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 .
The Response of 15 Sept. 2025 has been entered.
Claims 1-3, 5, 8-14, 22, 23 and 25-27 are currently pending and are considered here with respect to the elected species of Taq ligase as the DNA ligase, Phusion as the DNA polymerase, T5 as the exonuclease and a protein encoded by the circularized DNA as the end product.
Any rejection not reiterated herein has been withdrawn.
Response to Arguments
Applicant's arguments filed 15 Sept. 2025 have been fully considered but they are not persuasive.
Applicant argues that the claims are distinguishable from Chen on the basis of the buffering solution consisting essentially of the claimed ingredients and the two-stage incubation recited in the claims. These limitations are addressed in the modified prior art rejection(s) below.
With respect to the incubation times/temperatures, Applicant argues that the following passage in Chen teaches away from the claimed method: “the preferred reaction conditions are 50 °C reaction for 1 h. If some restriction enzymes have low enzymatic activity at 50 °C, they can be reacted at their optimum temperature (such as 37 ° C) for 0.5-1 h, and then reacted at 50 °C for 1 h.” This is not persuasive because Chen teaches the two stage reaction for essentially the same reason as the claimed method; i.e. an initial stage at 37 °C for restriction enzyme linearization, followed by a second stage at 50 °C for the polymerase/joining reaction. Since Chen teaches the same general reaction for the same purpose, it would have been obvious to discover the optimum and/or workable ranges for the various aspects of the reaction (including incubation times/temperatures) by routine experimentation. The prior art’s mere disclosure of more than one alternative does not constitute a teaching away where the disclosure does not criticize, discredit, or otherwise discourage the solution claimed (see MPEP 2145, X. D.). The suggestion by Chen to add additional incubation time for the restriction enzyme stage does not criticize, discredit, or otherwise discourage using a lesser time for the stage at 37 °C (such as the about 15 mins claimed vs 30-60 mins taught by Chen) in a manner that would arise to teaching away from the claimed method.
Claim Rejections - 35 USC § 112(a) (new matter)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 1-3, 5, 8-14, 22, 23 and 25-27 are rejected under 35 U.S.C. 112(a) as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claims 1 and 11 have been amended to recite incubating for about 15 mins at 37 °C, followed by more than 15 minutes at 50 °C. The instant specification describes various times for the incubation at 50 °C ranging from 10-60 mins (Spec., [0015]; [0017]; Example 1). However, the specification as filed does not provide written description support for the claimed range of “more than 15 minutes”.
Claims 2 and 12 have been amended to recite a “high-fidelity ligase” and a “high-fidelity and thermostable ligase”. The specification describes thermostable ligases along with the specific examples “Taq DNA ligase (New England Biolabs), 9N DNA ligase (New England Biolabs) or Ampligase” and “T7 ligase, T4 DNA ligase, E. coli DNA ligase and Taq ligase” (Spec., [0012], [0033]). However, the specification does not recite the genera of a “high-fidelity ligase” or a “high-fidelity and thermostable ligase”.
Claim Rejections - 35 USC § 103
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 1-3, 5, 8 and 10 are rejected under 35 U.S.C. 103 as obvious over PCT Pub. WO2017066943 to Chen et al.
Regarding claims 1 and 10, Chen teaches a method of preparing a circular DNA molecule for use in preparing a nucleotide end-product, comprising: mixing in a reaction vessel at the same time a circular DNA vector, an amplified target DNA molecule to be inserted/cloned into the vector and at least one restriction enzyme in an amount sufficient to linearize the circular DNA vector; adding to the reaction vessel an incubating solution including a DNA polymerase, a 5′-3′ exonuclease, a buffering agent and a DNA ligase; and incubating for a sufficient time and temperature for linearization of the circular DNA vector and joining of the amplified linearized target DNA molecule and the linearized circular DNA vector for production of the circularized DNA molecule (entire doc, including under Summary of the invention; p. 3, last ¶ to p. 5, 6th ¶, Examples 1-3; citations herein are to the provided English machine translation). The nucleotide end-product can be a protein encoded by the circularized DNA vector, expressed in a host cell (Examples 1-3). The restriction enzyme can be any enzyme suitable for cleaving/linearizing a circular DNA of interest, including SalI (p. 4, 8th-10th ¶).
Regarding the recitation in claim 1 that the method involves use of a circular DNA vector along with a linearized target DNA molecule, Chen teaches that the method can be used to assemble any combination of circular and/or linear vector and target DNAs (p. 3, last ¶ to p. 4, 5th ¶; p. 5, 5th ¶; Figs. 1 and 2). Chen further exemplifies cloning a linear DNA fragment (GFP) into a circular plasmid vector (Example 2). Thus, one of ordinary skill reading Chen would have at once envisaged the use of a circular vector and a linearized amplified DNA fragment in the method of Chen and/or it would have been obvious in view of Chen to use a circular DNA vector (as vectors are typically obtained in circular form) along with linear amplified target DNA, such as linear PCR products.
Regarding the recitation in claim 1 that the method is a “one-pot” method, Chen teaches that the method comprises a single mixture/step including the vector, target DNA, restriction enzyme/endonuclease and reaction mixture (DNA polymerase, exonuclease and ligase) (p. 4, 4th ¶; p. 5, 4th-5th ¶; Examples 1-3).
Regarding the composition of the buffering solution in claims 1 and 8, Chen teaches that an incubating solution comprising 5% PEG 8000, 100 mM Tris-acetate pH 7.5, 0.2 mM dNTPs, 20 mM potassium acetate, 10 mM magnesium acetate, and 0.1 mg/ml BSA (p. 5, 1st ¶; Example 3; claims 7 and 10). Thus, the buffering solution of Chen differs from that of claims 1 and 8 only in having differing concentrations of potassium acetate and BSA (claim 1) and slightly different pH and concentrations of potassium acetate, magnesium acetate and BSA (claim 8). Since Chen teaches use of the same components (enzymes, salts, DNA constructs, etc.) for the same purpose of carrying out a one-step Gibson assembly process, it would have been obvious for one of ordinary skill in the art to discover the optimum and/or workable concentration ranges of the ingredients using routine experimentation (see MPEP 2144.05). The instant specification does not provide any evidence of criticality with respect to the specific concentrations recited in claim 1. Moreover, the concentrations recited in claims 1 and 8 are recited with respect to a “buffering solution” which is a subcomponent of the “incubating solution” in which the enzymatic reaction takes place, which argues against criticality for any of the recited concentrations (since the incubating solution that is functionally operable is not necessarily limited by the recited concentrations).
Regarding the recitation in claims 1 and 8 that the buffering solution “consists essentially of” the recited ingredients, the transitional phrase "consisting essentially of" limits the scope to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention (see MPEP 2111.03). Absent a clear indication in the specification or claims of what the basic and novel characteristics actually are, "consisting essentially of" will be construed as equivalent to "comprising." (MPEP 2111.03). The instant specification does not disclose any particular “basic and novel characteristics” of the claimed method relating to the components used in the incubating solution. Rather, the specification repeatedly lists the components of such solution as including “at least a crowding agent such as a PEG molecule, and other components including but not limited to” those claimed (e.g., Spec., [0014]). As such, the phrase “consists essentially of” in the instant claims is construed herein as “comprising”. Alternatively, since Chen teaches use of the same components (enzymes, salts, DNA constructs, etc.) for the same purpose of carrying out a one-step Gibson assembly process, none of the additional components listed in the solution of Chen (e.g., DTT, NAD+) is considered to materially affect the basic and novel characteristic(s) of the claimed invention. Moreover, the concentrations recited in claims 1 and 8 are recited with respect to a “buffering solution” which is a subcomponent of the “incubating solution” in which the enzymatic reaction takes place, and the incubating solution “comprises” the buffering solution. As such, the incubating solution can contain additional elements not expressly recited in the claim.
With respect to the recitation in claim 1 of incubating for about 15 mins at 37 °C for linearization, followed by more than 15 minutes at 50 °C for joining, Chen teaches a two-stage reaction with a first stage at 37 °C for 0.5-1 hours for the restriction enzyme reaction (i.e. linearization), followed by a second stage at 50 °C for 1 hour for the assembly (i.e. joining) reaction (p. 4, last ¶). Thus, the incubation times/temperatures used by Chen differ only in that the duration of the first linearization stage at 37 °C is slightly longer in Chen (0.5-1 vs. about 15 mins). Since Chen teaches use of the same components (enzymes, salts, DNA constructs, etc.) for the same purpose of carrying out a one-step Gibson assembly process as well as a two-stage reaction providing optimal temperatures for the same subprocesses as in the claimed method, it would have been obvious for one of ordinary skill in the art to discover the optimum and/or workable incubation times for the linearization and assembly processes using routine experimentation (see MPEP 2144.05). The instant specification does not provide any evidence of criticality with respect to the specific incubation times recited in claim 1.
Regarding claim 2, Chen teaches that the DNA ligase can be Taq DNA ligase (i.e. a thermostable ligase; see Spec. [0012]) (p. 4, 11th ¶; p. 5, 3rd ¶; Examples 1-3; claims 4 and 9).
Regarding claim 3, Chen teaches that the DNA polymerase can be Phusion DNA polymerase (i.e. a high fidelity polymerase; see Spec., [0013]) (p. 4, 11th ¶; p. 5, 3rd ¶; Examples 1-3; claim 9).
Regarding claim 5, Chen teaches that the exonuclease can be T5 exonuclease (p. 4, 11th ¶; p. 5, 3rd ¶; Examples 1-3; claims 4 and 9).
Claims 9, 11-14, 22, 23 and 25-27 are rejected under 35 U.S.C. 103 as being unpatentable over the combination of Chen, as applied to claims 1-3, 5, 8 and 10 above, in view of Ma et al., African Journal of Biotechnology 12.26 (2013): 4094.
Claims 9, 11-14, 22, 23 and 25-27 differ from Chen, as applied to claims 1-3, 5, 8 and 10 above, in that: the restriction enzyme is a combination of BamHI and SalI (claims 9 and 11).
Ma teaches that BamHI and SalI are known to be active in buffer suitable for T4 DNA ligase and can linearize pUC19 vector DNA (p. 4096, last ¶).
It would have been obvious to one of ordinary skill in the art at the time the invention was made to use the method of Chen to carry out a one-pot assembly of a linear target DNA into a circular DNA vector wherein the restriction enzymes used in the method are a combination of BamHI and SalI because it would have been obvious to combine prior art elements according to known methods to yield predictable results. One of ordinary skill would have been motivated to use a combination of two restriction endonucleases because Chen teaches that the method can be used to assemble multiple DNA fragments in mixed form, such as cloning a combination of linear DNA fragments and cloned fragments in circular vector format into a circular destination vector of interest (e.g., Tai, p. 4, 5th ¶; Example 2). Carrying out the method of Chen using a combination of BamHI and SalI as the restriction enzyme would have led to predictable results with a reasonable expectation of success because Chen teaches that any restriction enzyme(s) can be used in the method, including SalI. Moreover, Ma teaches that BamHI and SalI are known to be active in buffer suitable for T4 DNA ligase as used in Chen and can linearize pUC19 vector DNA (note that Chen exemplifies use of pUC19 vectors in Example 2). It would have been obvious in view of the cited combination to use any one or more restriction enzymes effective to linearize target DNA and/or the destination vector DNA, and there is no evidence that the use of the particular enzymes recited in claims 9 and 11 yield any new or unexpected results relative to that taught by the cited references.
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 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.
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/ROBERT J YAMASAKI/Primary Examiner, Art Unit 1657