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 .
DETAILED ACTION
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office Action.
Status of Claims
Claims 1-12 are currently pending. Claims 1, 3, 4 and 6 have been amended by Applicants’ amendment filed 04-28-2026. No claims have been canceled by Applicants’ amendment filed 04-28-2026. Claim 12 has been added by Applicants’ amendment filed 04-28-2026.
Applicant's election with traverse of Group I, claims 1-6, directed to a method for analyzing a target nucleic acid; and Applicant’s election of Species without traverse as follows:
Species (A): further comprising determining a property dependent on the target nucleic acid other than its sequence (claim 3); and
Species (B): wherein the nucleotides of the mutation sites of one, two, or more libraries (claim 6), in the reply filed December 10, 2025 was previously acknowledged.
Claims 7-11 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected invention, there being no allowable generic or linking claim. Applicant timely
traversed the restriction (election) requirement in the reply filed on December 10, 2025.
Claims 2 and 5 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected species, there being no allowable generic or linking claim.
The restriction requirement was deemed proper and was made FINAL.
The claims will be examined insofar as they read on the elected species.
A complete reply to the final rejection must include cancellation of nonelected claims or other appropriate action (37 CFR 1.144) See MPEP § 821.01.
Therefore, claims 1, 3, 4, 6 and 12 are under consideration to which the following grounds of rejection are applicable.
Priority
The present application filed June 15, 2022, is a 35 U.S.C. 371 national stage filing of
International Application No. PCT/EP2020/086800, filed December 17, 2020, which claims the benefit of European Patent Application EP19217297.1, filed December 18, 2019.
Acknowledgment was made of applicant's claim for foreign priority based on an application filed in Germany on March 26, 2020 including the certified copy of the European Patent Application EP19217297.1, filed December 18, 2019.
Withdrawn Objections/Rejections
Applicants’ amendment and arguments filed April 28, 2026 are acknowledged and have been fully considered. The Examiner has re-weighed all the evidence of record. Any rejection and/or objection not specifically addressed below are herein withdrawn.
Maintained Objections/Rejections
Claim Rejections - 35 USC § 112(b)
The rejection of claims 1, 3, 4 and 6 is maintained, and claim 12 is newly rejected, under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which applicant regards as the invention.
Claim 1 is indefinite for the recitation of the term “each site mutagenesis library” such as recited in claim 1, lines 4 and 9. There is insufficient antecedent basis for the term “each site mutagenesis library” in the claim because claim 1, line 4 recites the term “two or more site mutagenesis libraries.” The Examiner suggests that Applicant amend the claim to recite, for example, “each of the two or more site mutagenesis libraries.”
Claim 1 is indefinite for the recitation of the term “the probe” such as recited in claim 1, line 12. There is insufficient antecedent basis for the term “the probe” in the claim because claim 1, line 10 recites the term “a probe nucleic acid.” The Examiner suggests that Applicant amend the claim to recite, for example, “the probe nucleic acid.”
Claim 1 is indefinite for the recitation of the term “one or more library-specific mutation sites and adjacent nucleotides” such as recited in claim 1, line 13 because it is unclear which “adjacent nucleotides” are being referred to. For example, it is unclear whether the probe nucleic acids include the mutations and nucleotides adjacent to the mutation sites; or whether the probe nucleic acids comprise library-specific mutation sites and general “adjacent nucleotides” and, thus, the metes and bounds of the claim cannot be determined.
Claim 1 is indefinite for the recitation of the term “adjacent” such as recited in claim 1, line 13 because the term “adjacent” is a relative term that renders the claim indefinite. The term “adjacent” is not defined by the claim, and the Specification does not provide a standard for ascertaining the requisite distance, number and/or location of nucleotides as compared to some other value that qualifies as an “adjacent nucleotides”, such that one of ordinary skill in the art would not be reasonably appraised of the scope of the invention and, thus, the metes and bounds of the claim cannot be determined.
Claim 1 is indefinite for the recitation of the term “the mixture” such as recited in claim 1, line 15. There is insufficient antecedent basis for the term “the mixture” in the claim because claim 1, line 14 recites the term “one mixture.”
The rejection of claim 3 is maintained as being indefinite for the recitation of the term “step ii)” such as recited in claim 3, line 2. There is insufficient antecedent basis for the term “step ii)” in the claim because claim 1, line 6 recites the term “ii).”
Claim 3 is indefinite for the recitation of the term “its mutated sequence” such as recited in claim 3, line 4 because claim 3 depends from instant claim 1, wherein claim 1 does not recite the term “mutated sequence” and, thus, the metes and bounds of the claim cannot be determined.
Claim 4 is indefinite for the recitation of the term “the property determined…is a property of the protein encoded by the mutated sequence…and the property is selected from one or more of…cell wall, excretion” such as recited in claim 4, lines 4-29 because claim 4 depends from claim 1 and 3, wherein claim 3 already recites what the property is dependent upon, which is “a property dependent on the target nucleic acid mutated at one or more library-specific mutation sites other than its mutated sequence” as recited in claim 3, lines 2-4, such that dependent claim 4 cannot then recite that the property is something entirely different (e.g., a property of the protein encoded by the mutated sequence of the at least one isolated member…) and, thus, the metes and bounds of the claim cannot be determined.
Claim 4 is indefinite for the recitation of the term “expression” and “location in the cytoplasm, spore, cell membrane, cell organelle lumen or membrane, cell wall, excretion” such as recited in claim 4, lines 18 and 25-26 because the terms “expression” and “location in the cytoplasm, spore, cell membrane, cell organelle lumen or membrane, cell wall, excretion” are duplicate terms also recited in lines 6 and 16-17, wherein the duplicate terms did not appear in the previous version of the claims, filed 06-15-2022, and where claim 4 is not marked to show amendments to claim where these duplicate terms have been added and, thus, the metes and bounds of the claim cannot be determined.
The rejection of claim 6 is maintained as being indefinite for the recitation of the term “the target nucleic acid sequence” such as recited in claim 6, lines 3 and 5. There is insufficient antecedent basis for the term “the target nucleic acid sequence” in the claim because claim 1, line 1 recites the term “a target nucleic acid.”
The rejection of claim 6 is maintained as being indefinite for the recitation of the terms “one or more arbitrary nucleotides;” “one or more degenerate nucleotides;” and a “predefined set of nucleotide sequences” such as recited in claim 6, lines 3-8. There is insufficient antecedent basis for the term “one or more arbitrary nucleotides” in the claim. Moreover, claim 6 depends from claim 1, wherein claim 1 does not recite the presence of arbitrary nucleotides; degenerate nucleotides; and predefined set of nucleotide sequences and, thus, the metes and bounds of the claim cannot be determined.
Claim 12 is indefinite for the recitation of the term “the property is a property of the target nucleic acid mutated at the one or more library-specific mutations sites” such as recited in claim 12, lines 1-2 because claim 12 depends from claims 1 and 3, wherein claim 3 already recites what the property is dependent upon, which is “a property dependent on the target nucleic acid mutated at one or more library-specific mutation sites other than its mutated sequence” as recited in claim 3, lines 2-4, such that dependent claim 12 cannot then recite that the property is something entirely different and, thus, the metes and bounds of the claim cannot be determined.
Claim Rejections - 35 USC § 112(d)
The rejection of claims 3, 4 and 6 is maintained, and claim 12 is newly rejected under 35 U.S.C. 112(d) 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.
Claim 3 recites (in part): “further comprising determining, for at least one of the isolated members in step ii)…other than its mutated sequence” in lines 1-4 because claim 3 depends from instant claim 1, wherein claim 1 does not recite a “step ii)” and/or a mutated sequence. Thus, claim 3 is an improper dependent claim 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.
Claim 4 recites (in part): “wherein the mutated sequence of each isolated member encodes a protein…cell wall, excretion” in lines 1-29 because claim 4 depends from instant claims 1 and 3, wherein claim 3 already recites what the property comprises, e.g., “a property dependent on the target nucleic acid mutated at one or more library-specific mutation sites other than its mutated sequence” as recited in claim 3, lines 2-4, such that dependent claim 4 cannot then recite that the property is something entirely different (e.g., a property of the protein encoded by the mutated sequence of the at least one isolated member…). Thus, claim 4 is an improper dependent claim 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.
Claim 6 recites (in part): “wherein the one or more library-specific mutation sites,…a predefined set of nucleotide or nucleotide sequences” in lines 2-8 because claim 6 depends from claim 1, wherein claim 1 does not recite arbitrary nucleotides, degenerate nucleotides, and/or a predefined set of nucleotide sequences. Thus, claim 6 is an improper dependent claim 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.
Claim 12 recites (in part): “wherein the property is a property of the target nucleic acid mutated at the one or more library-specific mutation sites” in lines 1-3 because claim 12 depends from instant claims 1 and 3, wherein claim 3 already recites what the property comprises, e.g., “a property dependent on the target nucleic acid mutated at one or more library-specific mutation sites other than its mutated sequence” as recited in claim 3, lines 2-4, such that dependent claim 12 cannot then recite that the property comprises something entirely different (e.g., a property of the target nucleic acid mutated at the one or more library-specific mutation sites). Thus, claim 12 is an improper dependent claim 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.
Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
Claim Rejections - 35 USC § 102
Please Note: the references were modified slightly in view of Applicant’s amendments and arguments, in the reply filed 04-28-2026.
The rejection of claims 1, 3, 4 and 6 is maintained, and claim 12 is newly rejected under 35 U.S.C. 102(a1)/102(a2) as being anticipated by Cozens et al. (hereinafter “Cozens”) (Nucleic Acids Research, 2018, 46(8), 1-13; of record; and Supplementary Information, 2018, 1-27).
Regarding claim 1, Cozens teaches a new library assembly method to uncompromisingly address all four quality bottlenecks of simultaneous multiple site saturation mutagenesis, wherein Darwin Assembly is a simple, fast, low-cost and flexible platform capable of delivering large (>108 transformations), user-defined libraries with any desired combinations of mutations anywhere in a gene of interest, or in multiple genes/features in a plasmid, which takes a single working day and is both highly effective and efficient (all clones mutated at all positions targeted) (pg. 2, col 1, first full paragraph). Cozens teaches that the Darwin Assembly was applied to different genes, including chloramphenicol acetyl transferase (CAT), Saccharomyces cerevisiae tryptohanyl tRNA synthetase (ScWRS), Thermococcus gorgonarius DNA polymerase (TgoT), Thermococcus kodakaraenis DNA polymerase (KOD) and T7 RNA polymerase (T7RSS), which differ in length as well as composition (interpreted as two or more sites mutagenesis libraries, claim 1(i)) (pg. 7, col 1, last partial paragraph). Cozens teaches that scanning libraries introduce a single point mutation at different sites in a target gene, where alanine scanning is a traditional approach to identify functionally important residues in enzymes but scanning libraries can also be used to map the local functional neighborhood of an enzyme or even to generate datasets for deep mutational scanning-guided rational protein design, wherein mutant generation can be laborious when mutations are introduced individually by site-directed mutagenesis (interpreted as site mutagenesis; interpreting scanning libraries as a set of two or more libraries; and providing isolated library members, claim 1(i) (pg. 8, col 1, second full paragraph). Cozens teaches that Darwin Assembly can efficiently generate scanning libraries by combining oligo-nucleotides targeting the same binding site, but each introducing a different mutation, such that using the CAT gene as the model, an alanine scan library was assembled around residues Thr101 and Ser104, using Leu105 (CTC→CTG) recording as an assembly control, such that five inner oligonucleotides were designed: four introducing an alanine mutation (Thr101Ala, Phe102Ala, Ser103Ala, or Ser104Ala) and one wild-type, such that all five oligonucleotides introduced the CTC→CTG control mutation at the Leu105 codon (interpreted as selecting one member of each site mutagenesis library; and obtaining a probe nucleic acid of the target mutated nucleic acid, claim 1(ii) and 1(iii)) (pg. 8, col 1, third full paragraph). Cozens teaches that the five oligonucleotides were mixed in a 1:1:1:1:1 ratio in the assembly reaction to create a library where each variant was expected to be 20% of the final population, wherein assembly was successful and over 103 transformants were isolated, pooled, their plasmid DNA purified and deep sequenced––generating nearly 80,000 reads, such that the control Leu105 (CTC→CTG) mutation was present in 99.7% of the samples, a frequency comparable to sites that had not been targeted for mutagenesis (see Table 2) (interpreted as isolating and mixing probe nucleic acids; and sequencing the probe nucleic acids of the mixture obtained in step iv in parallel, claim 1(iv) and 1(v)) (pg. 8, col 1, fourth full paragraph). Cozens teaches Illumina sequencing and data analysis, wherein deep sequencing was carried out on an Illumina MiSeq at the UCL Genomics Center using an 150 cycle MiSeq Reagent Kit v3 (interpreted as sequencing in parallel, claim 1(v)) (pg. 6, col 2, second full paragraph). Cozens teaches that single-stranded plasmid DNA was mixed with mutagenic oligonucleotides and boundary assembly oligonucleotides, the plasmid and oligonucleotide mixtures were annealed, treated with Darwin Assembly enzyme mix, incubated, and purified with streptavidin-coated paramagnetic beads; followed by PCR amplification and purification of assembled DNA (interpreted as mixing and isolating members of mutagenesis libraries by purification, claim 1) (pg. 4, col 2, last partial paragraph; pg. 5, col 1; and pg. 6, col 1, first full paragraph, lines 1-3). Cozens teaches a library targeting residues Arg96, Lys98, Glu207, Glu222, Asn748, Pro759, wherein the 2724 bp assembly, targeting six codons used five inner oligonucleotides introducing degeneracy at each of the target positions, such that assembled libraries were amplified, cloned and transformed, with approximately 1 × 107 transformants isolated, followed by sequencing the pooled transformants, which confirmed that at each position, all possible variants were introduced (See, Figure 4, and Supplementary Table S6) (interpreted as isolating and mixing probe nucleic acids; and one or more degenerate oligonucleotides, claims 1 and 6) (pg. 10, col 1, first and second full paragraphs; pg. 9, Figure 4, and Supp. Info., pg. 15, Supplementary Table S6). Cozens teaches that Darwin assembly of KOD 6G12 transformants were isolated and sequence-verified to confirm the incorporation of all targeted mutations (interpreted as mixing probe nucleic acids, isolating probe nucleic acids, and sequencing, claim 1) (Supplementary Info, pg. 3, Figure 2).
Regarding claims 3 and 4, Cozens teaches that natural enzymes are optimized to their in vivo setting and are often unsuited for the synthesis of biological or synthetic compounds in vitro, such that expression and functional optimization, or more radical engineering is often required to generate the desired enzymatic activity, whether boosting an existing activity or changing enzyme function altogether, where those needs have led to the flourishing of directed evolution and protein engineering and it has repeatedly proven possible to enhance a number of protein properties, including expression, folding, thermostability, substrate specificity and catalytic efficiency (interpreted as a property of the target nucleic acid selected from expression, activity, etc., claims 3 and 4) (pg. 1, col 1, first partial paragraph; and col 2, first partial paragraph).
Regarding claim 6, Cozens teaches that Darwin Assembly can efficiently generate scanning libraries by combining oligonucleotides targeting the same binding site, but each introducing a different mutation, such that using the CAT gene as the model, an alanine scan library was assembled around residues Thr101 and Ser104, using Leu 105 (CTC→CTG) recording as an assembly control, such that five inner oligonucleotides were designed: four introducing an alanine mutation (Thr101Ala, Phe102Ala, Ser103Ala, or Ser104Ala) and one wild-type, such that all five oligonucleotides introduced the CTC→CTG control mutation at the Leu105 codon (interpreted as differ from the target nucleic acid sequence by one or more arbitrary nucleotides; and are selected from a predefined set of nucleotide sequences, claim 6) (pg. 8, col 1, third full paragraph).
Regarding claim 12, Cozens teaches a library targeting residues Arg96, Lys98, Glu207, Glu222, Asn748, Pro759, wherein the 2724 bp assembly, targeting six codons used five inner oligonucleotides introducing degeneracy at each of the target positions, such that assembled libraries were amplified, cloned and transformed, with approximately 1 × 107 transformants isolated, followed by sequencing the pooled transformants, which confirmed that at each position, all possible variants were introduced (See, Figure 4, and Supplementary Table S6) (interpreted as a property of library-specific mutations sites, claim 12) (pg. 10, col 1, first and second full paragraphs; pg. 9, Figure 4, and Supp. Info., pg. 15, Supplementary Table S6). Cozens teaches that Darwin assembly of KOD 6G12 transformants were isolated and sequence-verified to confirm the incorporation of all targeted mutations (interpreted as a property of library-specific mutations sites, claim 12) (Supplementary Info, pg. 3, Figure 2).
Cozens meets all the limitations of the claims and, therefore, anticipates the claimed invention.
Response to Arguments
Applicant’s arguments filed April 28, 2026 have been fully considered but they are not persuasive. Applicants essentially assert that: (a) Cozens does not teach providing isolated members of a set of two or more site mutagenesis libraries, selecting one of the isolated members of each site mutagenesis library as described in the instant Application (pg. 4, lines 28-34) (Applicant Remarks, pg. 8 through pg. 9).
Regarding (a), although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26USPQ2d 1057 (Fed. Cir. 1993). Additionally, MPEP § 2112.01(I) states that,
where the claimed and prior art products are identical or substantially identical in structure or composition, or are produced by identical or substantially identical processes, a prima facie case of either anticipation or obviousness has been established. In re Best, 562 F.2d 1252, 1255, 195 USPQ 430, 433 (CCPA 1977). "When the PTO shows a sound basis for believing that the products of the applicant and the prior art are the same, the applicant has the burden of showing that they are not." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990).
Applicant’s assertion that Cozens does not teach providing isolated members of a set of two or more site mutagenesis libraries, selecting one of the isolated members of each site mutagenesis library, is not found persuasive. As an initial matter, instant claim 1 is very broadly recited, such that no specific method of providing, number and/or identity of isolated members, number and/or location of sites of mutagenesis, number of libraries, target nucleic acids, number and/or location of mutations sites, method of selecting, method of obtaining, number and/or identity of probe nucleic acids, method of mixing, method of sequencing, etc. The Examiner contends that Cozens teaches all of the limitations of the claims. To that end –
Cozens teaches:
Designing a library targeting residues Arg96, Lys98, Glu207, Glu222, Asn748, Pro759, wherein the 2724 bp assembly, targeting six codons used five inner oligonucleotides introducing degeneracy at each of the target positions, such that assembled libraries were amplified, cloned and transformed, with approximately 1 × 107 transformants isolated, followed by sequencing the pooled transformants, which confirmed that at each position, all possible variants were introduced (See, Figure 4, and Supplementary Table S6) (interpreted as mixing and isolating probe nucleic acids; selecting members; and sequencing, claim 1) (pg. 10, col 1, first and second full paragraphs; pg. 9, Figure 4, and Supp. Info., pg. 15, Supplementary Table S6).
The five oligonucleotides were mixed in a 1:1:1:1:1 ratio in the assembly reaction to create a library where each variant was expected to be 20% of the final population, wherein assembly was successful and over 103 transformants were isolated, pooled (interpreted as selecting, mixing, and isolating library members, claim 1) (pg. 8, col 1, fourth full paragraph).
Single-stranded plasmid DNA was mixed with mutagenic oligonucleotides and boundary assembly oligonucleotides, the plasmid and oligonucleotide mixtures were annealed, treated with Darwin Assembly enzyme mix, incubated, and purified with streptavidin-coated paramagnetic beads; followed by PCR amplification and purification of assembled DNA (interpreted as mixing, isolating and selecting members of mutagenesis libraries by purification, claim 1) (pg. 4, col 2, last partial paragraph; pg. 5, col 1; and pg. 6, col 1, first full paragraph, lines 1-3).
Darwin assembly of KOD 6G12 transformants were isolated and sequence-verified to confirm the incorporation of all targeted mutations (interpreted as mixing probe nucleic acids, isolating, selecting, and sequencing library members, claim 1) (Supplementary Info, pg. 3, Figure 2).
Darwin Assembly was applied to different genes, including chloramphenicol acetyl transferase (CAT), Saccharomyces cerevisiae tryptohanyl tRNA synthetase (ScWRS), Thermococcus gorgonarius DNA polymerase (TgoT), Thermococcus kodakaraenis DNA polymerase (KOD) and T7 RNA polymerase (T7RSS), which differ in length as well as composition (interpreted as two or more site mutagenesis libraries that are subsequently selected, isolated, obtained, mixed, and sequenced, claim 1(i)) (pg. 7, col 1, last partial paragraph).
Cozens teaches all of the limitations of the claims. Thus, the claims remain rejected.
Conclusion
Claims 1, 3, 4, 6 and 12 remain rejected.
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 AMY M BUNKER whose telephone number is (313) 446-4833. The examiner can normally be reached on Monday-Friday (6am-2:30pm).
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/AMY M BUNKER/Primary Examiner, Art Unit 1684
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