DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application is being examined under the pre-AIA first to invent provisions.
Status of the Application
The Amendment and Response filed October 14, 2025, and the Request for Continued Examination filed October 28, 2025, are each acknowledged.
Claims 30, 34 and 36-52 were pending. Claims 30, 34, 36-49 and 51-52 are being examined on the merits. Claim 50 is canceled.
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 October 14, 2025 has been entered.
Response to Arguments
Applicant’s arguments filed October 14, 2025 have been fully considered.
The previously made objections are WITHDRAWN in view of Applicant’s claim amendments.
The following rejections are MODIFIED in view of Applicant’s arguments and amendments to the claims.
Prior art rejections
Response to arguments regarding prior art rejections
The prior art rejections below are modified in view of Applicant’s arguments and claims amendments. However, to the extent that Applicant’s arguments relate to the modified rejections, the Examiner notes the following.
Applicant argues that the prior art rejections of, in particular, independent claim 30, which has been amended to incorporate the limitations of now-canceled claim 50, should be withdrawn for several reasons (Remarks, p. 9).
First, Applicant argues that Van Eijk does not comprise a plurality of subsets of primers because the primers are expended in the amplification process, and thus are no longer present in the pooled library (Remarks, p. 9). Applicants additionally argue that if the primers were still present in the pool it would undermine the disclosed function of using the tag to determine the origin of the PCR sample, and argues that “the post-pooling samples has no tagged primers at all (by design)” (Remarks, pp. 9, 10).
The Examiner disagrees. Van Eijk does not teach that the primers are completely consumed in the amplification process, nor has Applicant cited any evidence to that effect. Further, in reference to the design of the Van Eijk method, Applicant asserts that they know the intent of the Van Eijk inventors (i.e., that they designed the method to have no tagged primers in the post-pooling sample). Van Eijk does not teach this and it is not clear how Applicant could otherwise have knowledge of the Van Eijk inventors’ intent. Finally, it is not clear why or how having primers still present in the post-pooling samples would undermine the disclosed function of using the tag to determine the origin of the PCR sample, because sequence data derived from residual primers is easily trimmed in the sequence data processing workflow (e.g., Fig. 4).
Second, Applicant disagrees that the combination of the Van Eijk and Brenner teachings would result in increasing the throughput of screening, and also asserts that there would be no reason to expect that such tagging would offer any improvement over the tagging already disclosed in Brenner (Remarks, p. 10).
The Examiner disagrees. Van Eijk teaches pooling samples, as noted below, and also teaches that high-throughput screening “allows a researcher to effectively screen large amounts of sample simultaneously”. It is understood in the art that pooling multiple samples before sequencing is more efficient than sequencing individual samples in parallel. However, data generated from such pooled data must be deconvoluted after sequencing, and the Van Eijk sequence tags allow such deconvolution, as taught by Van Eijk (p. 8, ll. 5-19). Nevertheless, in the interest of advancing prosecution, the rejection below has been modified to clarify this issue.
Finally, Applicant argues that there are various “[c]onclusory assertions not otherwise explained” and reiterates their prior arguments (Remarks, p. 11).
The Examiner disagrees for the reasons noted above.
Claim Interpretation
Claim 30 recites the limitation “nucleic acids each comprising a region of interest (ROI)”. Since the specification does not define “ROI” in any way that structurally or functionally distinguishes it from the nucleic acid within which it is comprised, any sub-sequence of the “nucleic acid[]” can be considered a ROI, up to an including the entire nucleic acid itself.
Claim 30 also recites “a primer binding site”. Since any sequence in any nucleic acid sequence can bind to a primer, any nucleic acid sequence can be considered “a primer binding site”. Further, the specification does not define “a primer binding site” in any way that structurally or functionally distinguishes the term from any nucleic acid sequence. Similarly, the term “amplification site” (e.g., claim 37) can be any nucleic acid.
Further, since any known sequence can be used as a universal sequence, any corresponding known sequence can be used as a “binding site for a universal primer” (e.g., claim 39).
Finally, any nucleic acid can be excised, thus, any nucleic acid sequence can be an “excision site” (e.g., claim 41).
Claim Rejections - 35 USC § 103
The following is a quotation of pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter 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 under pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
Claims 30, 34, 36-49 and 51-52 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Brenner1 (WO 96/12039) in view of Van Eijk2 (WO 2007/073165) and Binladen (The Use of Coded PCR Primers Enables High-Throughput Sequencing of Multiple Homolog Amplification Products by 454 Parallel Sequencing, PLoS One, 2(2): e197, 2007), as evidenced by Schmid3 (Three new endonucleases MaeI, MaeII and MaeIII from Methanococcus aeolicus, Nucleic Acids Research, 12(6), 2619-2628, 1984).
Regarding independent claim 30, Brenner teaches …
An amplification reaction mixture, comprising: a plurality of nucleic acids each
comprising a ROI and a primer binding site (p. 18, ll. 30-38: a primer containing a tag sequence is attached to each target polynucleotide in a population through PCR; thus, the ordinary artisan would understand that each ROI/target polynucleotide primer comprises a primer binding site for a corresponding primer; p. 32, ll. 14-28);
and a plurality of primers, wherein each primer: i) hybridizes to the primer binding site
of a nucleic acid of the plurality of nucleic acids, and ii) comprises a unique identifier (p. 18, ll. 30-38: a primer containing a tag sequence is attached to a target polynucleotide through PCR; thus, the ordinary artisan would understand that the primer hybridizes to the primer binding site of the target polynucleotide; p. 32, ll. 14-28);
wherein the plurality of primers comprises a subset of primers, wherein the subset of
primers comprises primers that (i) hybridize to nucleic acids in the plurality of nucleic acids
comprising the same ROI, and (ii) comprise different unique identifiers such that amplification products produced by extension of the subset of primers in the amplification reaction mixture are distinguishable from one another by the respective unique identifiers (p. 18, ll. 30-38 through p. 19, ll. 1-29; p. 32, ll. 14-28).
Further, regarding the limitation reciting “a subset of primers”, Brenner more specifically teaches that the reaction mixture will comprise a very large population of tag-polynucleotide conjugates, where at least some of the polynucleotides will have the same polynucleotide sequence but with a different tag, which will result in a polynucleotide being processed/sequenced twice (p. 18, ll. 30-38 through p. 19, ll. 1-29). Thus, the primer molecules in Brenner which hybridize to those polynucleotides with the same sequence but different tags constitute the “subset of primers” recited in the claims.
Van Eijk additionally teaches a plurality of subsets of primers, and teaches that each primer subset hybridizes to a different ROI in the (target) nucleic acids. Specifically, Van Eijk teaches amplifying target nucleic acids (comprising ROIs) with primer pairs where at least one primer of the pair comprises an identifier tag, and then teaches pooling libraries derived from multiple samples (p. 12, ll. 24-40 through p. 13, ll. 1-14). Thus, after pooling the reaction mixture will comprise a plurality of subsets of primers, where each subset hybridizes to a different sample/target nucleic acid/ROI.
Finally, Binladen teaches PCR primers which are tagged with unique nucleotide sequence tags such that a unique tagged primer can be applied to each specific DNA template source. The amplicons comprising the unique tags are \sequenced, and the tags are then used to sort the sequences into their original template source (Fig. 1; p. 2, left col., para. 3 through p. 2, right col., para. 1). Binladen also teaches that using such tagged primers increases the throughput and cost-effectiveness of sequencing nucleic acids derived from different sources (p. 1: Background; p. 1, left col., para. 2 through p. 1, right col., para. 1; p. 2, right col., para. 1; p. 8, right col., para. 3).
Prior to the date of the instant invention, it would have been prima facie obvious to modify the Brenner reaction mixture with the Van Eijk plurality of subsets of primers, where each subset hybridizes to a different ROI. The ordinary artisan would have been motivated to modify the Brenner reaction mixture by including additional subsets of primers specific to different targets, as taught by Van Eijk, with the expectation that doing so will result in the advantage of a high-throughput sequencing reaction mixture which is capable of being used to pool different samples/target nucleic acids/ROIs, which improves efficiency and cost-effectiveness, as taught by Binladen. The ordinary artisan would have had an expectation of success as adding multiple/different tags/identifiers to target nucleic acids is known in the art, as taught by Van Eijk and Binladen.
Regarding dependent claims 34 and 36, Brenner additionally teaches that the unique identifiers comprise a degenerate sequence (p. 24, ll. 2-3), as recited in claim 34, and that the unique identifiers are selected from a diverse, pre-defined set of sequences (p. 9, Table 1; p. 10, Table II), as recited in claim 36.
Regarding dependent claims 37, 40 and 41, Brenner additionally teaches that the amplification products comprise at least one amplification site, which is positioned to allow amplification of the ROI and unique identifier, and one or more excision sites. Specifically, as noted above in the claim interpretation section, any sequence can be an amplification site and any sequence can be an excision site. Thus, in the amplification product on p. 19, l. 36, the underlined sequence can be used as either/both an amplification site and/or an excision site. In addition, since the underlined sequence is 5’ of the ROI (i.e., all of or a portion of the cDNA) and unique identifier, then that sequence is positioned to be used an amplification site which is positioned to allow amplification of the ROI and the unique identifier. In addition, as evidenced by Schmid (abstract), the sequence 5’ – CTAG – 3’ is a restriction site for MaeI endonuclease.
Similarly, regarding dependent claims 38-39 and 42-43, since any sequence can be an amplification site, the underlined sequence in p. 19, l. 36 plus any sequence 3’ of the portion of the cDNA that is the ROI of that same sequence, are two amplification sites flanking the ROI and the unique identifier, as recited in claim 38. As to claim 39, any sequence can be a binding site for a universal primer and any sequence can be a binding site for a primer specific to a subset of the plurality of nucleic acids. Further, since any sequence can be an excision site, the underlined sequence in p. 19, l. 36 plus any sequence 3’ of the portion of the cDNA that is the ROI of that same sequence, are two excision sites flanking the ROI and the unique identifier, as recited in claim 42, any sequence 3’ of the portion of the cDNA that is the ROI of that sequence, is an excision site adjacent to the site of the ROI, as recited in claim 43.
Regarding dependent claims 44-45, Brenner additionally teaches that the plurality of nucleic acids is in solution, or immobilized on a solid support, respectively. That is, Brenner teaches that the target polynucleotides may or may not be used with a solid support (i.e., p. 5, l. 4: “when used in combination with solid phase supports”).
Regarding dependent claims 48-49, Brenner additionally teaches primers for a two-primer PCR (p. 32, ll. 28; p. 33, ll. 13-16), as recited in claim 48. Regarding claim 49, any primer pair can be used in asymmetric PCR (which differs from “regular” PCR only in the concentrations of primers used), thus the primer pairs taught by Brenner for PCR also teaches the claim 49 limitation.
Regarding dependent claim 51, Brenner additionally teaches that a primer from one subset and a primer from a different subset comprise the same unique identifier. Specifically, Brenner teaches that the same tag can be on two different polynucleotides (p. 19, ll. 3-4).
Regarding dependent claim 52, Brenner additionally teaches that the subset of primers comprises at least five different primers, each with a different unique identifier (p. 18, ll. 30-38 through p. 19, ll. 1-29; p. 32, ll. 14-28; p. 9, Table 1; p. 10, Table II)
Regarding dependent claim 46, Brenner teaches that the plurality of nucleic acids is
isolated from a biological sample (e.g., p. 19, ll. 30-31: mRNA). Further, regarding claims 46-47, Van Eijk teaches that the nucleic acids comprise a rare sequence variant, specifically a minor allelic variant (p. 4, l. 19, p. 19, ll. 13-14, p. 2, ll. 10-11; p. 4, ll. 15-18).
Prior to the date of the instant invention, it would have been prima facie obvious to use the Van Eijk target of a nucleic acid comprising a rare sequence variant, in the modified Brenner reaction mixture, discussed above. Brenner teaches that the method is useful for high throughput screening of nucleic acids. It would have been obvious for the ordinary artisan to try the Van Eijk target nucleic acids in the Brenner assay, with the expectation that doing so would result in a high throughput method of detecting those particular target nucleic acids. The ordinary artisan would have had an expectation of success as Brenner does not limit the types of templates that may be used in the reaction mixture.
Conclusion
Claims 30, 34, 36-49 and 51-52 are being examined and are rejected. No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CAROLYN GREENE whose telephone number is (571)272-3240. The examiner can normally be reached M-Th 7:30-5:30 EST.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Gary Benzion can be reached at 571-272-0782. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/CAROLYN L GREENE/Examiner, Art Unit 1681
1 Brenner was cited in the Information Disclosure Statement submitted March 17, 2023.
2 Van Eijk was cited in the Information Disclosure Statement submitted March 17, 2023.
3 Schmid was cited in the PTO-892 Notice of References Cited mailed May 14, 2025.