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 .
Claim Status
Claim 18 is now cancelled, while claim 1-14, and 16-17 were previously cancelled. Claims 15, and 19-29 are pending and under examination. Claim 15 has been amended. Claim 15 is the only independent claim.
Response to Arguments
The declaration of Min Chen, filed 04/27/2026 in response to the prior Office Action, has been fully considered. The rejection of claims 15 and 18-29 as unpatentable under 35 USC 103 over Zheng from the prior office action has been withdrawn. The rejections of claim 15 and 19-29 set forth below relies in part on Yan (CN 106906210 A) for the limitation directed to the single reverse primer (R), a reference not previously of record. The Declarations is not sufficient to overcome the rejection of claims 15 and 19-29 stated below over Zheng in view of Yan.
The Declaration does not establish a prober comparison against the closest prior art.
The declaration states in paragraphs 7 that the four-component comparative scheme’s “primer structure is consistent with the Zheng, comprising barcode primer, forward primer (F2), reverse outer primer (R1) and reverse inner primer (R2).” However the declaration provides no molar ratios among the four primer species and gives no indication that the specific conditions disclosed in Zheng (e.g. the molar ratio of Barcode primer F1 to downstream outer primer R1 to upstream primer F2 to downstream inner primer R2 of 6:(10-6):(1-3):(1-3), or Zheng’s disclosed two-stage annealing temperature gradient, were actually replicated in the comparative experiment. The declaration states, “One-step amplification (Example 3 of the present application) was employed to integrate sequencing adapters, barcodes, and molecular barcodes.” However, Embodiment 3 of the present application uses different thermocycler amplification parameters from those disclosed by Zheng and does not disclose the molar ratios utilized for a four-primer system. Zheng expressly acknowledges that its four-primer architecture requires precise molar-ratio management to suppress inter-primer competition and prevent artifact generation (see Zheng [0103]). The molar ratio of F1:R1:F2:R2 must be maintained at 6:(10-6):(1-3):(1-3). Furthermore, Zheng disclosed the importance of the gradient PCR cycles that are not part of Embodiment 3 of the present application (see Zheng [0103]). Absent disclosure of these parameters, the Examiner is unable to confirm that the four-component comparative scheme constitutes a faithful embodiment of Zheng as actually disclosed, rather than an unspecified four-primer variant designed by the declarant. A showing of unexpected results must be made against the closest prior art as actually disclosed, not against an unspecified or potentially non-representative embodiment. See In Re Burckel, 592 F.2d 1175, 201 USPQ 67 (CCPA 1979). As such, the experiments should be conducted using Zheng’s model system following Zheng’s protocol compared to the Applicant’s model system following the disclosure of the instant application. Since the Declaration uses Embodiment 3 as the experimental protocol, and did not disclose the molar ratios of each primer used for a four-primer system, the Examiner concludes that the comparison was not made to the nearest prior art as disclosed.
Even setting aside the foregoing methodological concerns, an analysis of the Declarations own reported data reveals that the four-component comparative arm does not perform as Zheng’s disclosed, optimized four-primer system would be expected to perform, and that the observed variability in the four-component arm is itself evidence that the comparative experiment did not faithfully implement Zheng’s disclosed conditions.
The Declaration reports the following yield ratios for the four-component arm across its three FFPE samples: 6.17%, 62.87%, and 39.97% across samples 1, 2, and 3 respectively. The range across these three samples is 56.7 percentage points. By contrast the three-component arm reports yield ratios of 71.1%, 84.68%, and 82.65% for samples 1, 2, and 3 respectively. The range of only 13.58 percentage points across the three samples. This disparity in inter-sample variance is itself probative. A properly optimize, well-characterized library construction protocol applied to three FFPE samples of comparable quality should produce reasonably consistent results across samples. The three-component arm does precisely this, with tight range of 13.58 percentage points. The four-component arm, by contract produces results ranging from near complete failure (6.17%) to results approaching the three-component arm’s performance (62.87%), a 56.7 percentage point spread across only three samples. This degree of inter-sample variability is not consistent with a well-optimized, faithfully implemented version of Zheng’s disclosed protocol. It is consistent with an improperly optimized or inconsistently implemented protocol in which uncontrolled experimental variables, including but not limited to incorrect molar ratios among primer species, improper two-stage annealing temperature parameters, or other deviations from Zheng’s disclosed conditions, are dominating the outcome.
Sample 1’s four-component result is particularly telling. The Declaration reports a library concentration of 0.0389 ng/µL and a yield ratio of 6.17% for this sample, values the Declaration itself acknowledges “failed to meet the minimum requirements for sequencing”. A library concentration of 0.0389 ng/µL represents near total reaction failure, not merely a reduced yield. Zheng’s own specification demonstrates successful library construction from FFPE samples across all ten subjects in Example 3, with amplification products “concentrated in the range of 160-230 bp”, the expected specific target amplicon size, and mutation detection results consistent with independently validated Sanger sequencing and SureSelect capture results across all samples tested. A properly implemented Zheng system does not fail catastrophically on FFPE samples; Zheng’s entire stated purpose is the reliable detection of mutations in FFPE-derived DNA. Sample 1’s near zero yield in the four component arm is therefore not attributable to the four primer architecture as Zheng discloses it, it is attributable to a failure of the Declaration’s specific implementation of that architecture, most plausibly traceable to the molar ratio and thermocycling deviations discussed above.
The Reported Results Are Not Unexpected in View of the Rationale underlying the rejection
The results reported in the Declaration, (e.g., increased library yield/concentration and reduced background noise) are not unexpected results that rebut the prima facie case of obviousness established in Zheng in view of Yan. Rather, they are consistent with, and are affirmatively predictable both mathematically and mechanistically grounded consequences of reducing primer count in a one-step PCR amplicon library system, consequences that were bother predicted by peer-reviewed experimental data available in the prior art well before the effective filling date of the present application and confirmed by the cited references’ own disclosure.
The Prior Art Establishes That Primer Dimer Formation Is a Direct, Unavoidable Function of Primer Count
Brownie1 experimentally demonstrated, using gel electrophoresis and fluorescence quantification, that primer dimer (PD) formation in PCR is a direct and unavoidable function of the number of primer species present in the reaction. Specifically, Brownie showed that any combination of two different primers in a multiplex amplification refractory mutation system (ARMS) reaction produces PDs “irrespective of any primer complementarity” under standard PCR conditions; and that in multiplex reactions containing many primers at high concentrations, “it becomes impossible to eliminate PDs using” conventional methods including careful primer design, hot-start PCR, and touchdown PCR. Brownie further established experimentally that the solution to this primer-number driven PD problem is precisely the tailed-primer/Tag architecture that both Zheng and the claimed invention employ: genome-specific inner primers present at low concentration carrying a 5’ universal tail sequences, driven in later cycles by a single high-concentration Tag primer matching that tail, such that “low concentrations of specific primers can therefore be used in combination with high concentrations of Tag to produce a wide range of specificities without introducing large quantities of primer.” Using this architecture, Brownie demonstrated that PD accumulations is suppressed by a factor of up to 109 relative to a reaction that retains multiple primer species throughout amplification, a result directly attributable to driving bulk amplification with a single Tag primer rather than multiple genome specific primer species.
The Combinatorial Mathematics Quantifies the Expected Improvement From Reducing Four Primers to Three
The relationship between primer count and primer dimer formation is quadratic. For PCR reactions containing N primer species, the total number of possible pairwise dimer forming combinations, including bother heterodimers between distinct primers, and homodimers formed by a primer interacting with itself, is N(N+1)/2. This relationship is consistent with Brownie’s experimental finding that “every possible combination of two different primers” give rise to PDs irrespective of complementarity, must be treated as potential dimer forming interactions. Applying this formula, a four primer system (N=4) presents N(N+1)/2 = 10 possible dimer forming combinations; a three primer system (N=3) presents N(N+1)/2 = 6 possible dimer forming combinations. The elimination of exactly one primer species from a four primer system therefore reduces the number of possible pairwise dimer forming interactions by 4 out of 10, a 40% reduction. Critically, this 40% reduction in dimer forming combinations is disproportionate to the 25% reduction in primer count itself, going from four to three primers eliminates 25% of the primers but 40% of potential dimer interactions, because of the quadratic scaling of inter-primer combinations means each additional primer species contributes more dimer forming combinations than the last. A person of ordinary skill in the art familiar with Brownie’s experimental data and the mathematical relationship between primer count and dimer formation would therefore have expected a meaningful, quantifiable reduction in PD formation from eliminating one primer species from Zheng’s four-primer system, and would have expected that reduction in PD formation to translate directly into improved on-target amplification yield and reduced background, because PDs compete with the desired target amplicon for the same finite pool of dNTPs, polymerase, and primer resources, and every PD forming event represents substrate consumed nonproductively. This causal chain, fewer primer species [Wingdings font/0xE0]fewer pairwise dimer interactions[Wingdings font/0xE0] less substrate consumed by off-target PD extension [Wingdings font/0xE0] more substrate available for on-target amplification [Wingdings font/0xE0] higher library yield and lower background, was established in the prior art and was fully predictable before the filling of the present application.
Zheng’s Own Disclosures Confirm This Prediction
This prediction is not merely theoretical, Zheng’s own specification confirms, in the context of the identical one-step PCR amplicon library platform, that the four-primer architecture’s inter-primer competition is an identified, active problem requiring affirmative engineering controls. Zheng discloses the molar ratio of F1:R1:F2:R2 must be maintained at 6:(10-6):(1-3):(1-3) specifically because without this control, the copresence of inner and outer primers across all reaction cycles would result in off target amplification products. Zheng specifically explains that this constraint is necessary because without it “the subsequent 19 cycles [would not be] amplified within the molecular tag.” And additional off-target molecular tag additions would occur during amplification (see Zheng [0103]). Zheng further requires a non-standard two-stage annealing temperature program (gradient annealing for the first two cycles then a different set of conditions for the remaining 19 cycles) precisely because the four primer species have differential annealing behaviors requiring sequential engagement. A one-stage program would result in all four primers competing simultaneously, producing lower on-target yield and higher background (see Zheng Table 2, [0103]). These are Zheng’s own admission, in its own specification, that its four primer architectures inter-primer interactions are a source of reduced on-target yield and increased background noise that active, precisely calibrated concentration management and thermocycling engineering are required to suppress. Yan independently confirms that eliminating this inter primer complexity on the reverse side produces a library in which “the DNA chain composition of the obtained library is simple and clear,” attributing lower background complexity directly to the simplified two-primer architecture and requiring no differential molar ratio management whatsoever, using instead uniform concentrations for both primer species, a direct and practical consequence of the elimination of inter primer competition (see Yan [0093]). The claimed three-primer combination, by replacing Zheng’s nested R1/R2 reverse pair with Yan’s single fusion reverse primer, removes the inter-primer competition source that Zheng itself identifies as requiring suppression, and does so in exactly the manner Yan demonstrates is viable and effective.
Chen Provides Experimental Confirmation at Scale
Chen2 provides experimental confirmation, in a controlled NGS amplicon library sequencing comparison, that reducing the number of unique primer species competing in a multi-primer PCR amplicon system produces statistically significant, reproducible reduction in background and improvement in yield. Che compared a three step PCR protocol in which primers from earlier rounds carry over into subsequence rounds, increasing the effective number of competing primer species in the final amplification step, against a two step PCR protocol that reduced this carryover and competition. The result was a 64% reduction in undetermined/background reads, from 22.8% to 8.19%. replicated across ten independent MiSeq runs at two independent sequencing facilities, with read counts up to 4.74 fold higher in the reduced primer competition system. Chen explicitly attributed this improvement to primer carryover and inter-primer competition, identifying “primers carried over from the previous amplification reaction consumed in the current PCR cycles” as a primary cause of decreased PCR efficiency and increased undetermined reads, precisely the mechanism established by Brownie, and confirmed by Zheng’s own molar ratio and thermocycling disclosure.
The Chen data and the combinatorial mathematics are two independent lines of support that converge on the same conclusion, and each must be understood on its own terms before connecting them. Chen’s 64% empirical reduction in background reads reflects a larger effective primer count reduction than the single primer elimination at issue in the present case which is why Chen’s empirical figure exceeds the 40% combinatorial prediction for the three vs four comparison. The two numbers are not the same figure applied to the same comparison. Rather, they are consistent with each other in exactly the way the quadrative relationship between primer count and dimer forming combination predicts, removing more primer species produces a proportionally larger reduction in dimer forming combinations and a correspondingly larger reduction in background, while removing fewer primer species produces a smaller but still substantial and quantitatively predictable 40% reduction pairwise dimer forming opportunities. The 40% figure therefore does not come from Chen’s numbers, and Chen’s number do not validate the 40% figure directly. Instead Chen provides experimental confirmation that reducing inter-primer competition in a multi-primer PCR amplicon library system produces measurable, statistically significant, reproducible reduction in background and increases in yield, confirming that the mechanism is real, experimentally observable, and quantitatively meaningful at the scale Chen measures, while the combinatorial mathematics establishes that the specific one primer reduction from four to three primer produces a 40% reduction in pairwise dimer forming opportunities through the same confirmed mechanism. The 40% represents not a difference in kind from what Chen demonstrates, but a quantitatively smaller instance of the same phenomenon, scaled according to the mathematical relationship that the combinatorial formula expresses and Brownie’s experimental data establishes as a governing principle of PCR primer behavior. While Chen post dates the effective filing date of the present application and is therefore not itself prior art, it constitutes post-filing experimental confirmation that the mechanism and trajectory were real, consistent with how the art already taught this system would behave, and therefore cannot be characterized as unexpected. See MPEP 716.01.
The Declaration’s reported improvement in library yield and reduction in background noise are the predictable mechanistically grounded, and mathematically quantifiable consequences of reducing primer count from four to three in a one-step PCR amplicon library system. Brownie experimental establishes that primer dimer formation is unavoidable function of inter-primer combinations and that driving amplification with fewer primer species suppresses it by orders of magnitude. The combinatorial mathematics quantifies the specific 40% reduction in dimer forming combinations produced by three vs four primer reduction at issue. Zheng’s own specification confirms that the four-primer architecture’s inter-primer competition is an active problem requiring affirmative suppression and Yan confirms that the simplified single primer reverse side architecture eliminates that problem in practice. Che provides post-filling experimental confirmation that the mechanism produces statistically significant, reproducible improvements in yield and background at the scale of a real NGS amplicon sequencing experiments. The trajectory from four primer to three primers, through a 40% reduction in dimer forming combinations, to improved yield and lower background in not speculative, it is mathematically certain, mechanistically established in the prior art, confirmed by Zheng’s own admissions, and empirically validated by Chen at a larger scale. These results do not constitute unexpected results sufficient to rebut a prima facie case of obviousness.
When Sample 1 is recognized as a failed reaction attributable to implementation error rather than architectural inferiority, the remaining two four-component data points, 62.87% and 39.97%, tell a meaningfully different story than the Declaration presents. Samples 2 and 3 show that when the four-component arm produces a functional library at all, its yield ratios, while lower than the tree component arm are within the range expected for a functional one-step PCR amplicon library system rather than representing a categorical failure. The comparison between the three component arm (71.1%, 84.68%, 82.65%) and the functional four component results (62.87%, 39.97%) suggest a modest yield difference, not the categorically superiority the Declaration implied by averaging in Sample 1s near-zero failure. Excluding Sample 1’s failed reaction, the remaining four component results average approximately 51.4% yield, compared to the three-component arm’s average of approximately 79.5%, a difference that, while real, is consistent with the expected, predictable consequence of the 40% reduction in pairwise primer dimer-forming combinations established by combinatorial mathematics and confirmed by Brownie’s experimental data, and not evidence of unexpected results.
In sum, the Declaration’s comparative date, examined on its own terms, reveals: (1) the four-component arm’s extreme inter-sample variability (56.7 percentage points) is inconsistent with a properly optimized implementation of Zheng’s disclosed protocol; (2) Sample 1’s near zero yield represents a catastrophic reaction failure not attributable to the four-primer architecture as Zheng discloses and optimizes, and; (3) the functional four-component results, once the failed reaction is appropriately characterized, show a yield difference from the three component arm that is consistent in direction and approximate magnitude with the expected, predictable consequence of reducing pairwise dimer forming combinations by 40%. The Declaration therefore does not establish that the three-component primer combination produces results that are unexpected relative to a properly implemented version of Zheng’s four primer system. It establishes only that the three-component system outperforms an uncharacterized, inconsistently implemented four-primer system that deviated from Zheng’s disclosed conditions in undisclosed ways, a showing that does not rebut the prima facie case of obviousness. See In re Burckel, 592 F.2d 1175, 201 USPQ 67 (CCPA 1979).
The Declaration’s reported improvement in library yield and reduction in background noise are the predictable mechanistically grounded, and mathematically quantifiable consequences of reducing primer count from four to three in a one-step PCR amplicon library system. Brownie experimental establishes that primer dimer formation is unavoidable function of inter-primer combinations and that driving amplification with fewer primer species suppresses it by orders of magnitude. The combinatorial mathematics quantifies the specific 40% reduction in dimer forming combinations produced by three vs four primer reduction at issue. Zheng’s own specification confirms that the four-primer architecture’s inter-primer competition is an active problem requiring affirmative suppression and Yan confirms that the simplified single primer reverse side architecture eliminates that problem in practice. Chen provides post-filling experimental confirmation that the mechanism produces statistically significant, reproducible improvements in yield and background at the scale of a real NGS amplicon sequencing experiments. The trajectory from four primer to three primers, through a 40% reduction in dimer forming combinations, to improved yield and lower background in not speculative, it is mathematically certain, mechanistically established in the prior art, confirmed by Zheng’s own admissions, and empirically validated by Chen at a larger scale. These results do not constitute unexpected results sufficient to rebut a prima facie case of obviousness.
For the reasons above, the Declaration of Min Chen has been considered but does not overcome the rejection of claims 15 and 19-29 stated below.
Rejections Withdrawn
The rejection of claims 15 and 18-29 under 35 USC § 103 as being unpatentable over Zheng et al. (CN 107604045 A, published Jan. 19, 2018) is withdrawn following the applicants’ amendments. As demonstrated in the applicants’ remarks, Zheng’s method relies on the use of 4 primer system while the claim language has been amended and now states the claimed method consist of only 3 primers.
New Rejections
Specification
The disclosure is objected to because of the following informalities: typographical errors the brand AGILENT® is misspelled as “AGLIENT™” throughout the brief description of the drawings of figures 3 and 9-11, as well as throughout the specification.
Appropriate correction is required.
The specification is objected to because the use of improperly demarcated trademarks has been noted in this application. Although the use of trademarks is permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner that might adversely affect their validity as trademarks. See MPEP §608. 01(v). 9. One example of such an improperly demarcated trademarks “Eppendorf tube”, which appears in the present specification on page 10. Examiner notes that the provided example is not meant to be a complete list of improperly demarcated trademarks found in the present specification. Applicant should review the entire specification and correct all instances of improperly demarcated trademarks. Appropriate corrections required. Each letter of a trademark should be capitalized or otherwise the trademark should be demarcated with the appropriate symbol indicating its proprietary nature (e.g., ™ © ®) and accompanied by generic terminology. Applicants may identify trademarks using the USPTO's trademark database on the Internet at https://tmsearch.uspto.gov
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 15 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 15 recites the limitation "the reverse outer primer R" in line 13. There is insufficient antecedent basis for this limitation in the claim.
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.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], 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.
Claims 23 - 25 are 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. Claims 23 - 25, depend upon claim 21, which in turn depends on claim 15. Claim 15 already includes all of the limitations of each claim 23, 24, and 25. Claim 15 describes the F2 primer exactly as described in claim 23 including “the universal sequence, a molecular tag sequence, and a forward specific primer sequence of the target amplicon.” Claim 15 also includes the limitations of claim 24 describing the molecular tag, and claim 15 was amended to include all of the limitations from claim 25 already therefore the limitations stated in claim 25 are redundant. 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
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 15 and 19-29 are rejected under 35 U.S.C. 103 as being unpatentable over Zheng et al. (CN 107604045 A, published Jan. 19, 2018) in view of Yan et al. (CN 106906210 A, published Jun. 30, 2017).
In regards to claims 15, Zheng teaches a primer combination for preparing an amplicon library for detecting the variation of a target gene, comprising: a forward outer primer F1, a forward inner primer F2, and reverse primers designed according to a target amplicon (see pg. 1 ¶11); “wherein the forward outer primer F1 is sequentially composed of a sequencing adapter 1, a barcode sequence for distinguishing different samples, and a universal sequence” (see pg. 1, ¶12); “the forward inner primer F2 is sequentially composed of a universal sequence, molecular tag sequence, and a forward specific primer sequence of the target amplicon” (see pg. 1, ¶13); “wherein the molecular tag sequence comprises 6-30 bases.” Zheng teaches the molecular tag is composed of 10-12 random bases and teaches the use of a specific base for sequencing fidelity (see pg. 2, ¶8). While this isn’t the entire range of the claimed invention, the use of 12 bases would encode for over a trillion different possible combinations and solve the same multiplexing problem as using a 6-30 base molecular tag. One of ordinary skill in the art would know to modify the length of the tag as needed to account for sufficient multiplexing. Furthermore, the 10-12 bases taught by Zheng is within the claimed range of 6-30 bases reading on the limitations of the instant claim. Zheng further teaches “at least one set of specific bases; wherein each set of specific bases is among the random bases” (see pg. 1 ¶13, pg. 2 ¶6), as acknowledged in the applicant’s remarks dated 4/27/2026;
Zheng teaches a molecular tag sequence comprising random bases and at least one set of specific bases among the random bases, wherein the specific bases are “GAT”( see [0087]). Zheng teaches using the specific bases to “facilitate the analysis of the bioinformatics of the sequencing result, and to improve the efficiency of data screening” (see Zheng [0087]), a person of ordinary skill in the art at the time of filing would understand that any three bases would function similarly as long as the position and identity were known, as the use of specific bases in tags for improved fidelity had been well established prior to the time of filling.34567 Substituting the taught fixed sequence “GAT” with the claimed sequence “TGA” constitutes the simple substitution of one known sequence marker for another sequence marker performing the same function of providing a recognizable non-random sequence within a molecular tag for improved sequencing fidelity, yielding no more than the predictable result of maintaining accurate tag identification and sequence determination. The selection of any particular three-bases sequence to serve as a fixed identifier within an otherwise random molecular tag is a matter of routine design choice, not innovation, because the disclosed function of such a sequence does not depend on the specific identity of the bases chosen, only on the sequence being fixed, known, and distinguishable from the surrounding random-base background during computational analysis. Both GAT (as disclosed in Zheng and Yan) and TGA (as claimed) are three-base sequences drawn from the same finite, fully enumerable set of 64 possible trinucleotide permutations, and nothing in the specification identifies any chemical, structural, or functional property unique to “TGA” that would distinguish its performance from GAT or from any of the other 62 possible alternative 3 bases. Any of the 64 possible trinucleotide sequences would be expected to perform the identical identification function with substantially equivalent results since each is equally capable of being recognize by sequence-matching software during bioinformatic analysis, and a person of ordinary skill in the art , faced with Zheng’s express teaching of a fixed three-base sequence used “to facilitate the bioinformatic analysis of the sequencing results by identifying the fixed sequence.” Would have had every reason to expect that substituting any other specific trinucleotide for GAT, including TGA, would achieve the same result with the same degree of success. Absent some showing in the specification of unexpected or superior result specifically attributed to the use of TGA, that is not achieved by GAT or other three-base alternative, the selection of TGA is properly treated as one of a finite number of identified, predictable, solution to the same design problem, the selection of which, does not require an inventive leap. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007)
Zheng’s reverse side employs two primers: downstream outer primer R1, “sequentially composed of a sequencing linker 2 and a universal sequence 2,” and downstream inner primmer R2 “sequentially composed of a universal sequence 2 and a downstream specific primer sequence, connected through a shared internal universal sequence in a nested arrangement mirroring Zheng’s forward F1/F2 pair. The instant claim, by contrast, recites a single reverse primer R “sequentially composed of sequencing adapter 2 and a reverse specific primer sequence,” with no second reverse primer and no intervening universal sequence, therefore Zheng doesn’t teach the exact primer architecture for the reverse primer. However, Yan, assigned to the same corporate entity as Zheng (GENETRON HEALTH (BEIJING) CO LTD) and directed to the identical technical field of one-step PCR construction of NGS amplicon libraries, discloses fusion primer combination consisting of only two primers an upstream fusion primer and a downstream fusion primer (see Abstract, Claim 1). Yan’s downstream fusion primer architecture is sequentially composed of an adapter and a reverse specific sequence of the target amplicon (see Abstract). Yan’s primer combination, used together with no additional reverse-side primer, is sufficient by itself to complete one-step PCR construction of a sequencing-ready amplicon library (see Yan Example 1).
Yan thus discloses, within the same one-step PCR amplicon-library construction technology and assignee lineage as Zheng, that the reverse (downstream) side of a one-step PCR amplicon library primer combination can consist of a single primer combining an adapter sequence with a target-specific sequence, with no requirement for a second nested reverse primer or an intervening universal sequence. A person of ordinary skill in the art, present with Zheng’s fully-nested four-primer architecture (nested on both the F1/F2 forward side and the R1/R2 reverse side), and aware of Yan’s simultaneous teaching from the same group, that the reverse side of a comparable one-step PCR amplicon library system can instead consist of a single fusion primer with the adapter fused directly to the specific sequence, would have had reason to simplify Zheng’s R1/R2 reverse pair into the single primer R recited in the claim. The motivation for doing so is express in the shared design rationale of both references: reducing the number of unique primer species in the one-step PCR reaction reduces the opportunities for off-target primer-primer interaction and primer dimer formation, reduces oligonucleotide synthesis cost, and simplifies the reaction, while the claim’s retained forward-side architecture (F1/F2) preserves barcode-based sample identification and molecular tagging. This is the combination of known elements (Zheng’s nested four-primer system; Yan’s single-fusion-primer reverse-side architecture, from the same inventors and applied to the same one-step PCR amplicon library purpose) according to their established functions, yielding the predictable results of a functional, simplified amplicon library primer combination. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007).
For the reasons above, claim 15 would have been obvious over Zheng in view of Yan.
In regards to claims 19 and 20, Zheng teaches using sequencing adapters that correspond to different sequencing platforms, and specifically cites using i5 and i7 for Illumina platforms and A and P for Ion Torrent platforms (see pg. 2 ¶10-12).
In regards to claim 21, Zheng teaches using the primers described above to perform a one-step PCR amplification (see pg.2 ¶9).
In regards to claims 22, 27, and 29, Zheng teaches utilizing blood, urine, cerebrospinal fluid, or tissue samples (see pg. 2 ¶18).
In regards to claim 26 and 28, Zheng teaches using their method to detect mutation rates following the steps of preparing an amplicon library, then diluting to generate a sequencing library, followed by sequencing (see pg. 3 ¶4-5).
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Matthew H Raymonda whose telephone number is (703)756-5807. The examiner can normally be reached Monday - Friday 10:00 am - 4:00 pm.
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/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684
/AARON A PRIEST/ Primary Examiner, Art Unit 1681
1 Brownie et al. “The elimination of primer-dimer accumulation in PCR”, Nucleic Acids Research, Volume 25, Issue 16, 1 August 1997, Pages 3235–3241
2 Chen et al. “A Two-Step PCR Protocol Enabling Flexible Primer Choice and High Sequencing Yield for Illumina MiSeq Meta-Barcoding”, Agronomy 2021, 11(7), 1274
3 Brenner (US 5,604,097, published Feb. 18, 1997)
4 Kinde (“Detection and quantification of rare mutations with massively parallel sequencing”, Proc. Natl. Acad. Sci. U.S.A. 2011. 108 (23) 9530-9535)
5 Hamady (“Error-correcting barcoded primers allow hundreds of samples to be pyrosequenced in multiplex”, Nat Methods. 2008 Feb 10;5(3):235–237)
6 Ward et al. (US 2009/0099040 A1, published Apr. 16, 2009)
7 Vogelstein (US 2014/0227705 A1, published Aug. 14, 2014)
Prosecution Insights