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 February 03, 2026.
Claims 1-18 and 20-39 were previously pending. Applicant amended claims 1 and 10.
Claims 1-18 and 20-39 are currently pending, with claims 3-17, 22, 32-38 withdrawn.
Claims 1-2, 18,20-21, 23-31 and 39 are under consideration.
Applicant's claim amendments and arguments overcame the following rejections:
Rejections of claims 1-2, 18, 20-21, 23-31 and 39 under 35 U.S.C. 112(b);
Rejections of claims 1-2, 18,20-21, 23-31 and 39 under 35 U.S.C. 112(a).
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 1-2, 18,20-21, 23-31 and 39 is 05/03/2019, the filling date of the provisional application PRO 62/843,193. Because PRO 62/843,193 is the first to disclose "isothermal cycling" of P1 and P2 primers, required by independent claim 1.
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.
Regarding claim 1, it has been amended to recite an isothermal method comprising a hybridizing step, an extending step, and a "thermally denaturing" step, all performed at the " primer-cycling temperature."
While "thermally denaturing" of DNA is commonly understood in the art to require a temperature of 95°C or higher (see Non-Final Rejection - 11/03/2025, page 7 for detailed discussion), Applicant's remarks filed on February 03, 2026 explicitly disavow this conventional definition and provide a specific definition within the context of the claimed invention:
"Applicant has amended the hybridizing, extending, and thermally denaturing steps of claim 1 to recite: "hybridizing, at the primer-cycling temperature during the incubating, ... "; "extending, at the at the primer-cycling temperature during the incubating, ... "; and "thermally denaturing, at the primer-cycling temperature during the incubating…" to unambiguously clarify that these steps are all at the primer- cycling temperature.
As taught in the specification (see, e.g., at page 24, lines 11-15, teaching that in "isothermally accelerated amplification" ... "forward and reverse primers hybridize to the corresponding target strand, become extended by a DNA polymerase, and wherein their respective extension product(s) denature at the same "cycling" temperature (i.e., isothermally)"; see also page 33, lines 19-25; etc.), the primers are selected/designed such that they thermally denature at the primer-cycling temperature, which is compatible with hybridizing and extending at the primer-cycling temperature-and thus "thermally denaturing," as recited in claim 1, is clearly different from the classic PCR denaturation step (≥ 95°C), as would be immediately understood by one of ordinary skill in the art. " [emphasis added](Remarks, page 14)
Applicant's special definition provided in the remarks is accepted. Accordingly, in this application, "thermally denaturing" will be construed as denaturing at the same temperature as the hybridization and extension steps.
Claim 1 recites the term "isothermally-accelerated amplification," which is not explicitly defined in the applicant's disclosure. Page 23 of specification provides the following description regarding this term :
"The term "isothermally-accelerated amplification" collectively relates herein to the methods of the invention, wherein one of two (FIGS. 1 and 3), or both (FIG.2), forward and reverse primers hybridize to the corresponding target strand, become extended by a DNA polymerase, and wherein their respective extension product(s) denature at the same "cycling" temperature (i.e., isothermally). These primers (e.g., P1 and P2) can be referred to herein as "forward" and "reverse." This forward/reverse terminology, however, does not necessarily assign any special properties to the primers other than their relation to each other, the target nucleic acid sequence, and the target strands." (page 23, lines 19-27)
Therefore, the term "isothermally-accelerated amplification" is interpreted under BRI to encompass any assay that employ primers, target nucleic acid, and primer extension by DNA polymerase, wherein the extension product(s) are denatured at the same temperature as applied to primer hybridization and extension in the same assay.
Claim 1 recites the term "primer-cycling temperature," which is described by the applicant's disclosure in pages 23-24:
"In the methods, the phrase "incubating the reaction mixture at a primer Cycling temperature," as used herein, means an exposure of the reaction mixture to a temperature or temperature range that supports all three steps of the isothermal amplification reaction of the invention, i.e., (i) hybridization of a primer to a target template strand, (ii) extension of the primer by a DNA polymerase to produce a double-stranded target amplicon, and (iii) denaturation of the amplicon providing two target strands single-stranded, one of which serves as a primer template for another molecule of the primer in the next consecutive isothermal cycle (e.g. FIG.1). "
Therefore, under BRI, this term "primer-cycling temperature" is interpreted to mean a single temperature that is applied to hybridization, extension, and denaturation steps of a nucleic acid assay.
Claim 21 recites "DNA polymerase- compatible structural modification," which is not defined in the applicant's disclosure. Page 19 of specification provides the following descriptions regarding this term:
"In methods of the invention, the term "structural modifications" refers to any chemical substances such as atoms, moieties, residues, polymers, linkers or nucleotide analogs that are usually of a synthetic nature, and which are not commonly present in natural nucleic acids." (page 19)
"The ‘polymerase-compatible’ structural modifications refer to modifications that do not block DNA polymerase activity in extending the hybridized primers and/or that replicate the primer sequence incorporating these modifications." (page 19)
Therefore, the term "DNA polymerase- compatible structural modification" is interpreted under BRI to mean any type of modification that are not commonly present in natural nucleic acids, which also does not inhibit DNA polymerase activity.
Claim Rejections - 35 USC § 102 -- New Grounds
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-2, 18, 20-21, 23-30 and 39 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Piepenburg (Piepenburg et al.; US8062850B2- Methods for multiplexing recombinase polymerase amplification; 2011-11-22).
Regarding claim 1, Piepenburg teaches a method for isothermally-accelerated amplification (col 12, lines 36-50; Fig. 12) of a target nucleic acid sequence, comprising:
incubating a reaction mixture at a primer-cycling temperature (col 12, lines 36-50; Fig. 12; col 14, lines 39-42, e.g., 37°C), the reaction mixture being sufficient to support DNA synthesis and containing
active DNA polymerase (Fig.12; example 2, Bsu polymerase) ,
complementary, first target sequence template strand and second target sequence template strand (Fig.12; example 2) ,
a first oligonucleotide primer (P1) complementary to a P1 primer binding site on a 3'-terminal portion of the first target sequence template strand (Fig.12, primer hybridize to homologous sequences on target strand; example 2) ,
a second oligonucleotide primer (P2) complementary to a P2 primer binding site on a 3'-terminal portion of the second target sequence template strand (Fig.12, primer hybridize to homologous sequences on target strand; example 2),
the P1 and P2 primers each present in excess molar concentration relative to the first and second target sequence template strands (In an amplification reaction, the number of primers always exceed the number of input templates to support the amplification process, as a primer is “consumed” via incorporation, each time an amplified strand is created; for example see col 22, lines 2-31, the lowest primer input is 70nM in a 20µl reaction, which is 8.43* 10^11 copies, much higher than the highest template input of 10^4 copies ), respectively;
hybridizing, at the primer-cycling temperature during the incubating, P1 and P2 primers to the first and the second target sequence template strands, respectively (Fig.12; col 30; example 2 ; col 14, lines 39-42, e.g., 37°C);
extending, at the primer-cycling temperature during the incubating, the hybridized P1 and P2 primers to produce second and first target sequence template strands, respectively (Fig.12; col 30; example 2 ; col 14, lines 39-42, e.g., 37°C);
thermally denaturing, at the primer-cycling temperature during the incubating, the first and the second target sequence template strands to provide the first and the second target sequence template strands in P1- and P2-primable form, respectively (Fig.12; col 30; example 2 ; col 14, lines 39-42, e.g., 37°C); and
cyclically repeating, during the incubating, the hybridizing, extending and denaturing steps for the P1 and P2 primers isothermally at the primer-cycling temperature to provide isothermal P1 and P2 primer-driven cycling (Fig.12; col 12, lines 50-51),
wherein in each consecutive P1 and P2 isothermal cycle, at least some of the respective second and first target sequence template strands produced in and accumulated over all prior isothermal cycles serve as additional second and first target sequence template strands, to provide for isothermally-accelerated amplification of the target nucleic acid sequence (Fig.12; col 12, lines 50-51; col 30, example 2, “Repeated binding/extension events of opposing primers result in exponential DNA amplification”).
Regarding claim 2, Piepenburg teaches the P1 and the P2 isothermal cycles are symmetric (Fig.12, equal number of first and second strand produced).
Regarding claim 18, Piepenburg teaches prior to incubating the reaction mixture at the primer-cycling temperature, generating a polynucleotide comprising the target sequence in the reaction mixture by non-isothermal polymerase chain reaction (PCR) amplification (col 22, lines 3-5).
Regarding claim 20, Piepenburg teaches the P1 and the P2 primers that provide isothermal primer-driven cycling are used at a reaction concentration greater than 200 nanomolar (Fig. 1, 300nM primer concentration).
Regarding claim 21, Piepenburg teaches wherein the P1 or the P2 primer or both primer sequences incorporate at least one DNA polymerase-compatible structural modification (col 13, lines 22-41, cleavable primers comprising 3’ OH group for polymerase extension).
Regarding claim 23, Piepenburg teaches wherein products of the isothermally-accelerated amplification are detected (Fig. 12 C-D; col 30, example 2).
Regarding claim 24, Piepenburg teaches wherein the amplification and detection are performed simultaneously, in real time (Fig. 12 D; col 30, example 2).
Regarding claim 25, Piepenburg teaches determining an amount of the target nucleic acid in or from a sample (Fig. 12 D; col 30, lines 58-67).
Regarding claim 26, Piepenburg teaches wherein the reaction mixture further comprises a detectable label (Fig. 12 D; col 30, lines 60-64, Fluorescence upon intercalation of SybrGreenI into nascent product).
Regarding claim 27, Piepenburg teaches wherein the detectable label comprises a fluorescent label (Fig. 12 D; col 30, lines 60-64, SybrGreen).
Regarding claim 28, Piepenburg teaches wherein the reaction mixture comprises an oligonucleotide probe labeled with two dyes that are in FRET interaction, and wherein duplex formation of the probe with products of extension of first or second primers disrupts FRET resulting in a detectable signal (Fig. 7).
Regarding claim 29, Piepenburg teaches wherein at least one of the P1 and P2 primers is labeled with two dyes that are in FRET interaction, and wherein hybridization and extension of the primer during the amplification disrupts FRET resulting in a detectable signal (Fig. 10A, fret probe cleavable by Nfo., which can also function as primer, see col 18, lines 60-65).
Regarding claim 30, Piepenburg teaches wherein a distance, in nucleotides, between the 5' end of the P1 primer binding site on the first target sequence template strand and the 5' end of the P2 primer binding site on the second target sequence template strand within the complementary target sequence template strands in a hybridized state, is less than 20 (Fig 1, primer pairs with 45 residues length each, producing 100bp amplicon, thus the gap between the two primers sites on the amplicon is 10 residues; ladder with bp size is shown in Fig. 12C).
Regarding claim 39, Piepenburg teaches wherein a distance, in nucleotides, between the 5' end of the P1 primer binding site on the first target sequence template strand and the 3' end of a sequence on the first target sequence template strand that is complementary to the P2 primer binding site on the second target sequence template strand, is less than 20 (Fig 1, primer pairs with 45 residues length each, producing 100bp amplicon, thus the gap between the two primers sites on the amplicon is 10 residues; ladder with bp size is shown in Fig. 12C).
Claim Rejections - 35 USC § 103 -- New Grounds
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.
Claim 31 is rejected under 35 U.S.C. 103 as being unpatentable over Piepenburg (Piepenburg et al. US8062850B2- Methods for multiplexing recombinase polymerase amplification; 2011-11-22), in view of Zhang (Zhang et al. Isothermal Amplification of Long, Discrete DNA Fragments Facilitated by Single-Stranded Binding Protein. Sci Rep 7, 8497 (2017). doi.org/10.1038/s41598-017-09063-x).
The teachings of Piepenburg are recited above and applied as for base claim 1.
Regarding claim 31, while Piepenburg teaches the use of a DNA polymerase in its RPA reaction, it does not explicitly disclose a thermophilic polymerase. Instead, Piepenburg teaches Bsu DNA polymerase (Fig.12; example 2), which is mesophilic.
Zhang addresses this gap by teaching that all isothermal techniques, including RPA, rely on strand-displacing DNA polymerases to separate DNA strands. These strand-displacing DNA polymerase include Bsu (as taught by Piepenburg) and Bst DNA polymerase (which is thermophilic):
"Each isothermal technique relies on enzymatic activities or primer design to bypass the need for thermal denaturation of dsDNA. Approaches vary, but generally strand displacement activity by a DNA polymerase (typically the large fragment of Bsu, Bst, and E. coli DNA Polymerase I, or phi29 DNA polymerase) separates dsDNA after initiation at a primer, with the initiation step proving the key limit to speed and efficiency of an isothermal reaction. Strategies to facilitate initiation provide the main variance among isothermal methods. For example, initiation approaches include: creation of nicks by a nicking enzyme as in strand displacement amplification (SDA)3 and nicking enzyme amplification reaction (NEAR)4; facilitated strand invasion as in strand invasion-based amplification (SIBA)5, recombinase polymerase amplification (RPA)6 and helicase-dependent amplification (HDA)7; or thermodynamic invasion and annealing as in the initiating first round of SDA, NEAR, loop-mediated isothermal amplification (LAMP)8, multiple displacement amplification (MDA) and rolling circle amplification (RCA)9. "(page 1, para 2, lines 1-11).
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 substitute Bsu in Piepenburg's method with Bst DNA polymerase. Since both polymerases perform the same strand-displacement function in isothermal techniques, one can be substituted for the other to perform the same function with the predictable result of separating DNA strands. This rationale aligns with the principle of KSR for a simple substitution of one known element for another to obtain predictable results, see MPEP 2141.
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