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 .
Applicants' arguments filed on 2/13/2026, have been fully considered and are deemed to be persuasive to overcome some of the rejections previously applied. Rejections and/or objections not reiterated from previous office actions are hereby withdrawn. Claims 1, 4-20 are still at issue and are present for examination.
Terminal Disclaimer
The terminal disclaimer filed on 1/30/2026 disclaiming the terminal portion of any patent granted on this application which would extend beyond the expiration date of US 11,773,380 has been reviewed and is accepted. The terminal disclaimer has been recorded.
Election/Restrictions
Applicant's election without traverse of Group I, claims 1-10, to a polymerase composition in the paper of 2/27/2025, is acknowledged. Applicant's election without traverse of the following species: Species Group 1: F494, Species Group 2: 410, Species Group 3: Pyrococcus, in the paper of 4/18/2023, is acknowledged.
Claims 11-16 are withdrawn from further consideration by the examiner, 37 CFR 1.142(b), as being drawn to a non-elected invention.
Claim Objections
Claims 9, 10 and 18 are objected to because of the following informalities:
Claims 9, 10, and 18 are dependent on rejected claim 1.
Appropriate correction is required.
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.
The rejection of claim(s) 1, 2, 6, 7, 8, under 35 U.S.C. 103 as being unpatentable Easton et al. (WO 2019/148119) and Uniprot Accession No. A0A160VQZ9, April 7 2021 is withdrawn based upon applicants amendment of the claims and arguments presented in the paper of 7/2/2025.
Claim(s) 1, 4, 5, 6, 7, 8, 17, 19 and 20 is/are rejected under 35 U.S.C. 103 as being unpatentable Easton et al. (WO 2019/148119) and Tabor et al. (U.S. Patent No. 5,614,365), as evidenced by Uniprot Accession No. A0A5C0XMA4, Aug 2020.
This rejection was stated in the previous office action as it applied to previous claims 1, 4, 5, 6, 7, 8, 17, 19 and 20. In response to the rejection applicants have not amended the claims, but merely traverse the rejection as it applies to previous claims 1, 4, 5, 6, 7, 8, 17, 19 and 20.
For applicants convenience the original rejection is repeated herein.
Easton et al. (WO 2019/148119) teach methods for nucleic acid amplification and sequencing for research, diagnostics and treatment. Easton et al. teach such methods for example Next Generation Sequencing for providing large amounts of information on complex samples, genomes and other nucleic acid sources. Easton et al. teach a method of amplifying a target nucleic acid molecule, comprising contacting a sample comprising the target nucleic acid molecule, at least one amplification primer, at least one nucleic acid polymerase, and a mixture of nucleotides, wherein the mixture of nucleotides comprises at least one terminator nucleotide (e.g., 2-nitrobenzyl alkylated HOMedU triphosphates) which terminates nucleic acid replication by the polymerase, and amplifying the target nucleic acid molecule to generate a plurality of terminated amplification products, wherein the replication proceeds by strand displacement replication. Easton et al. teach the above methods comprising the use of a 3’ unblocked reversible terminator and any of a number of different polymerases (see claims 57-67 and supporting text). Easton et al. teach the above methods using any of a number of exonuclease minus DNA polymerases (see claims 65-66).
Tabor et al. (U.S. Patent No. 5,614,365) teach a number of DNA polymerases having modified nucleotide binding sites for DNA sequencing. Tabor et al. teach that 3’ to 5’ exonuclease activity can complicate the taught assays by making the DNA polymerase appear to discriminate more against a ddNTP than it actually does. While Tabor et al. disclose specific alterations in T7 DNA polymerase, E. coli DNA polymerase I and Taq DNA polymerase, they teach homologous regions in a number of other DNA polymerases. Tabor et al. teach a number of DNA polymerases for use in their sequencing methods which have a reduction in exonuclease activity including those listed in column 22, which include Pfu furiousus DNA polymerase mutated in the motif B region occurring at Leu489-Phe 494. The sequence of the Pfu DNA polymerase taught by Tabor et al. is 100% identical to instant SEQ ID NO:1 as evidenced by Uniprot Accession No. A0A5C0XMA4, Aug 2020 who teach the amino acid sequence of the wild-type Pfu DNA polymerase. Tabor et al. teach that modification of a single amino acid residue, even by the addition of a single hydroxyl group, as in the case of a phenylalanine to tyrosine change, provides a very large alteration (250-8,000 fold) in discrimination levels. Tabor et al. teach that those in the art will recognize that changes at this one site are not limiting in this invention, and changes at other sites that decrease the ability of a polymerase to discriminate can now be readily found by routine experimentation.
One of skill in the art before the effective filing date would have been motivated to substitute the Pfu DNA polymerase mutated at position F494 to another amino acid such as tyrosine, as taught by Tabor et al. for the DNA polymerases used by Easton et al. in the methods of nucleic acid amplification taught by Easton et al. as a means of amplifying a nucleic acid using an exonuclease negative DNA polymerase. In order to practice the methods obvious over Easton et al. and Tabor et al. one of ordinary skill in the art would have created a composition comprising a priming strand, a 3'-OH unblocked reversible terminator (e.g., 2-nitrobenzyl alkylated HOMedU triphosphates), and the polymerase and adding the composition to the template nucleic acid. Thus the obvious composition would both not comprise the template prior to adding to the template and comprises the template after adding to the template. While Tabor et al. does not teach that the taught DNA polymerase has an incorporation activity for the 3-OH unblocked reversible terminator of at least 2-fold higher than an incorporation activity of the DNA polymerase of SEQ ID NO:11, this is considered and inherent property of the DNA polymerase taught by Tabor et al. based upon the high degree of sequence identity to SEQ ID NO:1 and its reduced exonuclease activity. The expectation of success is high based upon the high level of skill in the art as exemplified by Easton et al. who teach all the methods necessary to practice the obvious methods.
Applicants Response
Applicants continue to traverse the rejection as in applicants previous response. Initially applicants present the technical importance of the 3'-OH unblocked reversible terminators recited in the claims and the considerations required when incorporating these nucleotides in sequencing-by-synthesis (SBS). Applicants submit that their architecture requires the polymerase to preserve natural Watson-Crick geometry, accommodate a bulky base-linked blocking group, and maintain cycle-to-cycle synchronization for accurate base calling. Incorporation efficiency, active-site accommodation, template binding and release, and deblocking compatibility are all affected by the unique structural demands of 3'-OH unblocked reversible terminators. Applicants submit that these considerations are specific to SBS and are neither addressed nor anticipated by the cited prior art.
Applicants then present a summary of the teachings of Easton and Tabor and the basis of the rejection that the examiner asserts that a person of skill in art would combine Easton and Tabor by substituting a Tabor-type mutation, such as one at Pfu residue F494, into the polymerases used in Easton, and would thereby arrive at a polymerase that inherently exhibits increased incorporation of 3'-OH unblocked reversible terminators. Applicant respectfully submits that this rationale lacks factual support.
Applicant submits that a person of skill in the art would not have turned to Easton to address the incorporation challenges associated with 3'-OH unblocked reversible terminators as applicants submit that Easton merely discloses that a terminator nucleotide may be present in an amplification mixture and that any polymerase may be used. Applicant submits that Easton does not teach polymerase engineering, does not identify any polymerase limitation, and does not discuss how the structure of a reversible terminator affects incorporation kinetics, fidelity, or polymerase dissociation. Applicant submits that because Easton is indifferent to active-site constraints and does not address SBS-specific performance variables, it cannot provide a motivation to modify any polymerase residue to improve incorporation of 3'- OH unblocked reversible terminators.
Applicant further submits that Tabor provides no teaching or suggestion applicable to the incorporation of 3'-OH unblocked reversible terminator as applicants submit that Tabor is directed to discrimination between dNTPs and ddNTPs, which differ at the sugar moiety. Applicant submits the substrates analyzed in Tabor do not contain a nucleobase-linked blocking group and therefore interact with the polymerase through an entirely different structural interface. Applicant submits nothing in Tabor suggests that mutations affecting ddNTP discrimination would influence incorporation of base-modified reversible terminators, nor that insights derived from sugar-based discrimination can be extrapolated to the steric and electronic demands of a bulky nucleobase modification.
Applicant submits that the examiner's assertion that mutation within the motif B region of Pfu polymerase would have been an obvious design choice extends Tabor beyond its teachings and overlooks the unpredictability of polymerase engineering. Applicant submits that although Tabor identifies a motif that influences ddNTP discrimination in certain polymerases, it does not teach which substitutions would affect nucleobase-modified substrates, nor does it address the distinct role of the base-binding pocket in accommodating 3'-OH unblocked reversible terminators.
Applicant submits because ddNTPs and reversible terminators perturb different regions of the active site, a person of skill in the art would not expect ddNTP-related mutations to yield improved incorporation of 3'-OH unblocked reversible terminators.
Applicant additionally submits that a person of skill in the art would not have had a reasonable expectation of success in substituting residues such as F494, or any of the enumerated positions recited in the present claims, to increase incorporation of 3'-OH unblocked reversible terminators. Applicant submits the examiner has not identified any disclosure in Tabor suggesting that mutation of Pfu polymerase at these residues would improve acceptance of bulky reversible terminators. Applicant submits the claimed functional requirement (enhanced incorporation relative to SEQ ID NO:11) is not inherent and cannot be inferred from ddNTP discrimination behavior in unrelated polymerase families.
Applicant further submits that the present claims recite polymerase mutants that provide coordinated improvements in incorporation efficiency, fidelity, and template dissociation in the presence of 3'-OH unblocked reversible terminators. Applicant submits these improvements arise from engineering directed specifically to the nucleobase-linked blocking group and are not predictable from the teachings of Easton or Tabor. Applicant submits the prior art provides no reasonable basis to expect that the claimed mutations would enhance incorporation of 3'-OH unblocked reversible terminators or support the cycle-synchronized behavior necessary for high-quality SBS performance.
Finally, Applicant submits that the examiner's characterization of the motif B region as a set of residues that a person of skill in the art would routinely mutate oversimplifies the technical complexity of polymerase engineering. Even within a defined region, individual residues contribute differently to substrate binding, geometric positioning, and catalytic transition states. Predicting which substitutions would enhance incorporation of 3'-OH unblocked reversible terminators requires empirical analysis and is not suggested by the prior art.
Applicants complete argument is acknowledged and has been carefully considered, however, is not found persuasive for the reasons previously made of record and for those reasons repeated herein.
Applicants summary of the technical importance of the 3'-OH unblocked reversible terminators recited in the claims and the considerations required when incorporating these nucleotides in sequencing-by-synthesis (SBS) is appreciated and has been considered in relation to the rejection.
Applicants summary of the teachings of Easton and Tabor and the basis of the rejection is acknowledged.
In response to applicants submission that a person of skill in the art would not have turned to Easton to address the incorporation challenges associated with 3'-OH unblocked reversible terminators as applicants submit that Easton merely discloses that a terminator nucleotide may be present in an amplification mixture and that any polymerase may be used this is not found persuasive, as Easton is used to teach methods of amplifying a target nucleic acid molecule, comprising contacting a sample comprising the target nucleic acid molecule, at least one amplification primer, at least one nucleic acid polymerase, and a mixture of nucleotides, wherein the mixture of nucleotides comprises at least one terminator nucleotide (e.g., 2-nitrobenzyl alkylated HOMedU triphosphates) which terminates nucleic acid replication by the polymerase, and amplifying the target nucleic acid molecule to generate a plurality of terminated amplification products, wherein the replication proceeds by strand displacement replication. Easton et al. teach the above methods comprising the use of a 3’ unblocked reversible terminator and any of a number of different polymerases (see claims 57-67 and supporting text). Easton et al. teach the above methods using any of a number of exonuclease minus DNA polymerases (see claims 65-66).
In response to applicants submission that Tabor provides no teaching or suggestion applicable to the incorporation of 3'-OH unblocked reversible terminator as applicants submit that Tabor is directed to discrimination between dNTPs and ddNTPs, which differ at the sugar moiety this is not found persuasive as Tabor et al. is used to teach a number of DNA polymerases for use in their sequencing methods which have a reduction in exonuclease activity including those listed in column 22, which include Pfu furiousus DNA polymerase mutated in the motif B region occurring at Leu489-Phe 494.
In response to above applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
In response to applicants submission that the substrates analyzed in Tabor do not contain a nucleobase-linked blocking group and therefore interact with the polymerase through an entirely different structural interface, while this is recognized, Tabor et al. disclose that the region, Leu489-Phe494 make up motif B conserved across six families of polymerases, DNA polymerases from Pol I, Pol alpha, and Pol beta families, DNA-dependent RNA polymerases, reverse transcriptases, and RNA-dependent RNA polymerases. Further Tabor et al. refer to the residue identified within the motif B as “the selectivity residue” referring to the nucleotide substrate. Accordingly one of skill in the art would look to those residues recognized as a selectivity residue as being involved in the selectivity of the nucleotide substrate no matter how the substrate was modified.
In response to applicants submission that the examiner's assertion that mutation within the motif B region of Pfu polymerase would have been an obvious design choice extends Tabor beyond its teachings and overlooks the unpredictability of polymerase engineering is not found persuasive as the predictability of polymerase engineering is not unpredictable as supported by Tabor et al. who teach that the “motif B” as well as other motifs are shared by multiple families of polymerases, including DNA polymerases from Pol I, Pol alpha, and Pol beta families, DNA-dependent RNA polymerases, reverse transcriptases, and RNA-dependent RNA polymerases.
While ddNTPs and reversible terminators may perturb different regions of the active site, a person of skill in the art would not expect those regions identified as having to do with substrate “selectivity” to be involved in substrate selectivity no matter the specific alteration of the nucleotide substrate. With regard to the interaction of the nucleotide substrate polymerase active site, one of skill in the art would recognize that various different modifications of the nucleotide substrate effect the nucleotride substrate as it interacts with the same active site and the same selectivity residues.
In response to applicants submission that the a person of skill in the art would not have had a reasonable expectation of success in substituting residues such as F494, or any of the enumerated positions recited in the present claims, to increase incorporation of 3'-OH unblocked reversible terminators, this is not found persuasive on the basis that the expectation os success is high based upon the high level of skill in the art as exemplified by Tabor et al. who teach all the methodology required to create the obvious polymerase mutant. While Tabor et al. does not teach that the taught DNA polymerase has an incorporation activity for the 3-OH unblocked reversible terminator of at least 2-fold higher than an incorporation activity of the DNA polymerase of SEQ ID NO:11, this is considered and inherent property of the DNA polymerase taught by Tabor et al. based upon the high degree of sequence identity to SEQ ID NO:1 and its reduced exonuclease activity.
In response to applicants final submission that the examiner's characterization of the motif B region as a set of residues that a person of skill in the art would routinely mutate oversimplifies the technical complexity of polymerase engineering and that predicting which substitutions would enhance incorporation of 3'-OH unblocked reversible terminators requires empirical analysis and is not suggested by the prior art, is not found persuasive for the reasons stated previously and repeated above.
Further as stated previously, although Tabor does not teach or suggest that Pfu DNA polymerase having a mutation in the region of Leu489-Phe494 have reduced exonuclease activity, Tabor et al. teach that 3’ to 5’ exonuclease activity can complicate the taught assays by making the DNA polymerase appear to discriminate more against a ddNTP than it actually does. While Tabor et al. disclose specific alterations in T7 DNA polymerase, E. coli DNA polymerase I and Taq DNA polymerase, they teach homologous regions in a number of other DNA polymerases also effect 3’ to 5’ exonuclease activity. Tabor et al. teach a number of DNA polymerases for use in their sequencing methods which have a reduction in exonuclease activity including those listed in column 22, which include Pfu furiousus DNA polymerase mutated in the motif B region occurring at Leu489-Phe 494. The sequence of the Pfu DNA polymerase taught by Tabor et al. is 100% identical to instant SEQ ID NO:1 as evidenced by Uniprot Accession No. A0A5C0XMA4, Aug 2020 who teach the amino acid sequence of the wild-type Pfu DNA polymerase. Tabor et al. teach that modification of a single amino acid residue, even by the addition of a single hydroxyl group, as in the case of a phenylalanine to tyrosine change, provides a very large alteration (250-8,000 fold) in discrimination levels. Tabor et al. teach that those in the art will recognize that changes at this one site are not limiting in this invention, and changes at other sites that decrease the ability of a polymerase to discriminate can now be readily found by routine experimentation.
Thus, Tabor et al. does suggest that Pfu DNA polymerase having a mutation in the region of Leu489-Phe494 has altered or reduced exonuclease activity.
Further Tabor et al. teaches that there are only six residues in this region (motif b) and so it is suggested that each of them may play a role in part and thus it is suggested that each of them be mutated accordingly. Tabor et al. teach that modification of a single amino acid residue, even by the addition of a single hydroxyl group, as in the case of a phenylalanine to tyrosine change, provides a very large alteration (250-8,000 fold) in discrimination levels. Tabor et al. teach that those in the art will recognize that changes at this one site are not limiting in this invention, and changes at other sites that decrease the ability of a polymerase to discriminate can now be readily found by routine experimentation.
In response to applicants submission that Easton fails to suggest that any modifications are needed to polymerases in order to increase their incorporation efficiency of 3' unblocked reversible terminators, this is not found persuasive on the basis that the rejection is not based on such a suggestion by Easton, but the rejection is based on one of skill in the art before the effective filing date would have been motivated to substitute the Pfu DNA polymerase mutated at position F494 to another amino acid such as tyrosine, as taught by Tabor et al. for the DNA polymerases used by Easton et al. in the methods of nucleic acid amplification taught by Easton et al. as a means of amplifying a nucleic acid using an exonuclease negative DNA polymerase. Further as stated previously and above, Easton et al. teach the above methods comprising the use of a 3’ unblocked reversible terminator and any of a number of different polymerases (see claims 57-67 and supporting text). Easton et al. teach the above methods using any of a number of exonuclease minus DNA polymerases (see claims 65-66).
Thus claim(s) 1, 4, 5, 6, 7, 8, 17, 19 and 20 is/are rejected under 35 U.S.C. 103 as being unpatentable Easton et al. (WO 2019/148119) and Tabor et al. (U.S. Patent No. 5,614,365), as evidenced by Uniprot Accession No. A0A5C0XMA4, Aug 2020.
Related Art not used in a Rejection:
US 5,614,365 teaches a number of DNA polymerases having modified nucleotide binding sites for DNA sequencing. US 5,614,365 further teaches the motif B region responsible for the dideoxyribose moiety is found in Pyrococcus furiosus DNA polymerase at positions Leu489-Phe494 (relative to instant SEQ ID NO:1).
US 11,034,942 teaches a mutant polymerase comprising K477W and A486V wherein said polymerase is capable of incorporating modified nucleotides.
US 8,435,775 teaches a mutant polymerase comprising Y546H wherein said polymerase is capable of incorporating modified nucleotides.
Remarks
No claim is allowed.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to RICHARD G HUTSON whose telephone number is (571)272-0930. The examiner can normally be reached on 6-3 EST Mon-Fri.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Robert Mondesi can be reached on (408) 918-7584. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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rgh
4/2/2026
/Richard G Hutson/
Primary Examiner, Art Unit 1652