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
This office action is in response to Applicant's Amendment filed on January 09, 2026.
Claims 1, 8, 14, 22-24, 27-40 were previously pending. Applicant amended claims 1 and 8. Claims 1, 8, 14, 22-24, 27-40 are currently pending, with claims 1, 8, 14, 22-24, 27-34 withdrawn.
Claims 35-40 are under examination. This is the first action on the merits.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 03/02/2023, 04/12/2023, 06/16/2023, 09/04/2024, 10/14/2025 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Election/Restrictions
Applicant's election without traverse of Group I (claims 1, 8, 14, 28-34, and 35-40) filed on July 31, 2025 is acknowledged 1.
Applicant’s election without traverse of the following species in the reply filed on January 09, 2026 is acknowledged:
Species of determining sequence of the template polynucleotide: E) determining sequence of the template polynucleotide comprises determining a variation of a pore current, and comparison of the variation of the pore current to a K-mer map (claim 35) 2;
Species of identifying an epigenetically modified base: H) identifying an epigenetically modified base in the determined sequence by an altered current relative to the non- modified base (claim 32,38).
Claims 1, 8, 14, 22-24, 27-34 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention.
Examination on the merits commences on claims 35-40.
Priority
The priority date of the instant claims 35-40 is 09/22/2021, filling date of the US provisional application NO. 63/247,155.
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, the term "variation of a pore current," as recited throughout the claims, is not expressly defined in the application's disclosure. Thus, under BRI, it is interpreted to encompass any change, shift, or variation observable in the pore current of a nanopore. This includes, for example, changes in pore current caused by blockage due to the introduction of a polynucleotide into a previously empty nanopore, temporal changes in pore current between two time points, or changes in pore current resulting from the application of different voltages across the membrane.
For the purpose of applying prior art, claim 35 recites "applied read potential," which is a term not expressly defined in the application's disclosure. The specification in paragraph [0141] provides the following relevant description:
"In another embodiment, since the applied electric potential difference may shift the position of the polynucleotide relative to the nanopore recognition zone, the regions of the polynucleotide sensed by the recognition zone may be shifted/offset when different electric potential differences are applied, and thus different maps may be used when different electric potential differences are applied. For example, at one applied voltage, the regions of the polynucleotide sensed by the recognition zone may include a 5-mer in the template strand and 3 nucleotides in the complementary strand; at another applied voltage, the regions of the polynucleotide sensed by the recognition zone may include a 5-mer in the template strand and 2 nucleotides in the complementary strand."
Thus, under BRI and in light of the specification, the term "applied read potential" is understood as voltage applied to the nanopore system, thereby allowing sensing of polynucleotides.
Claim 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.
Claims 35-37 and 39-40 are rejected under 35 U.S.C. 103 as being unpatentable over Akeson (Akeson et al. WO2008124107A1 - Compositions, devices, systems, and methods for using a nanopore; published on 2008-10-16; cited as Foreign Patent document # 10 in IDS filed on 03/02/2023), in view of Laszlo (Laszlo et al. Decoding long nanopore sequencing reads of natural DNA. Nat Biotechnol. 2014 Aug;32(8):829-33. doi: 10.1038/nbt.2950. Epub 2014 Jun 25. PMID: 24964173; PMCID: PMC4126851), as evidenced by
Clarke (Clarke et al. ; WO2019234432A1 – Method ; OXFORD NANOPORE TECH LTD; ; 12 December 2019; cited as Foreign Patent document # 16 in IDS filed on 03/02/2023) and
Goodwin (Goodwin et al. Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet. 2016 May 17;17(6):333-51. doi: 10.1038/nrg.2016.49. PMID: 27184599; PMCID: PMC10373632; cited as NPL # 25 in IDS filed 03/02/2023),
Deamer (Deamer et al.Three decades of nanopore sequencing. Nat Biotechnol. 2016 May 6;34(5):518-24. doi: 10.1038/nbt.3423. PMID: 27153285; PMCID: PMC673352; cited as NPL # 60 in IDS filed 09/04/2024) and
Vercoutere (Vercoutere et al.; Discrimination among individual Watson–Crick base pairs at the termini of single DNA hairpin molecules, Nucleic Acids Research, Volume 31, Issue 4, 15 February 2003, Pages 1311–1318, doi.org/10.1093/nar/gkg218; cited as NPL# 2 in IDS submitted on 10/14/2025).
A) Akeson (see [0088-0091] and Figure 4) discloses a method for nanopore-coupled
Sequencing by Synthesis (SBS), comprising measuring, in a nanopore, a duplex DNA comprising a single stranded portion and double stranded portion.
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Regarding claim 35, Akeson teaches a method of sequencing a template polynucleotide using a nanopore (Fig. 4), comprising:
incorporating a nucleotide to extend a complementary polynucleotide (Fig. 4) ;
determining a variation of a pore current when a single-stranded portion of the template polynucleotide and a duplex portion of the template polynucleotide and the complementary polynucleotide are held at an applied read potential in a position within the nanopore such that extension of the complementary polynucleotide cannot occur (Fig. 4; [0091] A DNA molecule with both doubled-stranded and single-stranded segments is captured in a nanoscale pore under an applied voltage. “The duplex terminus is pulled next to the pore's limiting-aperture where the identity of the added nucleotide is established. If no protected nucleotide has been added, the signal will be the same as in Step b. If this is the case, Steps d to f are repeated until the correct nucleotide is added and identified. “ Thus, a skilled artisan would understand the identity of the added nucleotide is determined via variation in pore current before and after the nucleotide addition);
determining a sequence of the template polynucleotide ([0088-0091] the sequence of template polynucleotide is determined via Sequencing by Synthesis).
The only distinction between Akeson' teaching of nanopore-coupled sequencing-by-synthesis and the subject matter of claim 35 is the use of a k-mer map.
Akeson discloses that, in its nanopore-based sequencing method, duplexes can be monitored for changes in pore ionic current, and that such changes may be used to identify the nucleotide incorporated during sequencing-by-synthesis ([0004]-[0005]; [0082] [0090], lines 7-13; [0091] lines 8-11). While Akeson does not expressly disclose the use of a k-mer map, the concept of using a k-mer map to associate distinct nanopore current signals with specific k-mer sequences is well-known in the art.
In the field of polynucleotide analysis using nanopore technology, “k-mer” is defined as the number of polymer units (e.g. nucleotides) measured by the nanopore as the polynucleotide moves through the pore. Shifts in current 3 are characteristic of the particular DNA sequence (or k-mer) in the pore. See Clark (page 34), See also in Goodwin (Figure 5 Ab; page 39, lines 16-20).
For example, in Goodwin, a review article covering next-generation sequencing technologies, it provides descriptions for k-mer, and further notes that Oxford Nanopore, a manufacturer of commercial stage Nanopore sequencing platforms already utilize k-mer map in their instruments.
“As the DNA translocates through the pore, a characteristic shift in voltage through the pore is observed. Various parameters, including the magnitude and duration of the shift, are recorded and can be interpreted as a particular k-mer sequence. As the next base passes into the pore, a new k-mer modulates the voltage and is identified.” (page 39, lines 16-20)
“To carry out sequencing, DNA is passed through a protein pore as current is passed through the pore (FIG. 5b). As the DNA translocates through the action of a secondary motor protein, a voltage blockade occurs that modulates the current passing through the pore. The temporal tracing of these charges is called squiggle space, and shifts in voltage are characteristic of the particular DNA sequence in the pore, which can then be interpreted as a k-mer. Rather than having 1–4 possible signals, the instrument has more than 1,000 — one for each possible k-mer, especially when modified bases present on native DNA are taken into account.” (page 8, para 2, lines 7-13)
Thus, the general principle that nanopores generate different ionic current signals corresponding to different polynucleotide k-mers is a foundational and well-understood feature of nanopore systems.
Laszlo fills the gap in Akeson and teaches a method for identifying polynucleotide sequences in nanopore sequencing using a k-mer map (see Abstract, "quadromer map").
Regarding claim 35, Laszlo teaches comparing measured current signals to values in a K-mer map (page 2, para 3; page 3, para 1-2, measured current level sequences were compared to predicted current levels based on the quadromer map.), the K-mer map comprising a plurality of entries in which each entry includes a K-mer sequence and a pore current (Supplementary Table 1), and determining a sequence of the template polynucleotide based upon the comparison of the pore current to the K-mer map (page 5, para 1).
Laszlo further suggests advantages of this k-mer map approach, such as "strong homology between quadromer-based current predictions and nanopore sequencing reads" (page 3, para 3, lines 1-2) and "highly predictive of ion current levels of previously unmeasured, complex, natural DNA sequences." (page 5, para 1, lines 4-5).
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 apply Laszlo's teaching of using a k-mer map to the nanopore coupled sequencing-by-synthesis method of Akeson. Both references relate to the same field ꟷ polynucleotide analysis using nanopores ꟷ and share the objective of identifying nucleotide sequence based on ionic current signals. Akeson already teaches sequencing-by-synthesis using controlled extension of a duplex and identifying the added nucleotide based on ionic current changes. Laszlo teaches how to generate and use a k-mer map to improve sequence identification based on the strong correlation between predicted and measured current signals.
A skilled artisan, in view of both references, would have been motivated to generate and apply a k-mer map for duplexes in order to improve base calling for each nucleotide added in Akeson's nanopore coupled sequencing-by-synthesis method, with high predictive confidence as suggested by Laszlo.
Although Akeson measures duplexes and Laszlo focuses on k-mer mapping in the context of single stranded DNA, a skilled artisan would appreciate ꟷ based on general knowledge of nanopore sequencing and the teachings of Akeson ꟷ that the addition of a nucleotide to a duplex results in a change in the pore current signal. Therefore, it would be reasonably expected that distinct duplex k-mers would also generate different and measurable ionic current signals, which can be used to construct a corresponding k-mer map for duplexes.
The person of ordinary skill would have had a reasonable expectation of success in combining these teachings because a skilled artisan in the field of nanopore sequencing would possess the knowledge and technical ability to measure ionic current signals from DNA duplexes and generate a duplex-specific k-mer map. Akeson already teaches measurement of duplex current signals, and Laszlo provides a straightforward method for generating and applying k-mer maps for sequence identification. The teachings are technically compatible and address the same analytical objective, making their combination predictable and well within the skill of the art.
B) Regarding claim 36, Akeson teaches the pore current is measured at more than one potential and the variation of the pore current is determined at more than one potential ([00269] lines 4-7; [0097] “ regulate the force on the macromolecule by varying the voltage acting across the pore”; [0090] “Strand capture and entry of the duplex segment into the pore vestibule can be confirmed based on current amplitude. Once this is achieved, the voltage is reduced under feedback control”).
Regarding claim 37, it recites "wherein the K-mer sequence of the K-mer map comprises at least two positions of the template polynucleotide in the single- stranded portion. "
This limitation is obvious in view of the combined teachings of Akeson and Laszlo.
Claim 37 requires the portion of polynucleotide being measured in the nanopore to comprise at least two single-stranded nucleotides.
Akeson teaches measuring a region of a polynucleotide comprising a double-stranded portion and single stranded portion (see Figure 4; see also [0090]), as the α-hemolysin Nanopore captures the single strand- double strand junction, this is because "[t]he nanopore is large enough to permit translocation of the ssDNA segment, but the double-stranded segment cannot translocate because its diameter is too large to fit through the narrowest part of the pore" ([0090]).
While Akeson does not explicitly teach the number of single stranded and double stranded (or paired) nucleotides, measured by the nanopore. The numbers claimed in claim 37 appear to be inherent given the known nature of the α-hemolysin Nanopore, utilized in Akeson ‘s method.
Akeson teaches measuring a region of a polynucleotide at its single strand- double strand junction, using a α-hemolysin Nanopore. The α-hemolysin Nanopore has a sensing region of about 12 nucleobases in length, as evidenced by Deamer (page 5, para 2, lines 6-8).
Vercoutere teaches that up to 9 base pairs of double stranded DNA can be measured within the α‐hemolysin Nanopore's sensing region, before reaching the narrowest part of the pore (page 1316, right-hand col, para 1).
Therefore, the polynucleotide junction being measured in Akeson’s method using α-hemolysin Nanopore having a sensing region of about 12 nucleobases in length, measures at least one double-stranded nucleotide pairs (e.g., 9) and at least 2 single-stranded nucleotides (e.g., 12 nt - 9nt =3nt ).
Regarding claim 39, Akeson teaches the nucleotide is incorporated to extend the complementary polynucleotide when the 3' end of the complementary polynucleotide is not sequestered within the nanopore (Fig. 4; [0088-0091]).
Regarding claim 40, Akeson teaches the variation of the pore current is determined when the 3' end of the complementary polynucleotide is positioned within the nanopore constriction zone (Fig. 4; [0088-0091]).
Claim 38 is rejected under 35 U.S.C. 103 as being unpatentable over Akeson, in view of Laszlo, evidenced by Clarke as applied to claim 35 above and further in view of Wang (Wang et al. Fast and precise detection of DNA methylation with tetramethylammonium-filled nanopore. Sci Rep 7, 183 (2017)doi.org/10.1038/s41598-017-00317-2; cited as NPL# 3 in IDS submitted on 10/14/2025) and
Ding (Ding et al. Differentiation of G:C vs A:T and G:C vs G:mC Base Pairs in the Latch Zone of α-Hemolysin. ACS Nano. 2015 Nov 24;9(11):11325-32. doi: 10.1021/acsnano.5b05055. Epub 2015 Oct 27. PMID: 26506108; PMCID: PMC4876701) as evidenced by
Yuen (Yuen et al. Systematic benchmarking of tools for CpG methylation detection from nanopore sequencing. Nat Commun 12, 3438 (2021). Published: 08 June 2021).
Regarding claim 38, Akeson teaches in its DNA sequencing method, measuring a region of a polynucleotide at its single strand- double strand junction, using a α-hemolysin Nanopore (Figure 4), and it further teaches its teaching could be applied to assaying DNA methylation state ([0134]).
It is well known in the art that modified nucleotide bases, such as methylcytosine, produces different pore current signals relative to non-modified bases (see CLARKE, page 82, para 2).
See also in Yuen:
"nanopore long-read technologies provide many distinct advantages. Individual DNA molecules, harboring base modifications, can be sequenced in their native state without any prior enzymatic or chemical treatment and without the need for PCR amplification8,9. As a single-DNA molecule travels through a pore, base modifications can be revealed by their unique signal shapes, which differ from the equivalent unmodified base8,9,10." (page 2, left-hand col, para 2)
Therefore, it would have been prima facie obvious to apply the DNA sequencing method in combined teaching of Akeson and Laszlo, which utilizes Nanopore and sequencing by synthesis, to assay methylated DNA, wherein the methylated base produces an altered pore current variation relative to the non-modified base.
Akeson teaches a sequencing method for analyzing DNA and further suggests that its teachings can be applied to assaying DNA methylation states. Specifically, Akeson suggests analyzing target DNA comprising methylated bases (as indicated in [0073] and [0134]), and demonstrates measuring target DNA comprising a duplex using Nanopore-coupled sequencing by synthesis (Figure 4).
A skilled artisan would have had a reasonably expectation of success in making this modification, as using nanopore technology to accurately detect DNA methylation in duplex is a known approach in the art as shown in Wang (see Abstract) and Ding (see Abstract).
The combination of these elements in the manner claimed does not impart any new or unexpected results beyond the teaching of Akeson and in the art. Given that each element performs a known function as per its prior art teaching, their combination to achieve a predictable result would have been obvious, as per MPEP 2143.
Double Patenting- Obvious Type
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 35 and 37-40 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 6, 9 and 134 of copending Application No. 17/947,877 (reference application). Although the claims at issue are not identical, they are not patentably distinct from each other because the instant claims are anticipated by the claims (filed on 09/23/2025) of the '877 application.
Instant claim 35 recites:
A method of sequencing a template polynucleotide using a nanopore, comprising:
incorporating a nucleotide to extend a complementary polynucleotide (‘877 Application, claim 1);
determining a variation of a pore current when a single-stranded portion of the template polynucleotide and a duplex portion of the template polynucleotide and the complementary polynucleotide are held at an applied read potential in a position within the nanopore such that extension of the complementary polynucleotide cannot occur (‘877 Application, claim 1);
comparing the variation of the pore current to a K-mer map (‘877 Application, claim 134),
the K-mer map comprising a plurality of entries in which each entry includes a K-mer sequence and a pore current variation at the applied read potential (‘877 Application, claim 134),
the K-mer sequence includes at least one position of the template polynucleotide in the single-stranded portion and at least one position of the template polynucleotide in the duplex portion (‘877 Application, claim 134); and
determining a sequence of the template polynucleotide based upon the comparison of the variation of the pore current to the K-mer map (‘877 Application, claim 134).
Therefore, instant claims 35 and 39-40 are anticipated by claims 1 and 134 the '877 application. Instant claims 37; 38 are anticipated by claims 6; 9 of the '877 application, respectively.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Conclusion
No claims are allowed.
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/TIAN NMN YU/Examiner , Art Unit 1681 /AARON A PRIEST/Primary Examiner, Art Unit 1681
1 Claims 22-24 and 27 are withdrawn as being drawn to non-elected group II.
2 Claims 1, 8, 14 and 28-34 are withdrawn as being drawn to non-elected species A-D.
3 Current (I) is directly proportional to voltage (V), see Ohm's Law V=IR, thus measurements in current and voltage appear to be used in the art.