotide at a concentration of from 5 nM to 15 nM,
which is a leak absorption oligonucleotide driving the deactivation of a signal sequence synthetized by leaky reactions and therefore allowing to avoid nonspecific amplification and
which has a 3' end which is complementary to the sequence amplified by the first oligonucleotide, and a 5' end that serves as a template to add a deactivating tail to the amplified sequence, and
a third oligonucleotide which is a target-specific conversion oligonucleotide converting the target biomolecule to a signal sequence and whose 3' end can bind to a target sequence and upon polymerization and nicking, the third oligonucleotide outputs a sequence able to activate the first oligonucleotide above the threshold adjusted by controlling concentration of the second oligonucleotide."
In this instant case, the above underlined portion in the claim are functional languages. Without specific structures in the invention that directly support or relate to these functional language, these functional languages are interpreted under BRI and are construed as descriptive statements that do not further distinguish the claimed oligonucleotides from the oligonucleotides in prior art mixtures that disclose all the structural and compositional features.
Furthermore, these functional languages do not meaningfully limit the claimed method, as the method does not require performing any of the described functions. While the claim later recites an amplification step in "d) isothermally amplifying the signal," this step does not require the use of any of the oligonucleotides in the mixture. As such, these descriptive statements do not further distinguish the claimed method from prior art methods.
Additionally, the claim defines:
a "first oligonucleotide" as "an amplification oligonucleotide";
a "second oligonucleotide" as "a leak absorption oligonucleotide"; and
a "third oligonucleotide" as "a target-specific conversion oligonucleotide."
However, the terms "amplification oligonucleotide," " leak absorption oligonucleotide," and "target-specific conversion oligonucleotide" do not contribute any structural features to the claimed oligonucleotides, as they are defined solely by their functional roles in the specification, with no defined structural features (see page 11) 1:
“In the context of the present invention, the term “amplification oligonucleotide” or “autocatalytic template” or “aT” designs an oligonucleotide which is able to exponentially amplifies the trigger sequence.”
“In the context of the present invention, the term “leak absorption oligonucleotide” or “pseudotemplate” or “pT” relates to the oligonucleotide which binds the amplified sequence more strongly than the autocatalytic template but only adds a few nucleotides at its 3′ end and thus deactivates it for further priming on the autocatalytic template (because its 3′ end is now mismatched on the autocatalytic template).”
“In the context of the present invention, the term “target specific conversion oligonucleotide” or “conversion template” or “cT” relates to an oligonucleotide which converts the target biomolecule to a universal trigger sequence (or also called herein “signal” or “signal sequence”).“
Regarding the "second oligonucleotide," the claim recites that it has a 3' end and a 5' end, with the 3' end being "complementary to the sequence amplified by the first oligonucleotide." Since the claim does not define the sequence amplified by the first oligonucleotide with any specific nucleotide sequence, it encompasses any sequence. Accordingly, the sequence complementary to any sequence also encompasses any sequence.
Therefore, under BRI, the oligonucleotides in the claimed mixture are interpreted as including: a first oligonucleotide encompassing any sequence, structure, and length; a second oligonucleotide comprising a 3' end and a 5' end, having any sequence and length, at a concentration of 5 nM to 15 nM; a third oligonucleotide having a 3'end, encompassing any sequence and length.
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.
For the purpose of applying prior art, claim 7 recites a "fifth oligonucleotide," described solely in functional language that outline intended use:
"a fifth oligonucleotide which is a cross inhibiting oligonucleotide for detecting and/or quantifying two or more biomolecules, wherein the fifth oligonucleotide is able to produce a leak absorption oligonucleotide of the opposite switch, thereby acting as a cross inhibitor for the amplification and mitigating unspecific crosstalk."
The terms "for", "able to" suggest intended use, without claiming specific structures in the oligonucleotide that directly support or relate to these functional language. The recited intended uses fail to distinguish the claimed oligonucleotide over the prior art oligonucleotide with the same physical components. See MPEP § 2111.05.
Claim Rejections - 35 USC § 112(b) -- New
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 "a 3' end which is complementary to the sequence amplified by the first oligonucleotide," which is indefinite because "the sequence amplified by the first oligonucleotide" lacks antecedent basis. The claim does not previously recite any sequence amplified by the first oligonucleotide with any specific sequence. It is unclear if this term refers to the "signal sequence" or an entirely different sequence not recited in the claim.
Claim 1 recites "a 5' end that serves as a template to add a deactivating tail to the amplified sequence," which is indefinite because "the amplified sequence" lacks antecedent basis. The claim does not recite any prior amplification steps, only functional descriptions for the oligonucleotides. And in the functional descriptions it is not clear if "the amplified sequence" refers to the products of the prior mentioned exponential amplification, nonspecific amplification, or something else.
Claim 1 also recites "the threshold," which lacks antecedent basis.
Claims 2-13 and 17-19 are rejected for depending from claim 1 and not remedying the indefiniteness.
B) Regarding claim 6, it recites "the trigger sequence," which lacks antecedent basis.
For the purpose of compact prosecution and applying prior art under 35 USC§ 102 and 103, “trigger sequence” is interpreted as encompassing any sequence.
Claim 18 is rejected for depending from claim 6 and not remedying the indefiniteness.
C) Regrading claim 12, it recites "the compartments receiving the fluorescent signal," which lacks antecedent basis.
Claim Rejections - 35 USC § 112(d) -- New
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 9 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Regarding claim 9, it depends from claim 8/1 and recites "wherein the size of droplet is between 0.001 and 100 pL." This claim is an improper dependent claim because it fails to further limit the scope of its base claim. Base claim 1 recites a narrower size range of 0.01 to 10 pL. By expanding the upper limit to 100pL, claim 9 broadens rather narrows the scope of its base claims. Accordingly, claim 9 fails to further limit the subject matter of claim 8/1, from which it depends.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103 -- New
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 1-13 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) ; and
Chang (WO2017209906A1 - Ac electrosprayed droplets for digital and emulsion pcr ; Published 2017-12-07).
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)."
Although Zhang teaches most of the claimed limitations, including an assay reaction mixture comprising buffer, enzymes, and oligonucleotides (pages 5-6; Fig. 1), it does not explicitly disclose the use of a first, second, and third oligonucleotide as required by the claim.
Rondelez fills this gap by specifically teaching isothermal assays with reduced background amplification (Abstract; Fig. 1; Fig. 2).
Regarding claim 1, Rondelez teaches an assay reaction mixture including buffer, enzymes, a first oligonucleotide (Fig.1, 2 ; claim 1); a second oligonucleotide at a concentration of from 5 nM to 15 nM (Fig.1, 2 ; claim 1; claim 6, “ threshold is adjusted by controlling a concentration of the second oligonucleotide”; [0044] lines 21-22, pseudo-template pTBe12T5SP is at concentration 6nM); and a third oligonucleotide (Fig.1, 2, 17, “capture template”; claim 1).
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 7, Rondelez teaches adding a fifth oligonucleotide (page 5, lines 1-5).
Regarding claim 8, Zhang teaches partitioning mixture into droplets (Scheme 1).
Regarding claim 9, it is obvious in view of the combined teachings of Zhang and Rondelez because it does not further limit the claimed method. See 35 USC § 112(d) section above for detailed discussion.
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 compartments receiving the fluorescent signal and the non-fluorescent compartments are counted and their ratio 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).
Double Patenting- Obvious Type -- New
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,
a first oligonucleotide (‘529 Patent, claim 1) which is an amplification oligonucleotide exponentially amplifying a signal sequence,
a second oligonucleotide (‘529 Patent, claim 1) at a concentration of from 5 nM to 15 nM (‘529 Patent, claim 9),
which is a leak absorption oligonucleotide driving the deactivation of a signal sequence synthetized by leaky reactions and therefore allowing to avoid nonspecific amplification and
which has a 3' end which is complementary to the sequence amplified by the first oligonucleotide, and a 5' end that serves as a template to add a deactivating tail to the amplified sequence, and
a third oligonucleotide (‘529 Patent, claim 1) which is a target-specific conversion oligonucleotide converting the target biomolecule to a signal sequence and whose 3' end can bind to a target sequence and upon polymerization and nicking, the third oligonucleotide outputs a sequence able to activate the first oligonucleotide above the threshold adjusted by controlling concentration of the second oligonucleotide;
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 first, second, and third oligonucleotide as claimed.
While ‘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 appear to be 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). Although the claims at issue are not identical, they are not patentably distinct from each other because the instant claims are anticipated by the claims (filed on 12/05/2025) of the '023 application.
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 (‘023 Application, claim 5),
a first oligonucleotide (‘023 Application, claim 1) which is an amplification oligonucleotide exponentially amplifying a signal sequence,
a second oligonucleotide (‘023 Application, claim 1) at a concentration of from 5 nM to 15 nM,
which is a leak absorption oligonucleotide driving the deactivation of a signal sequence synthetized by leaky reactions and therefore allowing to avoid nonspecific amplification and
which has a 3' end which is complementary to the sequence amplified by the first oligonucleotide, and a 5' end that serves as a template to add a deactivating tail to the amplified sequence, and
a third oligonucleotide (‘023 Application, claim 1) which is a target-specific conversion oligonucleotide converting the target biomolecule to a signal sequence and whose 3' end can bind to a target sequence and upon polymerization and nicking, the third oligonucleotide outputs a sequence able to activate the first oligonucleotide above the threshold adjusted by controlling concentration of the second oligonucleotide;
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).
Therefore, instant claims 1, 6, 8-10 are anticipated by claims 1, 5, 17 of the '023 application. Instant claims 2-3; 11; 13; 19 are anticipated by claims 11;18; 12; 6 of the '023 application, respectively.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Conclusion
The specification is objected to in this Office Action.
Claim 11 is objected to; claims 1-13 and 17-19 are rejected. No claims are allowed.
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/TIAN NMN YU/Examiner , Art Unit 1681 /AARON A PRIEST/Primary Examiner, Art Unit 1681
1 Although the specification includes descriptions indicating that some oligos need to be protected from degradation by adding modifications to the 5’ end (see for example, page 11, lines 12-17; 21-22), these descriptions do not appear to constitute an express definition of the terms. Therefore, under BRI, the oligonucleotides referred to by these terms "amplification oligonucleotide," " leak absorption oligonucleotide," and "target-specific conversion oligonucleotide" are understood as not being defined to necessarily include 5’ end modifications.