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
Application status
Claims 1-22 are pending in this application.
Priority
It is acknowledged that the instant application claims the benefit of U.S. Provisional Application No. 63304792, filed on 01/31/2022.
Election
Applicant's election of Group I, Claims 1-22, and species: sorbitol, in the response filed on 12/29/2025, is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
For the reasons provided above, this restriction requirement is deemed proper, and therefore, it is made final.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 07/14/2023 and 11/14/2024 are acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Claim Rejections - 35 U.S.C. § 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.
Claim 17 is 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 17 is recites the phrase “less than about” which is unclear.
The term “about” encompasses a range which includes values which are higher and lower than the recited reference value. The term “less than” implies that only values lower than the recited reference value are encompassed. Therefore, in the absence of a clear definition of what is encompassed by the term “about”, the term "less than about" is unclear and confusing since the term refers to values which are less than undefined values which are either higher or lower than the recited reference value. In essence, the term eliminates the relevance of the recited reference point because the reference value becomes variable and undefined. The Examiner suggests replacing the noted phrase with ---less than---.
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.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1-22 are rejected under 35 U.S.C. 103 as being unpatentable over Li et al. (Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC, Nature Methods volume 4, pages 251–256, 2007) in view of Benoit et al. (Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost, PLoS ONE 11(4): e0153158. https://doi.org/10.1371/journal.pone.0153158, 2016), Ozay et al. (A review of reaction enhancement strategies for isothermal nucleic acid amplification reactions, Sensors and Actuators Reports, Volume 3, November 2021, 100033), and KSR International Co. v. Teleflex Inc., 550 U.S.--, 82 USPQ2d 1385 (2007).
The instant claims are drawn to a method for the assembly of a plurality of double stranded DNA (dsDNA) fragments into a covalently bound circular dsDNA molecule, the method comprising:(a) combining a plurality of distinct dsDNA fragments with a reaction mixture comprising an exonuclease and a recombinase to form a DNA reaction mixture; wherein each individual dsDNA fragment comprises one or more terminal single- stranded nucleotides that are complementary to terminal single-stranded nucleotides of an independent dsDNA fragment;(b) subjecting the DNA reaction mixture to a hybridization incubation to form a hybridized DNA reaction mixture;(c) subjecting the hybridized DNA reaction mixture to a deactivation incubation to form a deactivated DNA reaction mixture;(d) transforming the deactivated DNA reaction mixture into a competent host cell; and (e) incubating the transformed competent host cell under conditions sufficient to assemble and replicate one or more covalently bound circular dsDNA molecules comprising the plurality of distinct dsDNA fragments.
Li et al. teach a method for the assembly of a plurality of double stranded DNA (dsDNA) fragments into a covalently bound circular dsDNA molecule, the method comprising: (a) combining a plurality of distinct dsDNA fragments with a reaction mixture comprising an exonuclease, i.e., T7 exonuclease, lambda exonuclease, or T4 polymerase has an inherent exonuclease activity, and a RecA recombinase (see abstract, i.e., SLIC with RecA) to form a DNA reaction mixture with ATP; wherein each individual dsDNA fragment comprises one or more terminal single-stranded nucleotides that are complementary to terminal single-stranded nucleotides of an independent dsDNA fragment, i.e., 20-40 bp overlaps (see page 254, left column, 2nd paragraph); (b) subjecting the DNA reaction mixture to a hybridization incubation to form a hybridized DNA reaction mixture, i.e., at 37 degrees Celsius for 30 min (see under METHODS SLIC on page 255); (c) transforming the DNA reaction mixture into a competent host cell, i.e., E. coli; and (d) incubating the transformed competent host cell under conditions sufficient to assemble and replicate one or more covalently bound circular dsDNA molecules comprising the plurality of distinct dsDNA fragments (see Figure 4a-e on page 254; page 252, left column, 1st and 2nd paragraphs), further comprising routine isolation of the covalently bound circular dsDNA molecules comprising the plurality of distinct dsDNA fragments from one or more competent host cells, and further comprising routine sequencing the covalently bound circular DNA molecule comprising the plurality of distinct dsDNA fragments following isolation from the competent host cell (see Figure 4de and left column on page 254). Li et al. further teach that the advantage of combining RecA in the reaction mixture is that it increases efficiency of the SLIC reaction, thereby allowing the use of very low DNA concentrations to catalyze homologous recombination (see Abstract). It is noted by the Examiner that the dsDNA fragments are “independent” because they are distinct from each other (see Figure 4d, with dsDNA inserts of different length and sequences represented by the rainbow colors.
Li et al. do not teach the deactivation incubation step, the use of T5 exonuclease, and ligase, wherein the reaction mixture further comprises one or more crowding agents, PEG, and/or one or more chaperone agents, sorbitol.
Benoit et al. teach the use of T5 exonuclease (see abstract), ligase (see abstract; Figure 5 on page 9), the deactivation incubation which is 80 degrees Celsius followed by 4 degrees Celsius for 15 min (see under “Gibson assembly” on page 11), wherein the reaction mixture further comprises PEG (see under “Gibson assembly” on page 11), to improve the efficiency of SLIC. Benoit et al. further teach that T5 exonuclease exhibited more than1000 fold increase in the number of positive colonies…so it is an excellent choice for SLIC (see page 7, last paragraph).
Ozay et al. teach that “[p]roline, glycine in combination with trehalose, betaine, sucrose, maltose, ectoine, hydroxyectoine, sorbitol, glycine betaine and homodeanol betaine have been shown to stabilize enzymes and they have been used as additives for isothermal amplification reactions (see page 7, right column 2nd paragraph).
It would have been obvious to a person of ordinary skill in the art (POSITA) prior to the effective filing date of the instant application to practice the method taught by Li et al. and replace the exonuclease with T5 exonuclease while adding ligase, PEG, sorbitol and further include a deactivation step as taught by Benoit et al. and Ozay et al.
A POSITA would have been motivated to practice such methods because Benoit et al. teach that T5 exonuclease exhibited more than1000 fold increase in the number of positive colonies…so it is an excellent choice for SLIC as it increases the efficiency of SLIC, while addition of ligase closes up the nicks and the use of PEG and sorbitol further enhances the SLIC reaction by improving crowing and stabilization of DNA enzymes as taught by Benoit et al. and Ozay et al.
As discussed in KSR International Co. v. Teleflex Inc., 550 U.S.--, 82 USPQ2d 1385 (2007), it is considered obvious to combine prior art elements known to be used in equivalent fields of endeavor together into a single combination. The references of Li et al, Benoit et al. and Ozay et al. clearly show that the use of deactivation step, T5 exonuclease, ligase, wherein the reaction mixture further comprises PEG and sorbitol, were known to be used in equivalent fields of endeavor, i.e., in performing and improving various methods of SLIC using DNA enzymes, and are routinely used together; thus, it is considered obvious to combine them together.
A POSITA would have had a reasonable expectation of success to practice such methods because all of the required biochemical reagents and techniques were readily available and rampantly used as evidenced by Li et al, Benoit et al. and Ozay et al. prior to the filing of the instant application.
For the reasons provided herein, the invention as claimed is prima facie obvious over the combined teachings of the prior art.
Conclusion
Claims 1-22 are rejected for the reasons as stated above. Applicants must respond to the objections/rejections in this Office action to be fully responsive in prosecution.
The instant Office action is non-final.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JAE W LEE whose telephone number is (571)272-9949. The examiner can normally be reached on M-F between 9:00-6:00.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Manjunath Rao can be reached on (571)272-0939. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/JAE W LEE/
Examiner, Art Unit 1656
/MANJUNATH N RAO/Supervisory Patent Examiner, Art Unit 1656