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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 05/01/2026 has been entered.
Claims Status
Applicant’s election with traverse of Group I (claims 1-3, 7-9, 11, 13, 15, 20-24, 28-30, 33, 35, 37 and 73-74), drawn to a method for identifying regions of genomic DNA bound to a protein, the method comprising: contacting genomic DNA with an adenine methyltransferase (A-MTase) to detect locations in the genomic DNA lacking methylated adenine residues to identify regions of genomic DNA bound to a protein.
Claims 1, 7-9, 11, 13, 20-23, 37 and 73-74 are pending and currently under examination.
Priority
This application is a 371 of PCT/US2021/025644 04/02/2021 which claims benefit of United States Provisional Application No. 63/004,361, filed on 04/02/2020. Accordingly, the priority date of the instant claims is determined to be 04/02/2020.
Specification
The abstract of the disclosure is objected to because the abstract is not presented on a separate sheet, apart from any other text. A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
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, 7,11,13, 21, 23 and 37 are rejected under 35 U.S.C. 103 as being unpatentable over Shipony et al. (“Shipony”; (2020). Long-range single-molecule mapping of chromatin accessibility in eukaryotes. Nature methods, 17(3), 319-327, Epub 2020 Feb 10. 2020.) in view of Drozdz et al. (“Drozdz”; Novel non-specific DNA adenine methyltransferases. Nucleic Acids Res. 2012 Mar;40(5):2119-30.)
Shipony discloses a method for profiling the accessibility of individual chromatin fibers, a single-molecule long-read accessible chromatin mapping sequencing assay (SMAC-seq), enabling the simultaneous, high-resolution, single-molecule assessment of chromatin states at multikilobase length scales. Our strategy is based on combining the preferential methylation of open chromatin regions by DNA methyltransferases with low sequence specificity, in this case EcoGII, an N6-methyladenosine (m6A) methyltransferase, and the ability of nanopore sequencing to directly read DNA modifications. We demonstrate that aggregate SMAC-seq signals match bulk-level accessibility measurements, observe single-molecule nucleosome and transcription factor protection footprints, and quantify the correlation between chromatin states of distal genomic elements. (Abstract)
Regarding claim 1, Shipony teaches a method comprising “use m6A methyltransferase EcoGII as an alternative/addition to CpG/GpC and use nanopore sequencing to generate single-molecule readouts of accessibility states over many kilobases… enabling the generation of methylation maps for individual DNA molecules, which can then be interpreted in terms of chromatin accessibility” (Pg. 319, Col. 2, Para. 1). “alternative” reads on instead of other option(s) as a single enzyme. “m6A methyltransferase EcoGII as an alternative” reads on a single enzyme that is m6A-MTase. “enabling the generation of methylation maps…in terms of chromatin accessibility” reads on m6A-MTase causes methylation of adenine residues at genomic sites not bound by protein and thus accessible. Nanopore sequencing reads long-read sequencing.
Regarding claim 1, Shipony teaches a method comprising “Nuclei were then treated with EcoGII” (Pg. 328, GM12878 SMAC-seq experiments) “Nuclei were then treated with EcoGII” reads on contacting genomic DNA with m6A-MTase. Shipony teaches a method comprising “Using m6A increases SMACseq’s resolution down to a theoretical limit of ~3 bp in all model organisms, and ensures proper coverage over all individual loci (Supplementary Figs. 1–12). We initially developed the method in S. cerevisiae”(Pg. 319, Col 2, last two para.) and “Using yeast gDNA or -λ DNA treated with a high dose of EcoGII (Supplementary Table 1).” (Pg. 320, Col 2, para. 1). “all model organisms”, “S. cerevisiae”, and “yeast gDNA” read on eukaryotic genomic DNA.
Shipony teaches a method comprising “single-molecule long-read accessible chromatin mapping sequencing (SMAC-seq), a single-molecule method that directly assays both open chromatin regions and nucleosome positioning within a single chromatin fiber at multikilobase scales. We use SMAC-seq to study chromatin architecture and coaccessibility states in the yeast Saccharomyces cerevisiae” (Pg. 319 Col. 1, para. 2). Shipony teaches a method comprising “SMAC-seq provides a high-resolution strand-specific view of genomic occupancy by DNA-binding proteins and complexes” (Pg. 324, Fig. 4, Legend Title). Furthermore, Shipony teaches Supplementary Figures 4 and 5 which depict “m6A-only, 1 bp resolution” (Supp. Fig. 4 and 5). “1 bp resolution” reads on single nucleotide resolution. Thus, Shipony suggests a method comprising: contacting the eukaryotic genomic DNA with a single enzyme, wherein the single enzyme is the N6-adenine methyltransferase (m6A-MTase); and conducting single-molecule long-read sequencing of the eukaryotic genomic DNA contacted with the single enzyme to detect locations in the eukaryotic genomic DNA lacking methylated adenine residues to identify at single nucleotide resolution regions of genomic DNA bound to a protein.
Furthermore, Shipony suggests that “ future applications of improvements on and extensions of the SMAC-seq approach to enable new insights into the dynamics of chromatin states in a wide variety of experimental systems” (Pg. 319 Col 1, last sent). Shipony also suggests that “EcoGII’s methylation efficiency is more difficult to estimate as fully methylated templates are known to be difficult to sequence … the limited number of observed reads exhibited ~50% methylation levels. We hypothesize these rates are underestimates, as biochemical reports suggest ≥50% methylation of gDNA after 5 min, increasing to ≥85% after an hour” (Pg. 320 col. 2- Pg. 321 col. 1). It would be obvious to the ordinary artisan from the teachings of Shipony that using m6A-only is a simple improvement on assay resolution over the previous CG/GC. One of skill in the art would be motivated to further assess the m6A methylation efficiency of EcoGII as well as other m6A-MTAses across various model organisms, including eukaryotic and/or -λ genomic DNA.
However, Shipony does not explicitly teach the claim limitations, wherein the single enzyme is the N6-adenine methyltransferase (m6A-MTase), Hia5, wherein Hia5 causes methylation of adenine residues in regions of the eukaryotic genomic DNA not bound to a protein.
Drozdz discloses “…Products of the hin1523, hia5 and nma1821 genes modify adenine residues to N(6)-methyladenine, both in vitro and in vivo. All of these enzymes catalyzed extensive DNA methylation; most notably the Hia5 protein caused the methylation of 61% of the adenines in λ DNA. Kinetic analysis of oligonucleotide methylation suggests that all adenine residues in DNA, with the possible exception of poly(A)-tracts, constitute substrates for the Hia5 and Hin1523 enzymes. Their potential 'sequence specificity' could be summarized as AB or BA (where B = C, G or T)…” (Abstract)
Regarding claim 1, Drozdz teaches Hia5 and other homologues tested “possess the characteristic signature motifs, including the catalytic motif IV (DPPY), which is typical for MTases that modify amino groups in various substrates… these enzymes catalyzed the transfer of methyl groups from an AdoMet donor to the substrate DNA, and that they modified only adenine residues, converting them into m6A” (Pg. 2128). Drozdz teaches a method wherein “The observed high level of adenine methylation strongly suggested that the Hia5… enzymes have minimal sequence specificity” (Pg. 2125-2126,Sequence preferences of MTases, Para. 1). Drozdz teaches that “These results were the first indication of the massive DNA methylation catalyzed by the Hia5… Corroborating evidence was obtained using an HPLC DNA methylation assay to evaluate the base composition of λ DNA methylated with Hia5 in vitro. This analysis revealed that as much as 61% of the adenine residues were converted to m6A.” (Pg. 2128, col. 2 para. 2). Moreover, Drozdz teaches that “Sequence searches with the Hia5 sequence against a non-redundant database (NCBI) revealed many closely related proteins, suggesting that there may exist more DNA : m6A MTases with similar properties, i.e. extremely relaxed substrate specificity” (Pg. 2129, col. 1, Para. 5) and suggests that “the discovery of a group of DNA:m6A MTases with extremely relaxed substrate specificity has significance due to their potential as tools in molecular biology research particularly in the study of the poorly characterized role of adenine methylation in eukaryotic DNA” (Pg. 2129, col. 2, Para. 3). One of skill in the art would be motivated to assess the role of adenine methylation in eukaryotic genomic DNA by the single enzyme m6A MTase, Hia5. Thus, Shipony and Drozdz suggest a method comprising wherein the single enzyme is the N6-adenine methyltransferase (m6A-MTase), Hia5, wherein Hia5 causes methylation of adenine residues in regions of the eukaryotic genomic DNA not bound to a protein.
Shipony and Drozdz suggest the method according to the limitations of claim 1.
Shipony and Drozdz are both considered to be analogous to the claimed invention because they are in the same field of non-specific m6A methyltransferases as a molecular biology tool. Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of methylating adenine residues in regions of the genome not bound by proteins using the nonspecific m6A-methyltransferase EcoGII as taught by Shipony to substitute the single nonspecific m6A-methyltransferase is Hia5 as suggested by Drozdz. Given these claim elements were known in the art, the non-specific m6A-adenine methyltransferases would be expected to function similarly and one of skill in the art could have combined these elements by known methods with no change in their respective function in order to yield a predictable result of identifying at single nucleotide resolution regions of eukaryotic genomic DNA bound to a protein and accessible unbound DNA regions using m6 adenine methylation only with similar bp resolution. According to the teachings in the prior art one of skill in the art would be motivated to further assess the adenine methylation efficiency of EcoGII as well as other m6A-MTAses across various model organisms, including eukaryotic genomic DNA. Additionally, one of skill in the art would be motivated to assess Hia5 adenine methylation on the eukaryotic genomic. A person of ordinary skill in the art would have had a reasonable expectation of success in substituting the non-specific m6A methyltransferase EcoII for Hia5 in order to methylate adenine residues in regions of the genomic DNA not bound to a protein to gain high, 1-bp resolution information on nucleosome positioning, chromatin architecture and accessibility and regulatory states, because both enzymes are were known in the art to be non-specific m6A-MTase that can produce high levels of adenine methylation.
The teachings of Shipony and Drozdz are documented above in the rejection of claim 1 under 35 U.S.C. 103. Claims 7, 11, 13, 21, 23 and 37 depends on claim 1.
Regarding claim 7, Shipony teaches a method comprising "GM12878 cells were washed … resuspended in … nuclei lysis buffer… Nuclei were then centrifuged, resuspended in … wash buffer…and centrifuged again … Finally, nuclei were resuspended in … reaction buffer ... Nuclei were then treated with EcoGII by adding 200 U of EcoGII (NEB) and SAM …" (Pg. 328, GM12878 SMAC-seq experiments, Para. 1). GM12878 cells read on eukaryotic genomic DNA. As stated in the rejection of claim 1 above, Shipony and Drozdz suggest a method comprising wherein the single enzyme is the N6-adenine methyltransferase (m6A-MTase), Hia5, wherein Hia5 causes methylation of adenine residues in regions of the eukaryotic genomic DNA not bound to a protein. Thus, Shipony and Drozdz suggest a method wherein the contacting comprises contacting isolated eukaryotic genomic DNA with Hia5.
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Regarding claim 11, Shipony teaches a method wherein at least 1 kb of genomic DNA was sequenced as shown in supplementary figure 73 below. (Supp. Fig. 73, Measurement of chromatin accessibility around transcription start sites using m6A-SMAC-seq human GM12878 cells.). With regard to supplementary figure 73, 500 bases in the plus and minus direction is interpreted as 1 kb. Thus, Shipony and Drozdz suggest a method wherein the sequencing is conducted on a stretch of genomic DNA that is at least 1 kilobase (kb) long or at least 3 kb long.
Regarding claim 13, Shipony teaches a method comprising “Nanopore sequencing” (Pg. 328, SMAC-seq analysis. Para. 1). Thus, Shipony and Drozdz suggest a method wherein the sequencing comprises translocating the genomic DNA through a nanopore.
Regarding claim 21, Shipony teaches a method comprising “GM12878 human lymphoblastoid cell lines” (Pg. 328, GM12878 cell culture, Para. 1). Thus, Shipony and Drozdz suggest a method wherein the eukaryotic genomic DNA is from a mammalian cell.
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Regarding claim 23, Shipony teaches a method wherein “sacCer3 reference genome” (Document Pg 10, Methods, Col. 1 Nanopore base calling, Para.1). SacCer3 reads on normal yeast cell. Shipony teaches a method further comprising “We use the m6A methyltransferase EcoGII as an alternative/… and use nanopore sequencing to generate single-molecule readouts of accessibility states over many kilobases. Nanopore sequencing allows direct detection of these modifications, enabling the generation of methylation maps for individual DNA molecules, which can then be interpreted in terms of chromatin accessibility. The addition of an m6A signal associated with accessible chromatin…" (Pg. 319, Col. 2, Para. 1-2; Figure 1). Mapped accessible and inaccessible regions to the A-MTase are shown in Figure 1. (Figure1; see m6A and accessibility footprint in Figure 1a and comparisons to SMAC-seq in 1h below). Shipony also teaches a method comprising "generated low-coverage SMAC-seq data for human GM12878 cells using only EcoGII and examined aggregate ‘m6ASMAC’ profiles around CTCF sites, open chromatin regions and TSSs. (Pg. 326, Discussion, Para.4; Suppl. Figures 71-73). GM12878 cells are interpreted as human lymphoblastoid cells often used as a reference/normal cells. Thus, Shipony and Drozdz suggest a method
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wherein the eukaryotic genomic DNA is from a normal cell and the method further comprises generating a chromatin accessibility map for the region of genomic DNA sequenced, wherein the map indicates regions of chromatin not bound to the protein and hence accessible to Hia5 and regions of the chromatin bound to the protein and hence inaccessible to Hia5.
Regarding claim 37, Shipony teaches a method wherein “We recovered the expected features of chromatin accessibility (strong nucleosome positioning…” (Pg. 326, Discussion, Para. 4). “Nucleosomes” reads on histones and DNA. Shipony teaches a method comprising “transcription factor” (Pg. 319, Col. 1, Para. 2). “transcription factor” reads on a transcriptional repressor or a transcriptional activator. Thus, Shipony and Drozdz suggest a method wherein the protein comprises nucleosomes, a transcriptional repressor, or a transcriptional activator.
Response to Arguments
Applicant' s arguments filed 05/01/2026 (Pg.5-13) with respect to claim 1-5, 7, 19-21, 25 and 26 have been considered but are not persuasive. To clarify some instances argued in the response filed 05/01/2026 see responses to each argument made by Applicant below:
Applicants’ argument: “None of the cited references teach or suggest a method for identifying at single nucleotide resolution regions of eukaryotic genomic DNA bound to a protein, the method comprising: contacting the eukaryotic genomic DNA with a single enzyme, wherein the single enzyme is the N6-adenine methyltransferase (m6A-MTase), Hia5..” (Pg. 6)
Response: Applicant' s arguments have been fully considered and found unpersuasive because in response to applicant's argument stated above, the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981).
Applicants’ argument: “Shipony does not teach or suggest a method for identifying at single nucleotide resolution regions of eukaryotic genomic DNA bound to a protein by using a single enzyme.” (Pg. 6)
Response: Applicant' s arguments have been fully considered and found unpersuasive because as stated above in the non-final action above on Pg. 4, “Shipony teaches a method comprising “use m6A methyltransferase EcoGII as an alternative/addition to CpG/GpC and use nanopore sequencing to generate single-molecule readouts of accessibility states over many kilobases… enabling the generation of methylation maps for individual DNA molecules, which can then be interpreted in terms of chromatin accessibility” (Pg. 319, Col. 2, Para. 1). “alternative” reads on instead of other option(s) as a single enzyme. “m6A methyltransferase EcoGII as an alternative” reads on a single enzyme that is m6A-MTase.” Furthermore, as stated above in the non-final office action, “Shipony teaches Supplementary Figures 4 and 5 which depict “m6A-only, 1 bp resolution” (Supp. Fig. 4 and 5). “1 bp resolution” reads on single nucleotide resolution”. Thus, Shipony does teach or suggest a method for identifying at single nucleotide resolution regions of eukaryotic genomic DNA bound to a protein by using a single enzyme.
Applicants’ argument: “Thus, in the section of Shipony cited for using a single enzyme (EcoGII), Shipony clearly states that using EcoGII generates a low-coverage SMAC-seq data and requires aggregating the sequence of multiple different DNA molecules. As such, Shipony fails to teach a method for identifying at single nucleotide resolution regions of genomic DNA bound to a protein by contacting the genomic DNA with a single enzyme. In other words, a person of ordinary skill in the art (POSITA) would not glean from this section of Shipony that EcoGII can be used by itself for identifying at single nucleotide resolution regions of genomic DNA bound to a protein..” (Pg. 7-8)
Response: Applicant' s arguments have been fully considered and found unpersuasive because the method of Shipony does teach 1-bp resolution data using the single enzyme EcoGII. The argument that the “Shipony clearly states that using EcoGII generates a low-coverage SMAC-seq data and requires aggregating the sequence of multiple different DNA molecules” is irrelevant to the claimed features upon which the applicant relies upon. Furthermore, the statement argued would actually be additional motivation for an ordinary artisan to improve upon the method in which the single m6A-MTase EcoGII is substituted for m6A-MTase Hia5 to further assess methylation in eukaryotic genomic DNA.
Applicants’ argument: “the term "1-bp resolution" does not imply that single nucleotide resolution was achieved.” (Pg. 9)
Response: Applicant' s arguments have been fully considered and found unpersuasive because 1-bp resolution reads on the claimed single nucleotide resolution.
Applicants’ argument: " Shipony teaches "Even with the addition of mGA methylation, the resolution of SMAC-seq still does not cover every nucleotide in the genome" (page 328, section bridging the left- and right-hand columns). Thus, Shipony teaches that even with methylation of CG, GC, and A (using EcoGII), single nucleotide resolution is not achieved.” (Pg. 10)
Response: Applicant' s arguments have been fully considered and found unpersuasive because resolution and coverage are not the same. Furthermore, coverage is not a claimed limitation and thus is irrelevant in arguing single nucleotide resolution is not achieved.
Applicants’ argument: “Drozdz provides no indication that (i) Hia5 can be used for methylating eukaryotic genomic DNA” (Pg. 10)
Response: Applicant' s arguments have been fully considered and found unpersuasive because Drozdz does suggest for Hia5 to be used as a tool in eukaryotic genomic DNA. (Pg. 2129, Discussion, last para.)
Applicants’ argument: “Drozdz does not teach or suggest that HiaS would be superior to EcoGII and could be used solely instead of using the combination of methylation of CG, GC, and A.” (Pg. 10)
Response: Applicant' s arguments have been fully considered and found unpersuasive because, in response to applicant's argument stated above, the fact that the inventor has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious. See Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985). Furthermore, differences between the claimed invention and the prior art may be expected to result in some differences in properties. The difference is not really unexpected as m6A MTAses are known to have different specificities and catalytic activities as suggested by Drozdz (2123-2125).
Claims 8-9 are rejected under 35 U.S.C. 103 as being unpatentable over Shipony et al. (“Shipony”; (2020). Long-range single-molecule mapping of chromatin accessibility in eukaryotes. Nature methods, 17(3), 319-327.) in view of Drozdz et al. (“Drozdz”; Novel non-specific DNA adenine methyltransferases. Nucleic Acids Res. 2012 Mar;40(5):2119-30) as applied to claim 1, and further in view of Vogel et al. (“Vogel”; (2007). Detection of in vivo protein–DNA interactions using DamID in mammalian cells. Nature protocols, 2(6), 1467-1478.).
The teachings of Shipony and Drozdz are documented above in the rejection of claims 1, 7,11,13, 21, 23 and 37 under 35 U.S.C. 103. Claims 8 and 9 depend on claim 1. Claim 9 depends on claim 8. Shipony does not explicitly teach the limitations of claims 8 and 9.
Vogel discloses DamID, an alternative technique to map genome-wide occupancy of interaction sites in vivo, that bypasses these limitations. DamID is based on the expression of a fusion protein consisting of a protein of interest and DNA adenine methyltransferase (Dam). This leads to methylation of adenines near sites where the protein of interest interacts with the DNA. These methylated sequences are subsequently amplified by a methylation-specific PCR protocol and identified by hybridization to microarrays. Using DamID, genome-wide maps of the binding of DNA-interacting proteins in mammalian cells can be constructed efficiently. Depending on the strategy used for expression of the Dam-fusion proteins, genome-wide binding maps can be obtained in as little as 2 weeks. (Abstract)
Regarding claim 8, Vogel teaches a method wherein “Stable cell lines and lentivirus transductions… are the preferred methods” and “For DamID, transfected/transduced cells are cultured …, cells expressing Dam” (Pg. 1469, Experimental design, Para. 2-3). “Cells expressing Dam” is interpreted as contact cell with genomic DNA. Thus, Shipony, Drozdz and Vogel suggest a method wherein the contacting comprises contacting a eukaryotic cell comprising the eukaryotic genomic DNA.
Regarding claim 9, Vogel teaches a method wherein “After transfection, cells are selected for stable genomic integration of the Dam-expressing vector using selective media” (Pg. 1469, Experimental design, Para. 1). Vogel teaches a method wherein “Stable cell lines and lentivirus transductions… are the preferred methods” and “For DamID, transfected/ transduced cells are cultured …, cells expressing Dam” (Pg. 1469, Experimental design, Para. 2-3). Thus, Shipony, Drozdz and Vogel suggest a method wherein the contacting comprises introducing into the eukaryotic cell a nucleic acid encoding Hia5 or wherein Hia5 is fused to a cell penetrating peptide that renders Hia5 plasma membrane permeable.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of methylating adenine residues in regions of the genome not bound by proteins as taught by Shipony and Drozdz to incorporate the method of contacting a cell comprising the genomic DNA with the m6A-MTase and introducing into the cell a nucleic acid into encoding the m6A-MTase as taught by Vogel to have yielded the predictable result of identifying regions of genomic DNA bound to a protein and accessible unbound DNA regions. According to the teachings in the prior art one of skill in the art would be motivated to further assess the adenine methylation efficiency of EcoGII as well as other MTAses across various model organisms, including eukaryotic genomic DNA. Additionally, one of skill in the art would be motivated to assess Hia5 adenine methylation on the eukaryotic genomic. It would be obvious to the ordinary artisan to in include contact a cell comprising the genomic DNA with the A-MTase and introduce nucleic acids encoding Hia5 into the cell of Vogel to the method of adenine methylation of unbound genomic DNA regions with the reasonable expectation of a identifying chromatin accessible and inaccessible regions in genomic DNA from cells.
Response to Arguments
Applicant's arguments filed 05/01/2026 have been fully considered but they are not persuasive. Arguments against Shipony and Drozdz on Pg. 6-12 are not persuasive as discussed above.
Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over Shipony et al. (“Shipony”; (2020). Long-range single-molecule mapping of chromatin accessibility in eukaryotes. Nature methods, 17(3), 319-327.) in view of Drozdz et al. (“Drozdz”; Novel non-specific DNA adenine methyltransferases. Nucleic Acids Res. 2012 Mar;40(5):2119-30) as applied to claim 1, and further in view of Flusberg et al. (“Flusberg”; (2010). Direct detection of DNA methylation during single-molecule, real-time sequencing. Nature methods, 7(6), 461-465.).
The teachings of Shipony and Drozdz are documented above in the rejection of claims 1, 7,11,13, 21, 23 and 37 under 35 U.S.C. 103. Claim 20 depends on claim 1. Shipony does not explicitly teach the limitations of claim 20.
Flusberg discloses direct detection of DNA methylation, without bisulfite conversion, through single-molecule, real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenine, 5-methylcytosine and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We used these kinetic signatures to identify adenine methylation in genomic samples and found that, in combination with circular consensus sequencing, they can enable single-molecule identification of epigenetic modifications with base-pair resolution. This method is amenable to long read lengths and will likely enable mapping of methylation patterns in even highly repetitive genomic regions. (Abstract)
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Regarding claim 20, Flusberg teaches a method wherein “In SMRT DNA sequencing, polymerase kinetics are measured alongside primary sequence determination…We showed that mA… in a DNA template alter incorporation kinetics …” (See Figure 1 below) and “By enabling repeated interrogation of individual molecules, circular consensus sequencing allows base-pair resolution and single-molecule sensitivity for detection of mA (Pg. 465, Discussion, Para. 1). Thus, Shipony, Drozdz and Flusberg suggest a method wherein the sequencing comprises single molecule real-time (SMRT) circular consensus sequencing (CCS).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of methylating adenine residues in regions of the genome not bound by proteins as taught by Shipony and Drozdz to incorporate the method comprising single molecule real-time (SMRT) circular consensus sequencing as taught by Flusberg to have yielded the predictable result of identifying regions of genomic DNA bound to a protein and accessible unbound DNA regions. It would be obvious to the ordinary artisan to in include single molecule real-time (SMRT) circular consensus sequencing of Flusberg to the method of adenine methylation of unbound genomic DNA regions with the reasonable expectation of a identifying chromatin accessible and inaccessible regions in genomic DNA from cells. Doing so would enable single-molecule identification of epigenetic modifications with base-pair resolution.
Response to Arguments
Applicant's arguments filed 05/01/2026 have been fully considered but they are not persuasive. Arguments against Shipony and Drozdz on Pg. 6-13 are not persuasive as discussed above.
Claims 22 and 73-74 are rejected under 35 U.S.C. 103 as being unpatentable over Shipony et al. (“Shipony”; (2020). Long-range single-molecule mapping of chromatin accessibility in eukaryotes. Nature methods, 17(3), 319-327.) in view of Drozdz et al. (“Drozdz”; Novel non-specific DNA adenine methyltransferases. Nucleic Acids Res. 2012 Mar;40(5):2119-30) as applied to claim 1, and further in view of Xie et al. (“Xie”; (2018). N6-methyladenine DNA modification in glioblastoma. Cell, 175(5), 1228-1243.).
The teachings of Shipony and Drozdz are documented above in the rejection of claims 1, 7,11,13, 21, 23 and 37 under 35 U.S.C. 103. Claims 22 and 73 depend on claim 1. Claim 74 depend on claim 73, which depends on claim 1. Shipony does not explicitly teach the limitations of claim 22, 73 and 74.
Xie discloses N6-methyladenine DNA modifications are enriched in human glioblastoma, and targeting regulators of this modification can inhibit cancer growth by altering heterochromatin landscapes and downregulating oncogenic programs.
Regarding claim 22, Xie teaches a method wherein “we elucidated the function of N6-mA in human glioblastoma. N6-mA levels were elevated in tumor relative to normal brain tissues and in GSCs compared with normal human astrocytes” (Pg1240, Discussion, Para. 1). Thus, Shipony, Drozdz and Xie suggest a method wherein the eukaryotic genomic DNA is from a cancer cell.
Regarding claims 73 and 74, Xie teaches a method wherein “we elucidated the function of N6-mA in human glioblastoma. N6-mA levels were elevated in tumor relative to normal brain tissues and in GSCs compared with normal human astrocytes” (Pg1240, Discussion, Para. 1). Glioblastoma is interpreted as a tumor that originates from astrocytes. Astrocytes are interpreted as glial cells. Thus, Shipony, Drozdz and Xie suggest a method wherein the genomic DNA is from a human cell; and wherein test cell are epithelial cells, white blood cells, glial cells, osteoblasts, or chondrocytes
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of methylating adenine residues in regions of the genome not bound by proteins and generating a chromatin accessibility map of genomic DNA in eukaryotic cells as taught by Shipony and Drozdz to incorporate a method comprising human cancer cells originating from glial cells as taught by Xie to have yielded the predictable result of identifying regions of genomic DNA bound to a protein and accessible unbound DNA regions in a cancer cell. It would be obvious to the ordinary artisan to include a cancer cell of Xie to the method of adenine methylation of unbound genomic DNA regions with the reasonable expectation of a identifying chromatin accessible and inaccessible regions in genomic DNA from cancer cells and furthermore glial derived cancer cells. Doing so would increase the resolution of chromatin accessibility mapping in the likely aberrant epigenetic landscape of cancer cells such as those comprised in glioblastoma to further understand the genomic landscape and transcriptional regulatory factors playing a role in the disease.
Response to Arguments
Applicant's arguments filed 05/01/2026 have been fully considered but they are not persuasive. Arguments against Shipony and Drozdz on Pg. 6-12 are not persuasive as discussed above
Conclusion
In view of the amendments, grounds of objection and rejection and above responses to arguments, no claims are in condition for allowance.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KENDRA R VANN-OJUEKAIYE whose telephone number is (571)270-7529. The examiner can normally be reached M-F 9:00 AM- 5:00 PM.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Winston Shen can be reached at (571)272-3157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/KENDRA R VANN-OJUEKAIYE/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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