DETAILED ACTION
Status of the Claims
Claims 1-3, 6-7, 9-10, 12-18, 20-21, and 25 are currently pending and are examined herein.
The following Office Action is in response to Applicant’s communication dated 12/12/2025. Rejection(s) and/or objection(s) not reiterated from previous office actions are hereby withdrawn. The following rejection(s) and/or objection(s) are either reiterated or newly applied. They constitute the complete set presently being applied to the instant application.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Modified Claim Rejections – 35 U.S.C. 103(a)
Necessitated by Amendments
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 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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
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 factual inquiries set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Miyazaki et al. and Dubey et al.
Claims 1-3, 6-7, 9-10, 12-18, 20-21, and 25 are rejected under 35 U.S.C. 103 as being unpatentable over Miyazaki et al. (Methods in Enzymology, 2011, 498:399-406, of record) in view of Dubey et al. (PLoS ONE, 2016, 11(3): e0152106, of record), as evidenced by Cadwell et al. (Genome Res., 1992, 2:28-33, of record) and Agilent (QuikChange Site-Directed Mutagenesis Kits, 2013).
Regarding claims 1 and 20-21, Miyazaki discloses a method for constructing a gene mutation library, comprising (1) synthesizing a pool of oligonucleotide sequences comprising mutant nucleotide(s) at one or more mutation sites;
(2) performing PCR amplification on the oligonucleotides in the synthesized pool of the oligonucleotide sequences comprising the mutant nucleotide(s) (e.g., preparing a mutated gene fragment as per page 401 as step 1 of the MEGAWHOP protocol, for example, from error-prone PCR as per Cadwell, which is specifically referenced by Miyazaki and will produce multiple, double-stranded megaprimers as PCR products);
(3) performing linear PCR amplification by using a plasmid comprising a methylated site and a gene to be mutated or a part thereof as a template and using the two strands of oligonucleotides in the pool of the amplified oligonucleotide sequences obtained in step (2) as primers to respectively extend to the head and tail ends of the gene to be mutated or a part thereof by controlling the extension time of the RCR reaction, (e.g., mixing the megaprimers and plasmid and performing plasmid PCR as per steps 2-3 of the MEGAWHOP protocol on page 401, which is linear amplification due to the lack of strand displacement activity of enzymes used by Miyazaki on p. 403 and/or as per Agilent who discloses linear amplification in commercially available site-directed mutagenesis kits, see p. 1 “Linear Amplification”); and
(4) using an endonuclease that recognizes and cleaves the methylated site to cleave the template plasmid present in the amplified system of step (3) (e.g., addition of DpnI as per step 4 of the MEGAWHOP protocol on page 402).
Regarding the limitation of wherein the molar ratio of the primers to the template in step (3) is 15:1 to 100:1 and the similar limitation in claim 17 wherein the molar ratio of the primers to template in step (3) is 15:1 to 50:1, , Miyazaki discloses “Typically, 0.2 mg of megaprimer (~750 bp) and 50 ng of template plasmid (~3.5 kb) with a PCR run of 24 cycles give satisfactory results”, which corresponds to ~0.41 pmol of primer and ~0.022 pmol of template, which is a ~19:1 ratio.
However, it is noted that Miyazaki is silent on the limitation of “(5) adding a forward primer and a reverse primer respectively corresponding to both ends of the gene to be mutated or the part thereof to the system obtained in step (4) and performing PCR amplification to obtain the gene mutation library comprising a plurality of genes containing the mutant nucleotide(s) or a part thereof”, as set forth in claim 1. Instead, Miyazaki discloses amplification of the final product by transformation into E. coli, as per page 402.
Dubey discloses a method of PCR-based subcloning technique to insert a DNA fragment into a plasmid vector for protein production (e.g., as per the Abstract and Fig. 1).
It would have been prima facie obvious to a person of ordinary skill in the art prior to the effective filing date of the application to take the product of step 4 of the MEGAWHOP protocol from Miyazaki and clone it into a vector as per Dubey. One of ordinary skill in the art would have been motivated to do so since Dubey teaches that their method can quickly and efficiently clone a gene of interest into a protein expression vector, which would be of interest to Miyazaki in cases, for example, where the library generation was performed in a vector not optimal for protein expression. Further, as per MPEP 2143(I)(A), the rationale to support a conclusion that the claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination yielded nothing more than predictable results to one of ordinary skill in the art. In the instant case, all of the elements of the gene mutation library construction were well known in the art, as per Miyazaki and Dubey, the mere combining of the individual elements in one embodiment in the manner of the claimed invention results in no change in the elements respective functions, and the combination yields nothing more than predictable results.
One of ordinary skill in the art would have had a reasonable expectation of success as of the application’s effective filing date in combining the teachings of the prior art references to arrive at the invention as presently claimed since all the techniques used in the process are well documented and routine enough to be well within reach for the skilled artisan.
Regarding claim 2, Miyazaki discloses the above wherein purification is not performed (e.g., as per the 2.1. MEGAWHOP protocol section).
Regarding claim 3, Dubey discloses the above, further comprising (6) recovering and purifying the amplified product of step (5) to obtain a final product (e.g., gel purification as per the PCR and cloning section on page 3).
Regarding claim 6, Miyazaki teaches that their “protocol guarantees a positive outcome with a 50–3000-bp megaprimer and a 2–8-kb template plasmid”, which encompasses the claimed range of 60-170 bp and in accordance with MPEP 2144.05(I), in the case where the claimed ranges “overlap or lie inside ranges disclosed by the prior art” a prima facie case of obviousness exists.
Regarding claims 7, 9-10, and 12-13, Miyazaki in view of Cadwell discloses wherein the pool of the oligonucleotide sequences synthesized in step (1) comprises mutant nucleotide(s) at 1 to 10 mutation sites, wherein the amino acid sequence encoded by a gene comprising the mutant nucleotide(s) has at least one amino acid difference compared to the amino acid sequence encoded by the gene to be mutated, wherein the at least one amino acid difference is selected from: substitution, addition or deletion, wherein the substitution is to substitute the original amino acid with an amino acid having different properties from the original amino acid, and wherein the properties are selected from acidity and alkalinity, polarity, charge characteristic and side chain group. Note that Miyazaki specifically references Cadwell, who teaches error-prone PCR, which would reasonably encompass such mutations and amino acid substitutions. See also the RESULTS section of Cadwell for typical mutation rates and types. It is noted that In re Best (195 USPQ 430) and In re Fitzgerald (205 USPQ 594) discuss the support of rejections wherein the prior art discloses subject matter which there is reason to believe inherently includes functions that are newly cited or is identical to a product instantly claimed. In such a situation the burden is shifted to the applicants to "prove that subject matter shown to be in the prior art does not possess characteristic relied on" (205 USPQ 594, second column, first full paragraph). In the instant case, the random nature of type and distribution of mutations occurring from error-prone PCR would be diverse enough to reasonably include the claimed substitution(s).
Regarding claim 14, Cadwell teaches the mutagenesis as above, wherein two primers respectively corresponding to both ends of the oligonucleotide sequences comprising the mutant nucleotide(s) at one or more mutation sites are used in the PCR amplification in step (2) (e.g., as per the MATERIALS section).
Regarding claims 15 and 18, Cadwell teaches the mutagenesis as above, wherein PCR is performed for 30 cycles and Miyazaki discloses 24 PCR cycles for step (3). However, it is noted that Cadwell specifically recites the number of cycles as a parameter that can be adjusted and optimized (e.g., middle column of page 31). Applicant is directed to In re Aller, Lacey, and Hall, 105 USPQ 233 (C.C.P.A. 1955), where the court found
"More particularly, where the general conditions
of a claim are disclosed in the prior art, it is
not inventive to discover the optimum or workable
ranges by routine experimentation."
Routine optimization is not considered inventive and no evidence has been presented that the selection of specific number of cycles was other than routine or that the results should be considered unexpected in any way as compared to the closest prior art.
Regarding claim 16, Miyazaki discloses the use of high-fidelity DNA polymerase (e.g., as per p. 403).
Regarding claim 25, Miyazaki discloses constructing gene mutation libraries as above followed by comparing the properties, regulation and/or function of the protein encoded by a mutant gene in the gene mutation library with the properties, regulation and/or function of the unmutated protein and analyzing the relationship between the amino acid at the mutation position and the properties, regulation and/or function of the protein (e.g., as per the several applications recited in 3. Applications of MEGAWHOP section on pages 403-405).
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Response to Arguments
The 12/12/2025 remarks argue: not all elements are taught.
Applicant's arguments have been fully considered but they are not persuasive for at least the following reasons.
Specifically, page 7 of the remarks argue that the PCR reaction of Miyazaki differs from the present claims, namely, that “[t]he obtained product is two single-stranded DNAs linked together by complementary regions in the middle, with the target amino acid sequence flanking the left and right sides (see paragraph [0089] of the description; and Figure 1 of the present application).” In response, it is noted that the claims do not exclude whole-plasmid PCR, rather they require extending the head and tail ends of the gene to be mutated via linear amplification and choice of primer:template ratios. Note that Miyazaki also discloses extending the head and tail ends of the gene to be mutated, discloses overlapping primer:template ratios, and results in linear amplification. Also note that the linear amplification results from the lack of strand displacement activity rather than from the extension time. Further, the remarks at page 8 allege some possible disadvantages of whole-plasmid PCR. In response to this argument, the fact that the inventor has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious. See Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985).
Next, the remarks at page 8 that “to implement the MEGAWHOP protocol of Miyazaki, it is necessary to transform the product obtained in step 3 into competent cells, perform gap repair within the competent cells, and screen for positive strains on a plate. To the best of the knowledge of those skilled in the art, plasmid extraction is typically required to extract mutant plasmids from the positive strains.” In response to 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).
Next, the remarks at page 9 assert that “[c]ontrary to the examiner's viewpoint, the disclosure of Dubey et al. is based on homologous recombination, which involves cloning DNA fragments with homologous arms at both ends into an expression vector for protein production via PCR. This method is not directly relevant to the technical solution of the present application, as the present application does not involve the step of cloning and inserting DNA fragments into a vector. Consequently, when confronted with the technical problem of the present application, a person skilled in the art would not attempt to combine Miyazaki with Dubey.” However, a careful review of the Dubey reference did not find anything to do with homologous recombination, and therefore these arguments are not persuasive.
Finally, the remarks contend that the whole-plasmid PCR method of Miyazaki is not linear amplification, as required by the newly amended claims. In response, and as addressed in the rejection supra, whole-plasmid based PCR site-directed mutagenesis, such as used by Miyazaki, is known to involve linear amplification, at least due to the lack of strand-displacing activity of a DNA polymerase used. Please refer to the Agilent reference which shows commercially available kits for such whole-plasmid based PCR site-directed mutagenesis methods, which are all denoted as being “linear amplification” (e.g., as per the table on p. 1).
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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 extension fee 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 date of this final action.
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/JEREMY C FLINDERS/Primary Examiner, Art Unit 1684