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 .
Application Status
The action is written in response to applicant’s correspondence received on 4/6/2026. Claims 1-34 are currently pending in the instant application.
Priority
This application is a 371 of PCT/US22/32777, which claim priority to US Provisional application 63/208,534, filed on 6/9/2021, is acknowledged. However, claims under examination that do not recite limitations of the provisional application will be granted the priority date of the PCT application, filed on 6/9/2022.
Election/Restriction
Applicant’s election of the inventions of Group III (claims 13, 23, and 24), drawn to a method for long-range linear amplification of a target double-strand DNA (dsDNA) comprising using a strand-specific or site-specific mesophilic nickase and a DNA polymerase, is acknowledged.
Claims 1-12, 14-22, and 24-27 have been withdrawn from consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected invention, there being no allowable or generic linking claim. Claims 28-34 are newly added in response to the restriction requirement. Based off the claims elected, priority will be granted to the PCT/US22/32777 application, with a filing date of 6/9/2022 as the provisional application claims an isothermal linear amplification of long DNA with primers, whereas the present invention is drawn to a method in the absence of primers. Election was made without traverse in the reply filed on 4/6/2026.
Applicant is reminded that upon the cancelation of claims to a non-elected invention, the inventorship must be corrected in compliance with 37 CFR 1.48(a) if one or more of the currently named inventors is no longer an inventor of at least one claim remaining in the application. A request to correct inventorship under 37 CFR 1.48(a) must be accompanied by an application data sheet in accordance with 37 CFR 1.76 that identifies each inventor by his or her legal name and by the processing fee required under 37 CFR 1.17(i).
Claims 13, 23-24, and 28-34 are currently under examination.
Claim Interpretation
Claim 13 recites a “long range” linear amplification of target double-strand DNA (dsDNA). Looking to the specification for guidance, “long range” or “long target” refers to a DNA sequence having a length of at least 300 bases, at least 1 kilobase, up to or at least 50 kilobases (see pg. 7, line 9 of the instant specification). Furthermore, the term “multiple turnover kinetics” refers to a property of enzymes that bind to substrate, are released from the product of the enzyme reaction, and are available to bind to substrate again (see pg. 7, line 14).
Claim Objections
Claim 23 is objected to because of the following informalities: Claim 23 recites “steps (a), (b), and (c) are performed in the of same reaction vessel” which is grammatically incorrect.
Appropriate correction is required.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 13, 23, 24, and 28-34 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Joneja et al. (Linear nicking endonuclease-mediated strand displacement DNA amplification, Analytical Biochemistry, Volume 414, Issue 1, pgs. 58-69, published 7/1/2011).
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Regarding claim 13, Joneja teaches a method for linear isothermal DNA amplification of dsDNA using nicking endonuclease-mediated strand displacement by a DNA polymerase (see abstract and Fig. 1). The DNA polymerase presented by Joneja is Sequenase 2.0, a genetically engineered form of T7 DNA polymerase, where, unlike the WT enzyme, it has virtually no exonuclease activity (see abstract, materials and methods, and detailed description from the ThermoFisher website, shown below).
Joneja teaches where the nicking of one stand of DNA target by the endonuclease produces a primer for the polymerase to initiate synthesis and the combined continuous repetitive action of nicking by the endonuclease and strand displacement synthesis by the polymerase results in linear amplification of one strand of the DNA molecule (see abstract). Joneja demonstrated that DNA templates up to five thousand nucleotides can be linearly amplified (see abstract) and where the nicking endonuclease has a seven base-pair recognition sequence (see abstract). Furthermore, in Fig. 1, Joneja shows where a DNA polymerase binds to the nicking site and extends from the 3’ OH group, displacing the downstream strand. The extension by the DNA polymerase regenerates the double-stranded recognition site for the nicking enzyme. The continuous combined actions of the nicking endonuclease and DNA polymerase result in linear amplification of one strand of the DNA, producing daughter strands, (see Fig. 1).
Regarding claim 23, the method of SDA taught by Joneja discloses the reaction steps with only one DNA template were prepared in a 25uL reaction volume on ice. The solution was divided into 5uL aliquots in give microtubes, and incubated at 37°C, therefore all steps of the reaction were performed in the same reaction vessel.
Regarding claim 24, the method of SDA taught by Joneja discloses where the 25uL reaction volume contains the DNA template, Sequenase 2.0 DNA polymerase, and Nt.BspQI endonuclease were mixed together in a tube and incubated at 37°C, indicating a one-step reaction (see ‘strand displacement amplification reactions’).
Regarding claim 28, Joneja teaches where the nicking endonuclease included a seven base-pair recognition sequence (see abstract), therefore the nickase is sequence-specific.
Regarding claim 29, Joneja teaches the use of engineered nicking enzymes Nt.BspQI and Nt.BbvCI both have seven base pair recognition sequences (see introduction).
Regarding claim 30, Joneja teaches where the nicking endonuclease included a seven base-pair recognition sequence (see abstract). Though the nicking endonuclease is considered sequence specific, as it has a recognition sequence, this sequence has the inherent function of binding to a specific site, absent evidence to the contrary. Looking to the specification for guidance, applicant often interchanges the two, reciting “a mesophilic nickase that is strand-specific, sequence-specific, and/or site-specific” (see instant specification, pg. 2, line 17).
Regarding claim 31, Joneja cites that another group, Turner et al. (Harnessing asymmetrical substrate recognition by thermostable EndoV to achieve balanced linear amplification in multiplexed SNP typing, Biochemistry and Cell Biology, Volume 84, Number 2, published 2006), exploited the nicking activity of the DNA mismatch-repair enzyme endonuclease V to enable linear SDA of target molecules, but did not demonstrate the ability to amplify long DNA molecules (see introduction of Joneja). Therefore, Joneja teaches that endonuclease V can be used in the method of strand displacement amplification, however, the results of amplification of long DNA fragments is uncertain.
Regarding claim 32, Joneja disclose the use of Sequanase 2.0, an engineered T7 DNA polymerase with no exonuclease (exo-) activity (see introduction and materials and methods).
Regarding claim 33 and 34, Joneja teaches where a library of randomly fragmented bacteriophage lambda genomic DNA was prepared and used as a model system to demonstrate the ability to amplify a complex library linearly by SDA. Joneja further teaches where the genomic DNA is fragmented and where both ends of the genomic DNA fragments were ligated to a duplex adapter containing the recognition site for nicking endonucleases. Furthermore, the duplex adaptor consists of 51-base oligonucleotide and a 50-base compliment from the 3’ end. The nicking sites for the endonucleases are located at 25 and 38 nucleotides respectively from the 5’ end of the top strand (see section titled ‘Genomic DNA library construction’).
In view of the foregoing, claims 13, 23-24, and 28-34 are anticipated by Joneja.
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.
Claim 29 is rejected under 35 U.S.C. 103 as being unpatentable over Joneja et al. (Linear nicking endonuclease-mediated strand displacement DNA amplification, Analytical Biochemistry, Volume 414, Issue 1, pgs. 58-69, published 7/1/2011) in view of Kuzmenko et al. (Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea, Nucleic Acids Research, Volume 47, Issue 11, 6/20/2019).
Regarding claim 29, Joneja teaches the method of claim 28, where a sequence specific nickase can be used to nick dsDNA for long range linear amplification. Furthermore, Joneja teaches the use of engineered nicking enzymes Nt.BspQI and Nt.BbvCI both have seven base pair recognition sequences (see introduction).
As claim 29 recites alternatives “selected from” and “and”, Joneja does not teach an alternative where the endonuclease is an argonaut protein having a guide DNA.
Regarding claim 29, Kuzmenko teaches argonaut (Ago) proteins from prokaryotes are programmable endonucleases that may potentially be used as a tool for DNA manipulations in vitro and in vivo, including molecular cloning and genome editing (see abstract and introduction). Kuzmenko teaches most pAgos only have one active site, so only one DNA strand can be cleaved by a single complex, however, several thermophilic pAgos were shown to perform slow guide-independent cleavage of dsDNA termed chopping (see introduction). Kuzmenko teaches that mesophilic Ago proteins from Clostridium butyricum (CbAgo) and cyanobacterium Limnothrix rosea (LrAgo) are DNA-guided DNA nucleases that function at much lower temperatures (37°C) compared to thermophilic counterparts and can perform precise-guide dependent cleavage of dsDNA when supplied with two guides targeting both strands of the dsDNA target (see introduction).
It would have been obvious to one with ordinary skill in the art, before the effective filing date of the claimed invention, to have combined the teachings of Joneja and Kuzmenko to arrive at a method of long range linear amplification of a target double-strand DNA, wherein the endonuclease is a mesophilic Ago protein with a guide DNA.
One would expect a reasonable chance of success as Kuzmenko shows guide-directed cleavage of double-stranded DNA by CbAgo and LrAgo (see results and section titled “double-stranded DNA cleavage by CbAgo and LrAgo”).
One would be motivated to combine these teachings as Kuzmenko teaches in contrast to other nucleases, CbAgo or LrAgo do not require the presence of any specific motifs in the guide or target DNAs which may enable DNA targeting with a single-nucleotide resolution. Furthermore, the short guides utilized by CbAgo and LrAgo are much easier to synthesize compared to longer RNA guides (see results section). Combined with Joneja, one would arrive at a method of amplifying dsDNA where Ago nucleases can bind to highly specific sites of dsDNA in order to cleave or create a nick where polymerase can then bind to amplify, in the absence of primers.
In view of the foregoing, claim 29 is rejected under 35 U.S.C. 103 as being prima facie obvious, before the effective filing date.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DAVID YU whose telephone number is (571)272-1118. The examiner can normally be reached Monday-Friday 7:30 am -5 pm.
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/D.T.Y./Examiner, Art Unit 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
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