DETAILED ACTION
Status of the Claims
Claims 1-32 are currently pending and are examined herein.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Information Disclosure Statement
The information disclosure statement(s) (IDS) submitted on 11/14/2025 and 08/19/2026 is/are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement(s) is/are being considered by the examiner.
Claim Rejections – 35 U.S.C. 102
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.
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.
Brown
Claims 1-2, 9-12, and 17-32 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Brown (WO 2022/035950 A2).
Regarding claim 1, Brown discloses a method of enriching a sample for sequences of interest (e.g., as per Example 5), comprising:
a) providing a sample comprising sequences of interest and targeted sequences for depletion (e.g., as mixture of 90% pBR322 and 10% E. coli genomic DNA as per para 00160);
b) contacting the sample with a phosphatase to remove 5' phosphates on the sequences of interest and the targeted sequences for depletion to generate a sample comprising sequences lacking 5' phosphates (e.g., with shrimp alkaline phosphatase as per para 00161-00165);
c) contacting the sample comprising sequences lacking 5' phosphates of step (b) with a plurality of nucleic acid-guided nuclease-guide nucleic acid (gNA) complexes, wherein the guide nucleic acids (gNAs) include a region complementary to targeted sequences for depletion, and whereby the targeted sequences for depletion are cleaved to generate a sample comprising sequences of interest and cleaved targeted sequences for depletion (e.g., treatment with Cas9 and two guide RNAs specific for pBR322 as per para 00160-00164 and 00173); and
d) contacting the sample of step (c) with at least one exonuclease capable of targeting 5' phosphate containing sequences for degradation and degrading the cleaved targeted sequences for depletion to generate a sample enriched for the sequences of interest (e.g., treatment with lambda exonuclease as per para 00162-00169 and 00173 leading to 23.35-fold enrichment of the E. coli genomic DNA as per para 00184).
Regarding claim 2, Brown discloses the above method, further comprising e) fragmenting the nucleic acids in the sample enriched for the sequences of interest from step (d) to generate a fragmented sample (e.g., fragmenting as per para 00162-00164 and 00170 and/or Fig. 4A-4C).
Regarding claim 9, Brown discloses the above method, further comprising ligating adapters to the sequences in the fragmented sample to generate adapter-ligated sequences (e.g., ligating adapters as per para 00161-00164 and 00171-00173).
Regarding claim 10, Brown discloses the above method, further comprising amplifying the adapter-ligated sequences using adapter-specific PCR, thereby further enriching for the sequences of interest (e.g., adapter-specific PCR as per para 00161 and 00171).
Regarding claim 11, Brown discloses the above method, further comprising generating a library comprising adapter-ligated sequences from the sample enriched for the sequences of interest (e.g., ligating adapters and generating a library as per para 00161-00164 and 00171-00173).
Regarding claim 12, Brown discloses the above method, further comprising sequencing the adapter-ligated sequences (e.g., as per Example 5 and para 00173-00184).
Regarding claims 17-18 and 22, Brown discloses the above method, further comprising treating the sample enriched in sequences of interest with an exonuclease capable of degrading single stranded DNA, wherein the exonuclease is ExoI (e.g., treatment with ExoI as per para 00169).
Regarding claim 19, Brown discloses the above method, wherein the nucleic acid-guided nuclease-gNA complexes are Crispr/Cas system protein-gRNA complexes (e.g., as per para 00160-00164 and 00173).
Regarding claim 20, Brown discloses the above method, wherein the phosphatase is shrimp alkaline phosphatase (SAP) or recombinant SAP (rSAP) (e.g., as per para 00161 and 00165).
Regarding claim 21, Brown discloses the above method, wherein the exonuclease in step (d) is lambda exonuclease (e.g., as per para 00162-00168).
Regarding claim 23, Brown discloses the above method, wherein the sequences of interest comprise less than 50% of the sample (e.g., 10% E. coli genomic DNA as per para 00160).
Regarding claim 24, Brown discloses the above method, wherein the sequences of interest and targeted sequences for depletion are DNA (e.g., E. coli genomic DNA and pBR322, respectively, as per para 00160).
Regarding claims 25 and 30, Brown discloses the above method, wherein the sample is selected from the group consisting of a human saliva sample (e.g., methods for “various applications to remove genome of one organism from another” such as from human saliva as per para 00185).
Regarding claim 26, Brown discloses the above method, wherein the sample comprises host nucleic acid sequences targeted for depletion and non-host nucleic acid sequences of interest (e.g., methods for “various applications to remove genome of one organism from another” such as from human saliva as per para 00185).
Regarding claims 27-28, Brown discloses the above method, wherein the non-host nucleic acid sequences comprise microbial nucleic acid sequences and are derived from bacteria, archaea, fungi, or a eukaryotic parasite (e.g., methods for “various applications to remove genome of one organism from another” such as from human saliva as per para 00185).
Regarding claim 29, Brown discloses the above method, wherein the gNAs include a region complementary to DNA corresponding to ribosomal RNA sequences, sequences encoding globin proteins, sequences encoding a transposon, sequences encoding retroviral sequences, sequences comprising telomere sequences, sequences comprising sub-telomeric repeats, sequences comprising centromeric sequences, sequences comprising intron sequences, sequences comprising Alu repeats, sequences comprising SINE repeats, sequences comprising LINE repeats, sequences comprising dinucleic acid repeats, sequences comprising trinucleic acid repeats, sequences comprising tetranucleic acid repeats, sequences comprising poly-A repeats, sequences comprising poly- T repeats, sequences comprising poly-C repeats, sequences comprising poly-G repeats, sequences comprising AT -rich sequences, or sequences comprising GC-rich sequences (e.g., as per para 0095).
Regarding claim 31, Brown discloses the above method, further comprising generating a double-stranded or single-stranded DNA library comprising the enriched sequences of interest (e.g., adapter-specific PCR as per para 00161 and 00171).
Regarding claim 32, Brown discloses the above method, further comprising amplifying, sequencing, or cloning the DNA library (e.g., as per Example 5 and para 00173-00184).
Claim Rejections – 35 U.S.C. 103
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.
Brown and Ignatov et al.
Claims 1-3, 9-12, and 17-32 are rejected under 35 U.S.C. 103 as being unpatentable over Brown (WO 2022/035950 A2) in view of Ignatov et al. (PLoS ONE, 2019, 14(4): e0210374).
Brown in relied on as above, however, the reference is silent regarding the fragmenting step is completed by contacting the sample of step (d) with a fragmentase to generate the fragmented sample, as set forth in claim 3.
Ignatov discloses the use of NEBNext dsDNA Fragmentase in fragmenting E. coli genomic DNA in the context of library preparation for NGS (e.g., as per the DNA fragmentation with NEBNext dsDNA Fragmentase section on p. 3).
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 use fragmentase as per Ignatov in the methods of Brown. One of ordinary skill in the art would have been motivated to do so since replacing Brown’s acoustic shearing for Ignatov’s fragmentase avoids the necessity for costly sonicator equipment and that an “additional disadvantage of acoustic shearing is that it can be a source of oxidative damage to DNA that may result in sequencing artifacts”, as per Ignatov in the paragraph bridging pages 1-2.
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 the fragmentase used by Ignatov is part of a commercially available kit complete with instructions and has been proven to be effective in such use cases.
Brown and Gourguechon
Claims 1-2, 4-7, 9-12, and 17-32 are rejected under 35 U.S.C. 103 as being unpatentable over Brown (WO 2022/035950 A2) in view of Gourguechon (WO 2020/210372 A1).
Brown is relied on as above, however, the reference is silent as to the limitation of the fragmenting step is completed by contacting the sample of step (d) with one or more CpG methylation-sensitive restriction enzymes to generate cut sites or nick sites in the DNA in the sample at enzyme recognition sites that lack CpG methylation to generate the fragmented sample, wherein the one or more CpG methylation-sensitive restriction enzyme comprises a restriction enzyme selected from the group consisting of AatII, AccII, AluI, Aor13HI, Aor51HI, BspT104I, BssHII, Cfr10I, ClaI, CpoI, DdeI, Eco52I, HaeII, HapII, HhaI, HpyCH4IV, MluI, NaeI, NotI, NruI, NsbI, Nt.CviPII, PmaCI, Psp1406I, PvuI, RsaI, SacII, SalI, SmaI, SnaBI, and Sau3AI, as set forth in claims 4-6.
Gourguechon teaches the use of HpyCH4IV, Sau3AI, and AluI (e.g., as per Table 3).
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 use one or more CpG methylation-sensitive restriction enzymes to generate cut sites or nick sites in the DNA in the sample at enzyme recognition sites that lack CpG methylation to generate the fragmented sample as per Gourguechon in the library depletion/enrichment methods of Brown. One of ordinary skill in the art would have been motivated to do so since this would provide a methylation-sensitive, tunable, and predictable way to generate fragments for subsequent library preparation.
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 the enzymes taught by Gourguechon are commercially available and predictable in their cleavage sites and frequency.
Brown in view of Gourguechon is relied on as above, however, the references are silent as to the limitation of the one or more CpG methylation-sensitive restriction enzymes comprising all five of AluI, DdeI, HpyCH4IV, RsaI, and Sau3AI, as set forth in claim 7. Instead, as detailed above, Brown in view of Gourguechon only disclose the use of AluI, HpyCH4IV, and Sau3AI.
It is noted that as of the effectively filed date, the CpG methylation-sensitive restriction enzymes DdeI and RsaI were well known in the art. It is further noted that as per MPEP 2144.06, "It is prima facie obvious to combine two compositions each of which is taught by the prior art to be useful for the same purpose, in order to form a third composition to be used for the very same purpose.... [T]he idea of combining them flows logically from their having been individually taught in the prior art" citing In re Kerkhoven, 626 F.2d 846, 850, 205 USPQ 1069, 1072 (CCPA 1980) (citations omitted).
Brown and Carpenter et al.
Claims 1-2, 8-12, and 17-32 are rejected under 35 U.S.C. 103 as being unpatentable over Brown (WO 2022/035950 A2) in view of Carpenter et al. (U.S. 10,774,365 B2, cited in the IDS of 11/14/2025).
Brown in relied on as above, however, the reference is silent as to the limitation of the fragmented sample is subjected to serial depletion by repeating steps (b) through (d) of claim 1 using the fragmented sample, as set forth in claim 8.
Carpenter is similarly drawn to methods of depletion of unwanted sequences from a sample prior to library preparation, but discloses that “[t]he compositions and methods of the invention can be utilized to serially process the nucleic acids in the sample to first deplete/partition out the host-DNA, and then subsequently deplete/partition out other known non-host nucleic acids that are not of interest, to arrive at a remaining pool of nucleic acids that represent a particular non-host of interest” (e.g., as per col. 21).
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 perform the serial or repeated depletion steps as per Carpenter in the depletion/enrichment methods of Brown. One of ordinary skill in the art would have been motivated to do so since this would further reduce the amount of unwanted DNA and further enrich the desired DNA.
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 Carpenter is similarly drawn to serial depletion and uses CRISPR and lambda exonuclease as per Brown.
Brown and Moreno et al.
Claims 1-2, 9-16, and 17-32 are rejected under 35 U.S.C. 103 as being unpatentable over Brown (WO 2022/035950 A2) in view of Moreno et al. (PLoS ONE, 2017, 12(6):e0178433).
Brown is relied on as above, however, the reference is silent as to the limitations of performing second strand synthesis on the sample of step (a) prior to step (b) by contacting the sample of step (a) with random hexamers and Klenow DNA polymerase, as set forth in claims 13-14 and wherein after step (b) the sample is further contacted with a blunt-end generating restriction enzyme EcoRV, as set forth in claims 15-16.
Brown discloses converting mRNA into cDNA before phosphatase then endo/exonuclease depletion (e.g., as per para 0026 and 0010). Moreno discloses the use of Klenow fragment and random hexamers as per the Random amplification, Sequence-Independent Single Primer Amplification (SISPA) method section on page 10. Brown teaches the use of EcoRV digestion following Cas9 treatment (e.g., as per Example 5 and para 00161).
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 combine these methods of Moreno and Brown to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to do so since this would allow the provide a double-stranded substrate for Brown’s downstream depletion of samples which came from RNA samples. 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 these methods were well known in the art, as per Brown and Moreno, 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.
Given the teachings of the prior art and the level of the ordinary skilled artisan at the time of the application’s effective filing date, it must be considered, absent evidence to the contrary, that said skilled artisan would have had a reasonable expectation of success in practicing the claimed invention.
Conclusion
No claims are allowed.
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/JEREMY C FLINDERS/
Primary Examiner, Art Unit 1684
