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 .
Election/Restrictions
Applicant’s election without traverse of species for the trimethyl lock wherein R1 is -CO- and R2 is I in the reply filed on 07/21/2025 is acknowledged.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 21-32, 35-38, 50-52, 55 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 21, 50 and 55 are vague and indefinite because it is unclear what the phrase a "chemically modified blocking primer". It is unclear of what are exactly the metes and bounds for the chemically modification. It is not stated what primer the disclosed chemically modified primer should be compared to in order to determine whether it is "chemically modified" or not, making it impossible to determine which primers fall within the scope of the claim. For the purpose of the search, any primer is considered chemically modified in relation to another arbitrarily selected primer.
Claims 21 and 50 further disclose a "blocking group acceptor" moiety within the blocking primer. Since the blocking group is undefined, and the features of the blocking group acceptor would depend on the chemistry of the blocking group, it is unclear which acceptor moieties would fall within the scope of said disclosure.
Claims 21 and 55 further disclose a method comprising the step of "ligating one or more of said plurality sticky ended DNA products together". It is unclear how one DNA product with one sticky end may be ligated "together".
Claims 22 and 55 disclose a step of "incorporating a thiophosphate group at a chosen position on said chemically modified blocking primer". It is unclear if said primer is already chemically modified prior to the incorporation of the thiophosphate group, or if the thiophosphate group represents the only chemical modification in the resulting primer. Furthermore, it is unclear to which part of the primer the thiophosphate group should be incorporated to without disrupting the annealing process.
Claim 38 discloses a method comprising a reaction carried out with "other reversible chemistry". This broad term encompasses many types of reactions, most of which would not result in decoupling of the blocking group as required by the method disclosed.
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.
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.
Claim(s) 21, 38, 50-51, 55 is/are rejected under 35 U.S.C. 103 as being unpatentable over Komiyama et al. US 2011/0009607 (hereinafter "Komiyama") in view of Behlke et al. US 2004/0248095 A1 (hereinafter "Behlke").
Regarding claim 21, 50 and 55, Komiyama teaches a method of direct synthesis of sticky ended DNA (abstract, a method for preparing a DNA fragment, in which a desired double-stranded DNA fragment having a sticky end is directly and easily obtained from an amplification product (an amplified fragment) after PCR without a restriction enzyme digestion) comprising the steps of:
generating a chemically modified blocking primer having a nucleotide sequence that anneals to a target region in a template DNA and an overhanging region at the 5' end; wherein said chemically modified primer includes a blocking group modification at a chosen position that prevents DNA polymerase from fully extending the complementary strand during PCR amplification resulting in a 5' overhang (para [0006], (1) A primer used for PCR, which is composed of a complementary DNA portion consisting of a nucleotide sequence complementarily binding to an amplification target region in a template DNA and a non-complementary DNA portion consisting of a nucleotide sequence that links to the 5' end of the complementary DNA portion but does not complementarily bind to the amplification target sequence, wherein at least a base corresponding to the 3’ end in the nucleotide sequence of the non-complementary DNA portion is modified with a protecting group capable of terminating the progression of DNA replication catalyzed by a DNA polymerase); and
running a polymerase chain reaction (PCR) protocol with said chemically modified blocking primer, and a template DNA wherein said end-product of said PCR is a plurality of sticky ended DNA products (abstract, (i) a step of performing a PCR reaction using a template DNA and specific primers to obtain an amplified DNA fragment; and (ii) a step of performing a prescribed treatment on the amplified DNA fragment to dissociate a protecting group from the fragment.; para [0089] and Figure 4.);
decoupling said blocking group from said plurality of sticky ended DNA products (para [0007], the protecting group can be dissociated from the modified base, for example, by a light irradiation treatment, an alkali treatment, an acid).
Komiyama does not specifically teach wherein one of the PCR primers is a replicable primer (not modified with blocker). Behlke teaches a polynomial PCR amplification comprising a replicable primer and a primer modified with blocking group (abstract, novel amplification methods, referred to herein as polynomial amplification, are provided. According to these methods, a nucleic acid molecule to be amplified
is contacted with at least two primer; a non-replicable primer which may hybridize to the nucleic acid molecule being amplified, and a replicable primer which may hybridize to a primer extension product generated from extension of the non-replicable primer.; para [0052]-[0053], FIG. 2 illustrates the general scheme of an exemplary 2-primer polynomial amplification method of the invention.....The synthesis of species F and species G in FIG. 2 do not progress beyond the non-replicable element in their corresponding template strands. Species F in the 2-primer scheme (FIG. 2) does not participate further in the primer extension reaction because the molecules do not have an effective binding site for the non-replicable primer(s).). Given that the carryover contamination from a PCR reaction is greatly reduced (Behlke, para [0031], Applicants have discovered that, by using such a combination of replicable and non-replicable primers, the amount of carry-over contamination is greatly reduced or even eliminated), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have performed polynomial PCR with the primer comprising a blocking group of Komiyama, leading to 5'-overhang at one end of the amplified product (Komiyama, Figure 1). One of ordinary skill in the art would have recognized that the amplified product with 5' sticky end can be ligated to another amplified product with complementary 5' sticky end, and thus to have obtained a long polynucleotide using the amplified product shaving 5' sticky end.
Regarding claim 38, Komiyama and Behlke teach the method of claim 21, wherein said step of decoupling said blocking group from said plurality of sticky ended DNA products comprises the step selected from the group consisting of: enzymatic deprotection, thermal deprotection, chemical deprotection, catalytic deprotection, photocage deprotection, or other reversible chemistry (Komiyama, para [0050], protecting groups that can be dissociated from the modified base by a light irradiation treatment, an alkali treatment, an acid treatment, an oxidation treatment, a reduction treatment, a desilylation treatment, a heat treatment, an esterase treatment or a phosphatase treatment, are preferable.).
Regarding claim 51, Komiyama and Behlke teach the method of claim 50, wherein said polymerase chain reaction (PCR) comprises a modified polymerase chain reaction (mPCR) (Behlke, para [0052]-(0053], FIG. 2 illustrates the general scheme of an exemplary 2-primer polynomial amplification method of the invention..... The synthesis of species F and species G in FIG. 2 do not progress beyond the non-replicable element in their corresponding template strands. Species F in the 2-primer scheme (FIG. 2) does not participate further in the primer extension reaction because the molecules do not have an effective binding site for the non-replicable primer(s).).
Claim(s) 30-31, 35-37 is/are rejected under 35 U.S.C. 103 as being unpatentable over Komiyama and Behlke, in view of Walker et al. "A method for generating sticky-end PCR products which facilitates unidirectional cloning and the one-step assembly of complex DNA constructs" Plasmid, May 2008, Vol. 59, No.3, pp.155-162 (hereinafter "Walker”).
Regarding claim 30, Komiyama and Behlke teach the method of claim 21, but do not specifically teach wherein said step of ligating said sticky ended DNA products together comprises the step of enzymatically ligating said sticky ended DNA products together. Walker teaches a method of unidirectional cloning (abstract, We have developed and tested a method for the restriction enzyme-independent generation of sticky-end PCR products..... The technique can be used to achieve unidirectional cloning of PCR products with an efficiency greater than 90%.) comprising the step of enzymatically ligating said sticky ended DNA products together (pg 160, col 1, para 1 to col 2, para 1, we attempted a one-step assembly of four DNA modules in a unique order to generate a circular plasmid.....Pairs of PCR products for each module, tailed with the sequences shown in Fig. 2A, were generated using primers homologous to the appropriate templates.....,Because of the way that the sticky-end molecules are formed, assembly of either the four 3'-overhang DNA molecules or the four 5'-overhang molecules will form identical plasmid products (Fig. 2B).; pg 157, col 1, para 5, Cloning of sticky-end molecules was performed as follows. Five microliters digested-plasmid DNA.....reannealed PCR products.....1 ul T4 DNA ligase.....). It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have applied the DNA comprising 5’ sticky end, as prepared by the teachings of Komiyama and Behlke, to the method of Walker, to facilitate the process of unidirectional cloning.
Regarding claim 31, Komiyama, Behlke and Walker teach the method of claim 30, wherein said step of enzymatically ligating said sticky ended DNA products together comprises the step of enzymatically ligating said sticky ended DNA products together using a ligase enzyme (Walker, pg 157, col 1, para 5, Cloning of sticky-end molecules was performed as follows. Five microliters digested-plasmid DNA.....reannealed PCR products.,,..1 ul T4 DNA ligase.....).
Regarding claim 35, Komiyama and Behlke teach the method of claim 21, but do not specifically teach wherein said overhanging region at the 5’ end may be complementary with another overhanging region at the 5' end on a DNA insert forming a recombinant double stranded DNA molecule. Walker teaches a method of unidirectional cloning (abstract, We have developed and tested a method for the restriction enzyme-independent generation of sticky-end PCR products.....The technique can be used to achieve unidirectional cloning of PCR products with an efficiency greater than 90%.) comprising annealing overhanging region at the 5’ end that is complementary with another overhanging region at the 5' end on a DNA insert forming a recombinant double stranded DNA molecule (pg 160, col 1, para 1 to col 2, para 1, we attempted a one-step assembly of four DNA modules in a unique order to generate a circular plasmid.....Pairs of PCR products for each module, tailed with the sequences shown in Fig. 2A, were generated using primers homologous to the appropriate templates.....Because of the way that the sticky-end molecules are formed, assembly of either the four 3'-overhang DNA molecules or the four 5'-overhang molecules will form identical plasmid products (Fig. 2B).). It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have applied the DNA comprising 5' sticky end, as prepared by the teachings of Komiyama and Behlke, to the method of Walker, to facilitate the process of unidirectional cloning.
Regarding claims 36-37, Komiyama and Behlke teach the method of claim 21, but do not specifically teach wherein the step of introducing 5’phosphate to said plurality of sticky ended DNA products. Walker teaches a method of unidirectional cloning (abstract, We have developed and tested a method for the restriction enzyme-independent generation of sticky-end PCR products.....The technique can be used to achieve unidirectional cloning of PCR products with an efficiency greater than 90%.) comprising introducing 5' phosphate to said plurality of sticky ended DNA products (pg 157, col. 1, para 6, Primer oligonucleotides for generation of the four sticky-end PCR products.....and manufactured with phosphorylated 5'-ends.). It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have applied the DNA comprising phosphorylated 5’ sticky end, as prepared by the teachings of Komiyama and Behlke, to the method of Walker, to facilitate the process of unidirectional cloning.
Claim(s) 32 and 52 is/are rejected under 35 U.S.C. 103 as being unpatentable over Komiyama and Behlke, in view of Zymergen Inc. WO2018/013990A1 (hereinafter "Zymergen”).
Regarding claim 32, Komiyama and Behlke teach the method of claim 21, but do not specifically teach wherein said step by step of ligating said sticky ended DNA products together comprises the step of ligating said sticky ended DNA products in vivo. Zymergen teaches the step of ligating sticky ended DNA products into cut sites in vivo (Claim 12, A method for editing the genome of a cell in vivo, said method comprising the steps of: a) introducing into the cell a Cpfl CRISPR system.....are expressed in the cell, and the Cpfl endonuclease cleaves the cell's genome at the first and second selected target sequences, thereby producing sticky ends on the cleaved ends of the cell’s genome.; Claim 13, a fourth, insert polynucleotide, wherein said insert polynucleotide is also cleaved by the Cpfl endonuclease, thereby creating sticky ends on the insert polynucleotide that are compatible with the sticky ends of the cell’s genome:.....anneal the sticky ends of the genome to the sticky ends of the insert polynucleotide; and.....ligate the annealed genome and insert sticky ends.). Given that incorporating sticky ended DNA into genome in vivo is known, as taught by Zymergen, It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have taken advantage of the known techniques and applied the sticky end PCR product of Komiyama and Behlke to the method of in vivo genome editing of Zymergen.
Regarding claim 52, Komiyama and Behlke teach the method of claim 50, but do not specifically teach the step of transforming a cell with said plurality of sticky ended DNA products. Zymergen teaches transforming a cell with said plurality of sticky ended DNA products (Claim 12, A method for editing the genome of a cell in vivo, said method comprising the steps of: a) introducing into the cell a Cpfl CRISPR system.....are expressed in the cell, and the Cpfl endonuclease cleaves the cell's genome at the first and second selected target sequences, thereby producing sticky ends on the cleaved ends of the cell's genome.; Claim 13, a fourth, insert polynucleotide, wherein said insert polynucleotide is also cleaved by the Cpfl endonuclease, thereby creating sticky ends on the insert polynucleotide that are compatible with the sticky ends of the cell's genome;.....anneal the sticky ends of the genome to the sticky ends of the insert polynucleotide; and.....ligate the annealed genome and insert sticky ends.). Given that incorporating sticky ended DNA into genome in vivo is known, as taught by Zymergen, It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have taken advantage of the known techniques and applied the sticky end PCR product of Komiyama and Behlke to the method of in vivo genome editing of Zymergen. Komiyama, Behlke and Zymergen wherein said plurality of sticky ended DNA products are ligated together by said cell's endogenous cellular DNA repair machinery forming a recombinant double stranded DNA molecule (Zymergen, para [0147], Existing techniques for targeted genome editing with CRISPR/Cas9 rely on the cell's native ability to repair double strand breaks via homologous recombination).
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JEZIA RILEY whose telephone number is (571)272-0786. The examiner can normally be reached 7:30-6:00pm.
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/JEZIA RILEY/ Primary Examiner, Art Unit 1681 20 November 2025