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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 June 26, 2026 has been entered.
Status of Claims / Response to Amendment
This office action is in response to an amendment filed on June 26, 2026.
Claims 1-9, 11, 14, 18-26 were previously pending. Applicant amended claims 1, 25-26; cancelled claim 24; claim 27 is newly added.
Claims 1-9, 11, 14, 18-23 and 25-27 are currently pending, with claims 3, 9, 11, 14, 20 withdrawn.
Claims 1-2, 4-8, 18-19, 21-23 and 25-27 are under consideration.
All of the previously presented rejections have been withdrawn as either being addressed or obviated by the amendment of the claims, which introduces a new combination of elements that were not previously considered in the prior rejection.
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
For the instant claims 1-2, 4-8, 18-19, 21-23 and 25-27 in this application, the applicant claims priority of the US provisional application 63/016,194, which has a filling date on April 27, 2020.
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.
For the purpose of applying prior art, claims 1 and 5 are drawn to a primer set comprising elements including "primer sequence a," "primer sequence b," "primer sequence e," "primer sequence f," and other positionally related components. These elements are interpreted in accordance with arrangement as shown in FIGs 1-3 of the current application's disclosure, reproduced below.
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For the purpose of applying prior art, claim 1 recites "cycling probe," which is defined by the application's disclosure in para [0170]:
"[0170] The term "cycling probe" that can be cleaved by an enzyme after annealing to a target nucleic acid sequence, wherein such cleavage releases an intact target nucleic acid."
Therefore, in light of the specification and under BRI, the term "cycling probe" is interpreted to encompass any probe cleavable by an enzyme.
For the purpose of applying prior art, claim 1 recites "modified base," which is defined by the application's disclosure in para [0167]:
"[0167] The term "modified base" is used herein to refer to a base that is not a canonical, naturally occurring base (e.g., adenine, cytosine, guanine, thymine, or uracil)."
For the purpose of applying prior art, claim 19 recites "RNase H2 cycling probe," which is not defined in the application's disclosure. Thus, under BRI the term "RNase H2 cycling probe" is interpreted to encompass any probe cleavable by RNase H2 enzyme.
New Grounds of 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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 4-8, 18-19, 21-23, 25-27 are rejected under 35 U.S.C. 103 as being unpatentable over Higuchi (WO2016100388A1 - Exponential base-greater-than-2 nucleic acid amplification; Published on 2016-06-23; cited as Foreign Patent Document #010 in IDS filed 10/31/2023), in view of
Yang (Yang et al., Eliminating primer dimers and improving SNP detection using self-avoiding molecular recognition systems, Biology Methods and Protocols, Volume 5, Issue 1, 2020, bpaa004, https://doi.org/10.1093/biomethods/bpaa004; Published: 10 February 2020), and
Han (US5763181 A - Continuous fluorometric assay for detecting nucleic acid cleavage; Published on: 1998-06-09; cited as U.S. Patent Document #004 in IDS filed 10/31/2023), as evidenced by Walder (US8911948 B2 - RNase H-based assays utilizing modified RNA monomers; Published on 2014-12-16).
A) The subject matter of claim 1 is an obvious combination of a known amplification oligonucleotide set taught by Higuchi with known approaches for improving amplification outcomes. These approaches include the SAMRS oligonucleotide modification scheme, which facilitates formation of preferred duplexes and prevents formation of undesirable duplexes between primers, as taught by Yang; and the use of cycling probes to improve signal-to-noise ratio in target detection, as taught by Han.
Higuchi teaches methods and compositions for highly efficient nucleic acid amplification with increased sensitivity and speed (entire document, Abstract for example). Higuchi teaches that its methods can be used to detect target nucleic acids such as SNPs ([0098] line 5).
Regarding claim 1, Higuchi teaches a nucleic acid primer set comprising oligonucleotides as shown in Fig 2 below.
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While Higuchi teaches that its primer sequences can include modified bases to manipulate nucleic acid duplex stability and achieve the desired nucleic acid complex configuration ([0091] c-a/c'-a' is more stable than a-b/a'-b'; [0060-0061]; [0102-0103]; page 27, lines 9-11, Tm can be adjusted by including stabilizing or destabilizing bases), it does not explicitly teach the positions of the modified bases, including:
a’ in c’-a’ (Figure 2) comprise a 2nd modified base, sequence a of the first outer primer comprise a 1st modified base, wherein the first modified base does not form a stable hydrogen-bonded base pair with the second modified base; and
a’ in c’-a’ (Figure 2) further comprise a sixth modified base, sequence a of the first outer primer comprises a fifth modified base, wherein the fifth modified base does not form a stable hydrogen-bonded base pair with the sixth modified base; and
the first modified base is a first number of bases, in primer sequence a, from the fifth modified base;
the second modified base is a second number of bases, in primer sequence a', from the sixth modified base;
the first number and the second number are the same;
wherein the first number and the second number of bases are unmodified bases.
Therefore, these claimed features together require that a’ in c’-a’ (Figure 2) comprise two modified bases that are flanking a number of unmodified bases, and sequence a of the first outer primer comprise two modified bases that are flanking the same number of unmodified bases, such that each the modifications are positioned in the primers so they pair with each other on separate strands. As shown in modified Fig 2 of Higuchi below:
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However, it would have been obvious to make such modifications in view of Higuchi and Yang. Higuchi already teaches the use of modified bases (stabilizing or destabilizing bases) to adjust the melting temperature (Tm) or stability of specific nucleic acid hybridization structures. This allows the primer regions to exhibit different hybridization preference within a complex.
Higuchi also present a specific primer configuration shown in Fig. 2, which supports a greater than two-fold increase in amplification efficiency (see para. [0090- 0091] and Fig 2) .
In this configuration, sequences a and a' are complementary, leading to the following possible hybridization schemes:
A.1. (c-a-b) with (c’-a’) or
A.2. (c-a-b) with (d’-a’-b’);
B.1. (a) with (c’-a’); or
B.2. (a) with (d’-a’-b’)
Fig. 2 illustrates the preferred configuration, A1 and B2, which supports the increased amplification efficiency. In this configuration, it is essential that both the inner primer b in (c-a-b) and the outer primer (a) bind to the target sequence (d’-a’-b’), forming a nested primer configuration. For this to occur, the hybridization of c-a with c'-a' must be more stable than a-b with a'-b' (i.e., A.1. is preferred over A.2.) (see [0091] line 1). This ensures that the outer primer binding site is not blocked by undesirable hybridization, allowing sequence a' in the target sequence to bind to the outer primer (a) (i.e., B.2. is preferred over B.1.).
Similarly, the hybridization of B2 [(a) with (d’-a’-b’)] need to be more stable than B1 [(a) with (c’-a’)]. Higuchi suggests potential approaches to achieve this preferred configuration, such as using of stabilizing or destabilizing bases. The general principle is to increase the relative stability of the preferred hybridization compared to the undesirable hybridization. This can be achieved by incorporating more stabilizing bases in the preferred hybridization, or more destabilizing bases in the undesirable hybridization.
Yang teaches an improvement to PCR amplification assays, known as “self-avoiding molecular recognition system” (SAMRS),” which eliminates PCR artifacts due to non-specific or undesirable binding when multiple probes and primers are used simultaneously (Abstract). SAMRS bases are modified nucleotide bases with "pseudo-complementarity" pairing properties. They bind stably to their natural DNA complements but do not form stable duplexes with other SAMRS bases, even if fully complementary. (Yang, page 2, left-hand col, para 3; Figure 1)
Accordingly, SAMRS bases incorporated into primers can prevent the formation of undesired duplexes between primers. In other words, undesirable hybridization can be reduced by modifying both hybridization partners with modified SAMRS bases at complementary positions.
Yang explores optimization of primers containing different numbers of SAMRS components placed at different positions, specifically to "identify SAMRS design rules to better discriminate single-nucleotide polymorphisms (SNPs) in template targets and facilitate faster development of multiplexed PCR by using SAMRS primers." (Yang, page 2, right-hand col, lines 1-21).
Yang specifically teaches that including unmodified nucleotide bases (indicated by upper case letters) between SAMRS modified bases (indicated by lower case bold letters) in primers improves amplification efficiency:
"Replacement of t by T was observed to modestly increase in PCR efficiency. As examples, upon tgtg going to TgTg, ctgt to cTgT, and gctg to gcTg, the appearance of product was faster by ∼0.3 cycles. Interestingly, replacement of t by T further delayed the formation of primer dimer by ∼4–9 cycles. This suggested that t might be less in preventing primer dimer formation than the rest of SAMRS components. Replacing one c by C (accc to acCc or ccct to cCcT) to avoid three consecutive c’s was improving amplification efficiency by ∼0.5 cycles." (page 10, right-hand col, para1)
Therefore, 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 primer complexes disclosed in Higuchi (Fig. 2) by incorporating the SAMRS destabilizing bases taught in Yang for SNP detection. Specifically, sequence (a) would include at least two SAMRS bases with unmodified base in between, and the corresponding complementary region in (c'-a') would also include at least two SAMRS base at nucleotide positions that pair with the SAMRS base in (a).
The skilled artisan in view of the teachings of Higuchi and Yang would appreciate that this modification would prevent the undesired hybridization of (a) with (c’-a’) (hybridization scheme B.1.) while allowing the preferred hybridization of (a) with (d’-a’-b’) (hybridization scheme B.2.), as shown in Fig. 2 of Higuchi. Such a modification with unmodified base between SAMRS bases would further allow for efficient amplification using the modified primers, when the primer sequences comprise Ts or consecutive Cs, as suggested by Yang.
A skilled artisan, understanding the specific configuration shown in Fig.2 of Higuchi, which is needed to achieve the predictable result of increased amplification efficiency, and in view of Higuchi's teaching that modified bases can be used to achieve the desired configuration, would have been motivated to apply the modification with SAMRS bases from Yang to the primers. This would ensure that the primer (a) binds more stably to the target sequence (d’-a’-b’) rather than to sequence (c’-a’), resulting in the desired configuration being the most stable form.
The person of ordinary skill would have had a reasonable expectation of success because the teachings of Higuchi and Yang are technically compatible and share the common objective of detecting SNP in a target nucleic acid.
This combination would have been obvious as it represents the KSR principle of predictable use of prior art elements (i.e., modified SAMRS bases in Yang) according to a known method (i.e., methods for nucleic acid amplification using a primer set comprising modified bases, disclosed in Higuchi) to yield predictable results (i.e., hybridized primer configuration in modified Fig. 2 of Higuchi). (See MPEP §2143).
The combined teachings of Higuchi and Yang teaches all the claim limitations, except for a cycling probe.
Han teaches methods and compositions for performing a fluorometric assay to detect nucleic acid cleavage, which can be used to enhance the efficiency and detection of well-known nucleic acid amplification techniques such as PCR (entire document; col 10, lines 21-25 for example).
Regarding claim 1, Han teaches the use of cycling probes with FRET in detecting specific DNA sequences (entire document; col 21-22: Example 3 for example), wherein the cycling probe comprises an enzyme cleavage site and a label (col 6, lines 31-34; entire document; col 21-22: Example 3 for example), enzyme mediated cleavage of the cycling probe within the probe target duplex results in release of the intact target sequence, which can repeatedly recycle through the reaction pathway (col 21, lines 32-35).
Han further suggests that cycling probes with FRET offer several advantages, such as preserving intact target for probe binding, which serves as a catalytic cofactor of continuous reaction. Additionally, the use of FRET-based cycling probes can further amplify the detection signal, resulting in an improved signal-to-noise ratio, thus enhancing detection efficiency:
"Catalytic hybridization amplification" (CHA), alternatively known as "cycling probe technology," is described in published PCT application WO 89/09284, and U.S. Pat. Nos. 5,011,769 and 4,876,187. Briefly, CHA is an improved hybridization assay method whereby the target sequence to be detected is able to capture many molecules of the probe in a repeating series of reactions (i.e., "cycling probe"). Essentially, enzyme mediated cleavage of the probe within the probe target duplex results in release of the intact target sequence, which can repeatedly recycle through the reaction pathway. The target sequence serves as a catalytic cofactor for the cleavage of a complementary, labeled nucleic acid probe that is hybridized to the target. The detectable signal in this reaction results from cleavage of the probe, e.g., after repeated CHA cycles, one measures the labeled probe cleavage product. The CHA method is useful in detecting specific DNA or RNA sequences.
The present inventors have reasoned that the last step of CHA (i.e., measuring the labeled probe cleavage product), could be more expeditiously and efficiently carried out by employing the presently disclosed fluorometric assay, based on FRET, for detecting DNA cleavage.
It is expected that the high efficiency of FRET will provide a means to amplify the detection signal. For example, if the donor fluorescence is quenched to 10% of its initial intensity, then complete cleavage of the oligonucleotide substrate (probe) by the RNase H enzyme used in CHA, will result in a 10 fold amplification of the signal. Moreover, only 10% cleavage of the probe will still result in a two fold increase in the detection signal. This intrinsic signal amplification will provide an excellent tool to improve signal-to-noise ratio and thereby increase the confidence in data interpretation." (col 21-22)
Therefore, instead of detecting each DNA amplicon only once, with the cycling probes the same DNA amplicon can be detected multiple times, therefore greatly increases the signal.
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 combine the teaching of a primer set comprising modified bases for PCR taught by Higuchi and Yang with the cycling probes using FRET taught by Han, because all references are in the same or overlapping field of nucleic acid amplification and detection, with the shared objective of improving the sensitivity and efficiency of detecting specific nucleic acid target sequences. More specifically, Higuchi teaches a method for highly efficient nucleic acid amplification using a primer set, while Han teaches the use of cycling probes with FRET to enhance nucleic acid detection approaches such as PCR by further amplifying the signal.
The person of ordinary skill would have had a reasonable expectation of success in combining these teachings because all three references teach sequence-specific nucleic acid detection via amplification, specifically with Higuchi teaching a PCR primer set and Han offering an additional cycling probe that further improves PCR and increases detection efficiency, thus demonstrating technical compatibility. Doing so would have yielded the predictable result of a primer set for enhanced nucleic acid detection by incorporating Han's cycling probes to further amplify detection signals in amplicon products in a PCR reaction.
The skilled artisan would have been motivated to combine the cycling probe technology of Han with the primer set taught by the combined teachings of Higuchi and Yang, to leverage the known advantages of FRET-based cycling probes, as highlighted by Han, resulting in reagent composition for a more sensitive and efficient assay.
B) Regarding claim 2, Higuchi teaches primer set additionally comprises at least one second primer (FIG. 3).
Regarding claim 4, Higuchi teaches the Tm of combined sequence c-a, in double-stranded form, is greater than that of combined sequence a-b, in double-stranded form ([0010]).
Regarding claim 5, The combined teachings of Higuchi, Yang and Han teaches its limitations. Higuchi teaches nucleic acid primer set comprising oligonucleotides as shown in Fig 3 below. The primer set in Fig. 3 is the “reverse” primer set to the “forward” primer set of Fig 2. (page 26, lines 14-20) It would have been obvious to modify primer sequence (e) and its complementary sequence in (g’-e’) for the same reasons discussed for claim 1 above.
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Regarding claim 6, Higuchi teaches the Tm of combined sequence g-e, in double-stranded form is greater than that of combined sequence e-f, in double- stranded form ([0015]).
Regarding claim 7, Higuchi teaches clamp sequence c is not capable of being copied during amplification ([0037]).
Regarding claim 8, Higuchi teaches clamp sequence c comprises) 2'-O-methyl RNA([0038]).
Regarding claim 18, Han teaches probe comprising a modified base (col 8, lines 4-8; col 10, lines 46-49) wherein the modified base is a modified form of a unmodified base selected from the group consisting of adenine, thymine, uracil, guanine, and cytosine (Col 10, lines 46-52, 5’ Cytosine of SEQ ID NO: 1 is modified with primary amine; col 16, lines 59-60, 5-Amino (12)-2'-deoxyuridine).
Regarding claim 19, Han teaches a cycling probe which is a RNase H2 cycling probe by teaching cycling probes consist of DNA-RNA-DNA strands (col 22, lines 15-16),which are substrates of RNase H2 as evidenced by Walder. Walder teaches RNase H2, specifically it teaches RNase H2 cleaves RNA ribonucleotide embedded within a DNA sequence (col 5, lines 19-22). Therefore, the probe taught by Han comprising RNA ribonucleotide embedded within a DNA strand is a RNase H2 cycling probe cleavable by RNase H2.
Regarding claim 21, Han teaches an RNase cycling probe (col 3, lines 58-60; col 6, lines 60-67; col 22, line 7, RNase H; claim 14).
Regarding claim 22, it recites:
"wherein the first modified base of primer sequence a is a first number of bases from the 5' end of primer sequence a; and the second modified base of primer sequence a' is the first number of bases from the 3' end of primer sequence a'. "
Thus, this limitation is interpreted to mean that the modified bases are complementary pairs located on the two strands of the sequences forming a hybridization duplex [(a) with (c’-a’)]. This limitation is obvious in view of the combined teachings of Higuchi, Yang, and Han, as discussed above for claim 1.
Claim 23 recites:
"wherein the Tm of primer sequence a of the first double-stranded primer sequence / primer sequence a' of the first double-stranded primer sequence, in double-stranded form, is greater than the Tm of primer sequence a of the first outer primer / primer sequence a' of the first double-stranded primer sequence, in double-stranded form."
This limitation is interpreted to mean that the hybridization of (c-a) with (c’-a’) in the region where sequence a hybridizes sequence a’, has higher Tm than (a) with (c’-a’) in the same region.
This is obvious in view of Yang's teaching that "the Tms of oligos having greater numbers of SAMRS components are lower than the Tms of oligos having less SAMRS (Supplementary Table S3)" (page 10, right-hand col, conclusion paragraph, lines 13-16).
As the duplex of (c-a) with (c’-a’) has less SAMRS modified bases (i.e. SAMRS only present on one strand) than the duplex of (a) with (c’-a’) (i.e., SAMRS present on both strands), a skilled artisan would readily understand that the hybridization of (c-a) with (c’-a’) in the region where sequence a hybridizes sequence a’, has higher Tm than (a) with (c’-a’) in the same region.
Regarding claim 25, it recites
"wherein the first number is at least two; and the first modified base is 5' of the fifth modified base and the second modified base is 3' of the sixth modified base or the first modified base is 3' of the fifth modified base and the second modified base is 5' of the sixth modified base."
Therefore, the claim further requires at least two unmodified bases between two modified bases. This feature would have been obvious because Yang suggests that the number of unmodified bases between modified bases is a result-effective variable subject to optimization.
For example, Yang teaches that replacing t with T in primer sequences ꟷ where lower case letters indicate SAMRS modified bases and uppercase letters indicate unmodified casesꟷ improves amplification efficiency (page 10, right-hand col, lines 14-15).
Therefore, for a primer sequence comprising two consecutive Ts, for example, TGTTG, routine optimization of the modification pattern would have yielded TgTTg, in which the two modified bases are separated by two unmodified bases. The bases would occupy the claimed positions because they pair with each other on opposite strands.
Regarding claim 26, it recites
"wherein primer sequence a of the outer primer in a double strand form with the first template strand sequence a' is more stable than primer sequence a of the outer primer in a double strand form with primer sequence a' of the second strand; and
wherein the first modified base of the first outer primer a is at a position complementary to the second modified base of the primer sequence a' of the second strand, and wherein the fifth modified base of the first outer primer a is at a position complementary to the sixth modified base of the primer sequence a' of the second strand. "
These limitations is interpreted to mean that a duplex in which only one strand comprises modified bases (i.e., in the duplex of "primer sequence a" | "first template strand sequence a' ", only primer sequence a comprises modified bases) is more stable than a duplex in which both strands comprise modified bases (i.e., in the duplex of "primer sequence a" | "primer sequence a' of the second strand," both strands comprise modified bases in view of combined teaching of Higuchi, Yang, and Han), wherein when both strands are modified and hybridized, the modified bases pair with each other.
In view of the combined teachings of Higuchi, Yang, and Han, the described stability and modified base positioning in claim 26 would have been an obvious and predictable characteristics of the primers having structures (e.g., Higuchi’s primers having modified bases) taught and suggested by the prior art, as discussed above for claim 1. Specifically, a skilled artisan would have readily understood, based on the SAMRS modification principle taught by Yang, that a duplex containing SAMRS modified bases on both strand is less stable than a duplex containing SAMRS-modified bases on only one strand.
Regarding claim 27, it is analogous to claim 26, but broader, because the limitation of claim 27 is fully encompasses by claim 26/1. Accordingly, claim 27 would also have been obvious in view of Higuchi, Yang, and Han for the reasons discussed above for claims 1 and 26.
Conclusion
No claims are allowed.
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/TIAN NMN YU/Examiner , Art Unit 1681
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