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 .
Status of Claims
Claims 1, 7, 13-16, 18, 19, 21 and 26-28 are currently pending. Claim 1, 7, 21, 27 and 28 have been amended by Applicants’ amendment filed 12-16-2025. No claims have been added or canceled by Applicants’ amendment filed 12-16-2025.
A complete reply to the final rejection must include cancellation of nonelected claims or other appropriate action (37 CFR 1.144) See MPEP § 821.01.
Therefore, claims 1, 7, 13-16, 18, 19, 21 and 26-28 are under consideration to which the following grounds of rejection are applicable.
Priority
The present application filed December 5, 2022 is a DIV of US Application 15/656,947 (now US Patent 11535883), filed July 21, 2017; which claims the benefit of US Provisional Patent Application 62/451305, filed January 27, 2017; and US Provisional Patent Application 62/365916, filed July 22, 2016.
Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 120 as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of the first paragraph of 35 U.S.C. 112. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed applications US Provisional Patent Application 62451305, filed January 27, 2017; and US Provisional Patent Application 62365916, filed July 22, 2016 fail to provide adequate support or enablement in the manner provided by the first paragraph of 35 U.S.C. 112 for one or more claims of this application. The specific method steps recited in independent claim 1 does not have support for; “wherein the indexed nucleic acid fragments remain attached to the transposases”. Therefore, the priority date for the presently claimed invention is July 21, 2017, the filing date of US Patent Application 15/656,947.
Applicants are invited to specifically indicate the location of the cited phrase pertinent to claim 1 of the instant application.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on February 13, 2026 has been considered. An initialed copy of the IDS accompanies this Office Action.
Withdrawn Objections/Rejections
Applicants’ amendment and arguments filed December 16, 2025 are acknowledged and have been fully considered. The Examiner has re-weighed all the evidence of record. Any rejection and/or
objection not specifically addressed below are herein withdrawn.
Specification Objections
The objection to the disclosure is withdrawn because the amendment to the Specification, filed December 16, 2025 includes the status of US15/656,947 (now US Patent No 11535883).
Claim Rejections - 35 USC § 112(b)
The rejection of claims 1, 7, 13-16, 18, 19, 21 and 26-28 is maintained under 35 U.S.C. 112(b) paragraph as being indefinite for failing to particularly point out and distinctly claim the subject matter which applicant regards as the invention.
The rejection of claim 1 is maintained as being indefinite for the recitation of the term “the other compartments” such as recited in claim 1, line 15. There is insufficient antecedent basis for the term “the other compartments” in the claim because claim 1, lines 9-10 recites the term “a first plurality of compartments”. Instant claim 1 does not recite any ‘other compartments’.
The rejection of claim 1 is maintained as being indefinite for the recitation of the term “remain attached to the transposase” such as recited in claim 1, line 20 because it is unclear how fragmenting nucleic acids leads to indexed nucleic acid fragments that remain attached to a transposase. Instant claim 1 does not recite that the indexed nucleic acids are attached to the transposases, and/or that a transposition reaction occurs, such that the indexed nucleic acids later “remain attached to the transposases”. Additionally, the as-filed Specification also does not teach that the indexed nucleic acids are attached to the transposases, and/or that a transposition reaction occurs and, thus, the metes and bounds of the claim cannot be determined.
The rejection of claim 1 is maintained as being indefinite for the recitation of the term “genome-wide uniform incorporation of the first index sequences that is not restricted to sites of chromatin accessibility” in claim 1, lines 23-24 because claim 1 recites that the indexed nucleic acids remain attached to transposases, where claim 1 does not recite: (i) nucleosome depletion in the presence of first index sequences as suggested in lines 22-24 (depletion occurs in step b, while incorporation occurs in step d); (ii) any specific reaction that leads to such “genome-wide incorporation”; (iii) sites of chromatin accessibility; and/or (iv) any restrictions on chromatin accessibility, such that it is unclear what the first index sequences are “incorporated into” genome-wide. Moreover, the instant as-filed Specification and original claims do not teach the term as recited in claim 1. Instead, the instant as-filed Specification teaches that nucleosome depletion produces genome-wide uniform coverage that is not restricted to sites of chromatin accessibility (See, as-filed Specification, pg. 7, lines 14-15), which refers to sequencing and mapping techniques (and, not to the incorporation of index sequences) and, thus, the metes and bounds of the claim cannot be determined.
Claim 7 is indefinite for the recitation of the term “prior to step (a): (i) isolated nuclei” such as recited in claim 7, lines 1-2 because claim 7 depends from claim 1, wherein claim 1 does not recite a “step (a)” and/or a “step (a): (i)” and, thus, the metes and bounds of the claim cannot be determined.
The rejection of claim 16 is maintained as being indefinite for the recitation of the term “step (i)” such as recited in claim 16, line 2 because claim 16 depends from claim 1, 14 and 15, wherein none of claims 1, 14 and 15 recite a “step (i)” and, thus, the metes and bounds of the claim cannot be determined.
Claim 16 is indefinite for the recitation of the term “each comprising” such as recited in claim 16, line 4 because it is unclear what the term “each” is referring to including whether the term is referring to each second index sequence, each compartment, each first universal primer, each second universal primer, etc. and, thus, the metes and bounds of the claim cannot be determined.
Claim 21 is indefinite for the recitation of the term “contacting the surface…to produce a plurality of amplification sites that each comprise a clonal population of amplicons” such as recited in claim 21, lines 5-7 because contacting a surface with dual-index fragments will not produce a clonal population of amplicons, such that it is unclear how a surface comprising a plurality of amplification sites can comprise a clonal population of amplicons and, thus, the metes and bounds of the claim cannot be determined.
Claim 27 is indefinite for the recitation of the term “have access to the plurality of amplification sites: such as recited in claim 27, line 3 because it is unclear what it means for the dual-index fragments to have “access” to the plurality of amplification sites; particularly given that claim 21 recites a surface comprising a plurality of amplification sites and, thus, the metes and bounds of the claim cannot be determined.
The rejection of claim 27 is maintained as being indefinite for the recitation of the term “the amplification sites” such as recited in claim 27, line 4. There is insufficient antecedent basis for the term “the amplification sites” because claim 21, line 2 recites the term “a plurality of amplification sites.” The Examiner suggests that Applicant amend the claim to recite, for example, “each of the plurality of amplification sites.”
Claim 28 is indefinite for the recitation of the term “simultaneously (i) transporting…to the plurality of amplification sites, and (ii) amplifying the dual-index fragments at the plurality of amplification sites” in claim 28, lines 2-4 because the two steps cannot be simultaneous given that the dual-index fragments cannot be amplified at the amplification sites if they have not yet been transported there. This is supported by the recitation in (ii) which recites that amplifying occurs at the plurality of amplification sites and, thus, the metes and bounds of the claim cannot be determined.
The rejection of claim 28 is maintained as being indefinite for the recitation of the term “the contacting comprises” such as recited in claim 28, line 1 because it is unclear whether the term refers to amended claim 1(c) and “contacting each subset with transposome complexes”; whether the term refers to amended claim 21 and “contacting the surface comprising the plurality of amplification sites” and/or whether the term refers to some other step of “contacting” and, thus, the metes and bounds of the claim cannot be determined.
Claims 13-16, 18, 19 and 26 are indefinite insofar as they ultimately depend from instant claim 1.
Response to Arguments
Applicant’s arguments filed December 16, 2025 have been fully considered but they are not persuasive. Applicants essentially assert that: (a) claim 1 has antecedent basis for the term “the other compartments” in the term “a first plurality of compartments” (Applicant Remarks, pg. 8, III); (b) regarding claim 1 and the term “remain attached to the transposase,” claim 1 recites “incorporating the first index sequence into at least one strand of the nucleic acid fragment” such that a transposition reaction occurs in claim 1 (Applicant Remarks, pg. 8, V); (c) regarding claim 1 and the term “genome-wide uniform incorporation…chromatin accessibility”, it is well known that chromatin is formed from multiple nucleosomes, the repeating units of a complex of protein and nuclear DNA, such that a person of ordinary skill would recognize the term "genome-wide uniform incorporation of the first index sequences that is not restricted to sites of chromatin accessibility" defines the metes and bounds of the claims (Applicant Remarks, pg. 9, VI); (d) regarding claim 16 and the term “step (i)” the preliminary amendment filed 12/5/2022 include an amendment that revised claim 1 to include step (i) (Applicant Remarks, pg. 9, VII); and (e) regarding claim 28 and the term “the contacting comprises” a person of ordinary skill would recognize that "the contacting comprises" refers to the contacting recited in claim 21 (Applicant Remarks, pg. 10, XIII).
Regarding (a), the term “the other compartments” as recited in claim 1 does not have proper antecedent basis in the claim. The Examiner suggests that Applicant amend claim 1 to recite, for example, “wherein each first index sequence within each compartment of the first plurality of compartments comprises a different nucleotide sequence.”
Regarding (b), regarding the term “remain attached to the transposase” as recited in claim 1, the Examiner notes that there are several different methods by which a first index sequence can be incorporated into a strand of a nucleic acid fragment including, for example, adapter ligation, PCR amplification, tagmentation, etc. Thus, there is no clear recitation in claim 1 to indicate what has occurred, and/or whether all methods of incorporating a first index sequence into a nucleic acid fragment will result in “indexed nucleic acid fragments that remain attached to transposases.” The rejection is maintained.
Regarding (c), regarding the term “genome-wide uniform incorporation…chromatin accessibility” as recited in claim 1 because the term is completely unclear and confusing. Additionally, claim 1 does not recite: (i) nucleosome depletion in the presence of first index sequences as suggested in lines 22-24 (depletion occurs in step b, while incorporation occurs in step d); (ii) any specific reaction that leads to such “genome-wide incorporation”; (iii) sites of chromatin accessibility; and/or (iv) any restrictions on chromatin accessibility, such that it is unclear what the first index sequences are “incorporated into” genome-wide. Moreover, the instant as-filed Specification teaches that nucleosome depletion produces genome-wide uniform coverage that is not restricted to sites of chromatin accessibility (See, as-filed Specification, pg. 7, lines 14-15). Thus, the as-filed Specification does not teach genome-wide uniform incorporation of first index sequences. The rejection is maintained.
Regarding (d), claim 16 is indefinite for the term “step (i)” because claim 16 depends from claims 1, 14 and 15, wherein none of claims 1, 14 and 15 recite a “step (i).” The Examiner suggests that Applicant amend the claim to recite, for example, “wherein the incorporation of the second index sequence as recited in claim 1(i).” The rejection is maintained.
Regarding (e), regarding claim 28 and the term “the contacting comprises,” it is noted that 35 USC 112(b) requires that the claim particularly point out and distinctly claim the subject matter which the applicant regards as his invention. Claim 28 depends from claims 1 and 21, wherein both claims 1 and 21 recite a step directed to “contacting.” Thus, the rejection is maintained.
Claim Rejections - 35 USC § 112(d)
The rejection of claims 16 is maintained under 35 U.S.C. 112(d) as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to
include all the limitations of the claim upon which it depends.
Claim 16 recites (in part): “wherein the incorporation of the second index sequence in step (i) comprises contacting the indexed nucleic acid fragments” in lines 1-2 because claim 16 depends from claim 1, wherein claim 1 does not recite a “step (i)”. Thus, claim 16 is an improper dependent claims for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
Response to Arguments
Applicant’s arguments filed December 16, 2025 have been fully considered but they are not persuasive. Applicants essentially assert that: (a) regarding claim 16 and the term “step (i)” the preliminary amendment filed 12/5/2022 includes an amendment that revised claim 1 to include step (i) (Applicant Remarks, pg. 11, second and third full paragraphs).
Regarding (a), instant claim 16 is an improper dependent claim. Claim 16 depends from claim 1, 14 and 15, where none of claims 1, 14 and 15 recite a “step (i)” [noting that claim 1 recites “(i)”]. Thus, the rejection is maintained.
Claim Rejections - 35 USC § 103
The rejection of claims 1, 7, 13-16, 18, 19, 21 and 26-28 is maintained under 35 U.S.C. 103 as being unpatentable Giresi et al. (hereinafter “Giresi”) (US Patent Application Publication No. 20160060691, published March 3, 2016; International Application WO2014189957, published November 27, 2014; effective filing date May 23, 2013) in view of Arrigoni et al. (hereinafter “Arrigoni”) (Nucleic Acids Research, 2015, 1, 1-13; and Supplementary Data, 2015, 1, 1-10) as evidenced by Gruenwald et al. (hereinafter “Gruenwald”) (US Patent Application Publication No. 20100120098, published May 13, 2010); and Adey et al. (hereinafter “Adey”) (Genome Research, 2014, 24, 2041-2049); and Kaper et al. (hereinafter “Kaper”) (PNAS, 2013, 110(14), 5552-5557); and Cusanovich et al. (hereinafter “Cusanovich”) (Science, 2015, 348(6237), 910-914); and Illumina (Illumina, 2010, 1-5); and Rio et al. (hereinafter “Rio”) (Cold Spring Harbor Protocol, 2010, 1-5); and Vaidyanathan et al. (hereinafter “Vaidyanathan”) (The Journal of Physical Chemistry B, 2014, 118, 2112-2123).
Regarding claim 1 (in part), Giresi teaches a method for analyzing polynucleotides such as genomic DNA, the method comprising: (a) treating chromatin isolated from a population of cells with a transposase and molecular tags to produce tagged fragments of genomic DNA; (b) sequencing a portion of the tagged fragments to produce a plurality of sequence reads; and (c) making an epigenetic map of a region of the genome of the cells by mapping information obtained from the sequence reads to the region, wherein the fragments of defined size range are nucleosome-free fragments (interpreted as a plurality of cells; fragmenting nucleic acids; gDNA; and nucleosome-depleted nuclei, claim 1) (paragraphs [0004]-[0005]). Giresi teaches that the cell sample can be isolated directly from a primary source such as fresh tissues; and/or that cell sample can be isolated directly from frozen tissues or from fixed tissues, wherein primary sources of cell samples include, but are not limited to, cells dissociated from tissues, blood cells, FFPE tissues, bacterial, viral, mitochondria, chloroplast, in vitro assembled protein DNA complexes, neutrophil extracellular traps (interpreted as fixed cells and FFPE tissues as comprising crosslinks, claim 1) (paragraph [0111]). Giresi teaches that the cell sample and/or polynucleotides can be sorted after the molecular tags are inserted into the polynucleotide, and sorting can be performed before the fragments are sequenced (interpreted as distributing a first subsets; and distributing a second subsets, claim 1) (paragraph [0113]). Giresi teaches that the treating step (a) can comprise isolating nuclei from a population of cells (interpreted as isolating cell nuclei); and combining the isolated nuclei with the insertional enzyme complex (interpreted as a transposome complex), wherein the combining results in both lysis of the nuclei to release the chromatin, and production of the tagged fragments of genomic DNA, such that the transposase can be derived from Tn5 transposase, from MuA transposase, or from Vibhar transposase including the use of custom Nextera PCR primers (interpreted as isolating nuclei from a plurality of cells; and a plurality of transposome complexes, each inherently comprising a transposase and two mosaic end sequences on the transposon; and indexed fragments inherently remaining attached to the transposase, claim 1) (paragraphs [0009]; and [0158], lines 1-4). Giresi teaches that all publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference (paragraph [0022]). Giresi teaches that ATAC-seq is a sensitive, accurate probe of open chromatin state, such that: (a) ATAC-seq reaction schematic, wherein transposase (green), loaded with sequencing adapters (red and blue), inserts only in regions of open chromatin (nucleosomes in grey) and generates sequencing library fragments that can be PCR amplified; and (b) approximate reported input material and sample preparation time requirements for genome-wide methods of open chromatin analysis; and (c) a comparison of ATAC-seq to other open chromatin assays at a locus in GM12878 lymphoblastoid cells displaying high concordance, wherein the lower ATAC-seq track was generated from 500 FACS-sorted cells (interpreted as ATAC-seq; encompassing distributing subsets of the nucleic into a plurality of first compartments; interpreting genome-wide methods as genome-wide uniform incorporation of index sequences in sites of chromatin accessibility; fragmenting the nuclei; interpreting PCR amplification to comprise subsets divided into a second plurality of compartments; pooling; and sequencing, claims 1 and 13) (paragraph [0024]; and Figures 1A-C), where it is known that the integrated method for ATAC-seq on 15,000 single cells involves the steps of: tagging nuclei in 96 wells with barcoded transposase complexes, they are pooled, diluted, redistributed 15 to 25 nuclei to each of the 96 wells of a second plate using a cell sorter, the nuclei are lysed, a second barcode is introduced during PCR with indexed primers complementary to the transposase-introduced adapters, all PCR products are pooled and sequenced as evidenced by Cusanovich (pg. 910, col 3, last full paragraph). Giresi teaches ATAC-seq probes chromatin accessibility with transposons, wherein hyperactive Tn5 transposase (Goryshin, J Biol Chem. 1998 273: 7367-7374; Adey, A. et al. Genome Biol 2010 11: R119, loaded in vitro with adapters for high-throughput DNA sequencing, can simultaneously fragment and tag a genome with sequencing adapters (previously described as "tagmentation") (interpreted as forming transposome complexes; fragmenting; tagging, claims 1, 14-16, 18 and 19) (paragraph [0172]-[[0173]). Giresi teaches a method for analyzing chromatin is provided, comprises: (a) treating chromatin isolated from a population of cells with an insertional enzyme complex to produce tagged fragments of genomic DNA, wherein the chromatin is tagmented including being cleaved and tagged in the same reaction using an insertional enzyme such as Tn5 or MuA that cleaves the genomic DNA in open regions in the chromatin and adds adaptors to both ends of the fragments, wherein methods for tagmenting isolated genomic DNA are known in the art (see, e.g., Caruccio Methods Mo!. Biol. 2011 733: 241-55; Kaper et al, Proc. Natl. Acad. Sci. 2013 110: 5552-7; Marine et al, Appl. Environ. Microbiol. 2011 77: 8071-9 and US20100120098) (interpreted as producing a plurality of transposome complexes, each inherently comprising a transposase and two mosaic end sequences on the transposon; fragmentation; generation of indexed fragments; and indexed fragments inherently remaining attached to the transposase, claims 1, 15 and 16) (paragraph [0091], lines 1-13), where it is known that method of tagmentation can result in whole-genome haplotyping as evidenced by Kaper (pg. 5552, col 2; last partial paragraph, lines 1-2); and where it is known that transposase-mediated library construction or “tagmentation” utilizes a hyperactive Tn5 transposase to both fragments and append universal adaptors in a single enzymatic step; and that an inherent property of the Tn5 transposase is that the transposase enzyme remains tightly bound to the target DNA after tagmentation as evidenced by Adey (pg. 2041, col 1; last partial paragraph, lines 1-3 and 12-15); and where it is known that transposons comprises a transferred strand, a non-transferred strand and two end sequence tags for insertion into the DNA fragments as evidenced by Gruenwald (paragraphs [0018]; [0062]; and Figure 4). Giresi teaches that the chromatin used in the method can be made by any suitable method, wherein the nuclei can be isolated, lysed, and the chromatin can be further purified, e.g., from the nuclear envelope; or the chromatin can be isolated by contacting isolated nuclei with the reaction buffer; and/or the isolated nuclei can lyse when it makes contact with the reaction buffer (which comprises insertional enzyme complexes and other necessary reagents), which allows the insertional enzyme complexes access to the chromatin, such that the method can comprise isolating nuclei from a population of cells; and combining the isolated nuclei with the transposase and adaptors, wherein the combining results in both lysis of the nuclei to release of said chromatin and production of the adaptor-tagged fragments of genomic DNA, wherein the chromatin does not require cross-linking as in other methods such as in ChIP-SEQ methods (interpreted as isolating nuclei; generating a pool of indexed nuclei; dissociating the transposase from the indexed fragments; combining indexed nuclei to form a pool of indexed nuclei; and encompassing treatment with SDS; and genome-wide uniform incorporation of index sequences in sites of chromatin accessibility, claim 1) (paragraph [0091], lines 19-35). Giresi teaches that the chromatin has been fragmented and tagged to produce tagged fragments of genomic DNA, at least some of the adaptor tagged fragments are sequenced to produce a plurality of sequence reads, wherein the fragments can be sequenced using any convenient method including being sequenced using Illumina's reversible terminator method, Roche's pyrosequencing method (454), Life Technologies' sequencing by ligation (the SOLiD platform) or Life Technologies' Ion Torrent platform; and examples of such methods are described in the references provided, wherein the primers used can contain a molecular barcode (an "index") so that different pools can be pooled together before sequencing, and the sequence reads can be traced to a particular sample using the barcode sequence (interpreted as generating a pool of indexed nuclei; and sequencing, claim 1k) (paragraph [0092]). Giresi teaches that the sequencing adaptors can further comprise a barcode label including barcode labels comprising a unique sequence, such that the unique sequences can be used to identify the individual insertion events, wherein any of the tags can further comprise fluorescence tags (e.g. fluorescein, rhodamine, Cy3, Cy 5, thiazole orange, etc.) (paragraph [0098]). Giresi teaches that a group of cells were permeabilized, and the chromosomal DNA was transposed with Tn5 transposase, where the cells were kept under conditions that prevented the resulting ATAC-seq fragments from leaving the cell nucleus (i.e. divalent cation was not chelated), and the individual cells were sorted into independent PCR reactions for library preparation, as described above, such that this workflow significantly simplified the workflow for single-cell analysis and provided two additional advantages including (1) this abrogated any effect of the sorting process on the chromatin state because transposition preceded sorting; and (2) it provided more robust ATAC-seq signal, as cells were sorted directly into a PCR master mix and amplified, such that using this workflow, -2,000-5,000 unique ATAC-seq reads per cell were generated, wherein these reads were enriched for known open chromatin sites in GM12878 cells (FIG.19) and displayed characteristic periodic enrichments indicative of nucleosomes (FIG. 20) (interpreting PCR to comprise a plurality of amplification sites, claims 1, 21, 27 and 28) (paragraph 0185]; [0205]-[0206]). Giresi teaches that the cell sample can be permeabilized to allow access for the insertional enzyme, wherein the permeabilization can be performed in a way to minimally perturb the nuclei in the cell sample, such that permeabilization agents include, but are not limited to; NP40, digitonin, tween, streptolysin, cationic lipids, hypotonic shock, ultrasonication, and/or the insertional enzyme can be highly charged, which can allow it to permeabilize through cell membranes (interpreted as encompassing any permeabilization agent including SDS; and unbinding nucleosomes from genomic DNA while maintaining integrity of the isolated nuclei, claim 1) (paragraph [0100]). Giresi teaches that amplifiable DNA fragments suitable for high-throughput sequencing are preferentially generated at locations of open chromatin (FIG. 1a), wherein the entire assay and library construction can be carried out in a simple two-step process involving Tn5 insertion and PCR; and in contrast, published DNase- and FAIRE-seq protocols for assaying chromatin accessibility involve multi-step protocols and many potentially loss-prone steps, such as adapter ligation, gel purification, and crosslink reversal (interpreted as crosslink reversal, claim 1) (paragraph [0173], lines 15-22). Giresi teaches that ATAC-Seq provides accurate and sensitive measure of chromatin accessibility genome-wide (interpreted as genome-wide incorporation, claim 1) (paragraph [0174]). Giresi teaches that ATAC-seq is an information rich assay, allowing simultaneous interrogation of factor occupancy, nucleosome positions in regulatory sites, and chromatin accessibility genome-wide (interpreted as genome-wide incorporation, claim 1) (paragraph [0181], lines 8-11).
Regarding claim 7, Giresi teaches that the cell sample can be isolated directly from a primary source such as fresh tissues; and/or that cell sample can be isolated directly from frozen tissues or from fixed tissues, wherein primary sources of cell samples include, but are not limited to, cells dissociated from tissues, blood cells, formalin-fixed paraffin-embedded (FFPE) tissues, bacterial, viral, mitochondria, chloroplast, in vitro assembled protein DNA complexes, neutrophil extracellular traps (interpreting cell samples such as fixed cells and FFPE tissues as isolated nuclei treated with a crosslinking agent; or a plurality of single cells treated with a crosslinking agent then nuclei isolated; and the crosslinking agent is formaldehyde, claims 1 and 7) (paragraph [0111]).
Regarding claim 13, Giresi teaches that ATAC-seq is a sensitive, accurate probe of open chromatin state, such that: (a) ATAC-seq reaction schematic, wherein transposase (green), loaded with sequencing adapters (red and blue), inserts only in regions of open chromatin (nucleosomes in grey) and generates sequencing library fragments that can be PCR amplified; and (b) approximate reported input material and sample preparation time requirements for genome-wide methods of open chromatin analysis; and (c) a comparison of ATAC-seq to other open chromatin assays at a locus in GM12878 lymphoblastoid cells displaying high concordance, wherein the lower ATAC-seq track was generated from 500 FACS-sorted cells (interpreted as ATAC-seq; encompassing distributing subsets of the nucleic into a plurality of first compartments; interpreting genome-wide methods as genome-wide uniform incorporation of index sequences in sites of chromatin accessibility; fragmenting the nuclei; interpreting PCR amplification to comprise subsets divided into a second plurality of compartments; pooling; and sequencing, claims 1 and 13) (paragraph [0024]; and Figures 1A-C), where it is known that the integrated method for ATAC-seq on 15,000 single cells involves the steps of: tagging nuclei in 96 wells with barcoded transposase complexes, they are pooled, diluted, redistributed 15 to 25 nuclei to each of the 96 wells of a second plate using a cell sorter, the nuclei are lysed, a second barcode is introduced during PCR with indexed primers complementary to the transposase-introduced adapters, all PCR products are pooled and sequenced as evidenced by Cusanovich (pg. 910, col 3, last full paragraph).
Regarding claim 14, Giresi teaches that the term "insertional enzyme complex," as used herein, refers to a complex comprising an insertional enzyme and two adaptor molecules (the "transposon tags") that are combined with polynucleotides to fragment and add adaptors to the polynucleotides, wherein such a system is described in a variety of publications, including Caruccio (Methods Mo!. Biol.2011 733: 241-55) and US20100120098 (Gruenwald), which are incorporated by reference herein (interpreting the transposome complex to inherently comprise a transposon and a transferred strand, claim 14) (paragraph [0078]), where it is known that transposons comprises a transferred strand, a non-transferred strand and two end sequence tags for insertion into the DNA fragments as evidenced by Gruenwald (paragraphs [0018]; [0062]; and Figure 4).
Regarding claims 15 and 16, Giresi teaches the use of an insertional enzyme such as Tn5 or MuA that cleaves the genomic DNA in open regions in the chromatin and adds adaptors to both ends of the fragments (interpreted as producing a plurality of transposome complexes, each inherently comprising a transposase and two mosaic end sequences on the transposon; and indexed fragments inherently remaining attached to the transposase, claims 1, 15 and 16) (paragraph [0091], lines 1-13). Giresi teaches ATAC-seq probes chromatin accessibility with transposons, wherein hyperactive Tn5 transposase (Goryshin, J Biol Chem. 1998 273: 7367-7374; Adey, A. et al. Genome Biol 2010 11: R119, loaded in vitro with adapters for high-throughput DNA sequencing, can simultaneously fragment and tag a genome with sequencing adapters (previously described as "tagmentation") (interpreted as forming transposome complexes; fragmenting; tagging, claims 1, 14-16, 18 and 19) (paragraph [0172]-[[0173]), where it is known that transposase-mediated library construction or “tagmentation” utilizes a hyperactive Tn5 transposase to both fragments and append universal adaptors in a single enzymatic step; and that an inherent property of the Tn5 transposase is that the transposase enzyme remains tightly bound to the target DNA after tagmentation as evidenced by Adey (pg. 2041, col 1; last partial paragraph, lines 1-3 and 12-15).
Regarding claims 18 and 19, Giresi teaches ATAC-seq probes chromatin accessibility with transposons, wherein hyperactive Tn5 transposase (Goryshin, J Biol Chem. 1998 273: 7367-7374; Adey, A. et al. Genome Biol 2010 11: R119, loaded in vitro with adapters for high-throughput DNA sequencing, can simultaneously fragment and tag a genome with sequencing adapters (previously described as "tagmentation") (interpreted as forming transposome complexes; and interpreted as comprising an anchor sequence and a capture sequence claims 1, 14-16, 18 and 19) (paragraph [0172]-[[0173]), wherein it is known that CPT-seq is a means of anchoring unplaced novel human contigs to the reference genome including sequences for anchoring as well as, for detecting misassembled sequences as evidenced by Adey (Abstract; and pg. 407, col 2, last partial paragraph).
Regarding claim 21, Giresi teaches that a group of cells were permeabilized, and the chromosomal DNA was transposed with Tn5 transposase, where the cells were kept under conditions that prevented the resulting ATAC-seq fragments from leaving the cell nucleus (i.e. divalent cation was not chelated), and the individual cells were sorted into independent PCR reactions for library preparation, as described above, such that this workflow significantly simplified the workflow for single-cell analysis and provided two additional advantages including (1) this abrogated any effect of the sorting process on the chromatin state because transposition preceded sorting; and (2) it provided more robust ATAC-seq signal, as cells were sorted directly into a PCR master mix and amplified (interpreting independent PCR reactions to comprise a plurality of amplification sites, claims 1 and 21) (paragraph 0185]; [0205]-[0206]).
Regarding claims 26, Giresi teaches that PCR reactions include qPCR (interpreted as exponential amplification, claim 26) (paragraph [0158], lines 7-8).
Regarding claims 27 and 28, Giresi teaches that the fragments can be sequenced using a high-throughput sequencing technique, wherein the terms "next-generation sequencing" or "high throughput sequencing" refer to the so-called parallelized sequencing-by-synthesis or sequencing-by ligation platforms currently employed by Illumina, Life Technologies, Roche, Ion Torrent, etc. (paragraphs [0070]; and [0095]), where it is known that Illumina sequencing technology including sequencing-by-synthesis (SBS) comprises sequencing template immobilized on a flow cell surface for the generation of single-molecule clusters as evidenced by Illumina (pg. 1, col 1, second full paragraph).
Giresi does not specifically exemplify treating crosslinked nuclei with SDS (claim 1, in part).
Regarding claim 1 (in part), Arrigoni teaches that chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a key technique in chromatin research, where in the initial steps of ChIP-seq, particularly chromatin shearing, are deemed to be exceedingly cell-type-specific, thus impeding any protocol standardization efforts, such that harmonization of ChIP-seq workflows across cell types and conditions is possible when obtaining chromatin from properly isolated nuclei are demonstrated, such that an ultrasound-based nuclei extraction method called nuclei extraction by sonication (NEXSON) is highly effective across various organisms, cells types, and cell numbers, wherein the method has the potential to replace complex cell-type-specific, but largely ineffective, nuclei isolation protocols, such that this fully standardized ChIP-seq allows even small cell numbers (~10,000 cells per ChIP) can be easily processed without application of modified chromatin or library preparation protocols (Abstract). Arrigoni teaches that a typical ChIP-seq workflow includes cell fixation to covalently bind proteins to the DNA, chromatin extraction, immunoprecipitation with the antibody of interest, library preparation and deep sequencing, wherein ChIP-seq procedures including nuclei isolation, nuclei lysis and chromatin sonication vary greatly across protocols and cell types (pg. 1, col 2, first full paragraph). Arrigoni teaches that problems with scarce samples such as patient-derived specimens and sorted cells, small amounts of input materials, and artifacts from PCR amplification derive from the insufficient extraction of nuclei from formaldehyde fixed cells, and we have developed a novel method to solve them, such that ChIP-seq workflow are completely independent of the cell type if chromatin is extracted from properly isolated nuclei (pg. 1, col 2, last partial paragraph; and pg. 2, col 1; first full paragraph, lines 1-6). Arrigoni teaches that NEXSON produces efficient nuclei isolation using a simple and reproducible procedure to generate high quality genome-wide chromatin maps across many different cell types (pg. 2, col 1, first full paragraph; lines 10-14). Arrigoni teaches that all cells types were fixed in 1% methanol-free formaldehyde in D-MEM at room temperature; and that to enhance nuclei extraction, incubation times were prolonged up to 1 hour, and other nuclei extraction buffer with different detergent compositions were tested (pg. 2, col 2, first and second full paragraphs). Arrigoni teaches that all NEXSON-isolated nuclei were resuspended in 1 ml of shearing buffer (10 mM Tris-HCl pH 8; 0.1% SDS; 1 mM EDTA) supplemented with Complete Protease Inhibitor Cocktail EDTA-free, and sheared for 15–20 min to a fragment size distribution of 100–800 bp (Covaris S220 focused ultrasonicator), wherein for protocol comparison, the NEXSON protocol was compared to other protocols including BLUEPRINT, Young and ENCODE (pg. 3, col 1, last partial paragraph), where it is known that SDS disrupts protein-nucleic acid interactions; and that SDS can be used as a solubilization buffer as evidenced by Rio (Abstract, lines 3-4; and pg. 2, Reagents); and it was known that current induced after cells were exposed to SDS increased membrane permeability at 0.2 and 0.14 mM as evidenced by Vaidyanathan (pg. 2118, col 2, last full paragraph). Arrigoni teaches that chromatin was sonicated, de-crosslinked and DNA purified to inspect the size distribution by capillary electrophoresis, wherein NEXSON gives an optimal size distribution in a desired range of 100-800 base pairs, wherein the BLUEPRINT protocol uses high SDS concentration (1% SDS) in the shearing buffer (Supplementary Data, pg. 9, Supplementary Figure S8).
“It is prima facie obvious to combine prior art elements according to known methods to yield predictable results; the court held that, "…a conclusion that a 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 would have yielded nothing more than predictable results to one of ordinary skill in the art. KSR International Co. v. Teleflex Inc., 550 U.S. ___, ___, 82 USPQ2d 1385, 1395 (2007); Sakraida v. AG Pro, Inc., 425 U.S. 273, 282, 189 USPQ 449, 453 (1976); Anderson’s-Black Rock, Inc. v. Pavement Salvage Co., 396 U.S. 57, 62-63, 163 USPQ 673, 675 (1969); Great Atlantic & P. Tea Co. v. Supermarket Equipment Corp., 340 U.S. 147, 152, 87 USPQ 303, 306 (1950)”. Therefore, in view of the benefits of standardizing nuclei isolation and obtaining chromatin as exemplified by Arrigoni, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of ATAC-seq including cell sample permeabilization, the isolation of cell nuclei, and/or the transposition of native chromatin including contacting the cell sample with a permeabilization agents including, but are not limited to; NP40, digitonin, tween, streptolysin, cationic lipids, hypotonic shock, and ultrasonication as disclosed by Giresi to be replaced by, or to include, the NEXSON or BLUEPRINT protocols comprising crosslinking and ultrasonication in a buffer comprising SDS as taught by Arrigoni with a reasonable expectation of success in producing a standardized method for effectively isolating nuclei from any type of fixed cell; in permeabilizing through cell membranes while minimally perturbing the nuclei; and/or in producing epigenetic maps using genome-wide methods of open chromatin analysis. Moreover, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to try choosing from a finite number of identified, predictable potential solutions including replacing the cell permeabilization buffers disclosed by Giresi with other permeabilization techniques and/or buffers such as sonication and/or SDS with a reasonable expectation of success in permeabilizing a cell ample to minimally perturb the cell nuclei, while allowing access for the insertional enzyme. Furthermore, it would have been obvious to try treating a fixed cell sample with ultrasonication and buffer (or treating with SDS alone) because a person of ordinary skill has good reason to pursue the known options within his or her technical grasp; and if this leads to the anticipated success, it is likely the product not of invention but of ordinary skill and common sense.
Thus, in view of the foregoing, the claimed invention, as a whole, would have been obvious to one of ordinary skill in the art at the time the invention was made. Therefore, the claims are properly
rejected under 35 USC §103 as obvious over the art.
Response to Arguments
Applicant’s arguments filed December 16, 2025 have been fully considered but they are not persuasive. Applicants essentially assert that: (a) the Examiner’s reasoning fails to mention the supporting documents Gruenwald, Adey, Kaper, Cusanovich, Rio, and Vaidyanathan and how the primary document Giresi would be modified in view of those supporting documents (Applicant Remarks, pg. 14, first partial paragraph); (b) regions of genomic DNA present in closed chromatin are not converted into library molecules, are not present in a sequencing library, and are not the result of "genome-wide uniform incorporation...that is not restricted to sites of chromatin accessibility" (claim 1, step d); and modifying Giresi in view of Arrigoni does not result in the claimed invention (Applicant Remarks, pg. 14, first full paragraph); and (c) even if the skilled person modified Giresi or Arrigoni to produce libraries that were not limited to open chromatin, such a modification would render Giresi and Arrigoni inoperative for its intended purpose (Applicant Remarks, pg. 14, last full paragraph).
Regarding (a), Applicant’s assertion that the Examiner’s reasoning fails to mention the supporting documents Gruenwald, Adey, Kaper, Cusanovich, Rio, and Vaidyanathan and how the primary document Giresi would be modified in view of those supporting documents, is not found persuasive. As indicated in MPEP 2124:
In certain circumstances, references cited to show a universal fact need not be available as prior art before applicant’s filing date. In re Wilson, 311 F.2d 266, 135 USPQ 442 (CCPA 1962). Such facts include the characteristics and properties of a material or a scientific truism. Some specific examples in which later publications showing factual evidence can be cited include situations where the facts shown in the reference are evidence "that, as of an application’s filing date, undue experimentation would have been required, In re Corneil, 347 F.2d 563, 568, 145 USPQ 702, 705 (CCPA 1965), or that a parameter absent from the claims was or was not critical, In re Rainer, 305 F.2d 505, 507 n.3, 134 USPQ 343, 345 n.3 (CCPA 1962), or that a statement in the specification was inaccurate, In re Marzocchi, 439 F.2d 220, 223 n.4, 169 USPQ 367, 370 n.4 (CCPA 1971), or that the invention was inoperative or lacked utility, In re Langer, 503 F.2d 1380, 1391, 183 USPQ 288, 297 (CCPA 1974), or that a claim was indefinite, In re Glass, 492 F.2d 1228,1232 n.6, 181 USPQ 31, 34 n.6 (CCPA 1974), or that characteristics of prior art products were known, In re Wilson, 311 F.2d 266, 135 USPQ 442 (CCPA 1962)." In re Koller, 613 F.2d 819, 824 n.5, 204 USPQ 702, 706 n.5 (CCPA 1980) (quoting In re Hogan, 559 F.2d 595, 605 n.17, 194 USPQ 527, 537 n.17 (CCPA 1977) (emphasis in original)). See also Amgen Inc. v. Sanofi, 872 F
The Gruenwald, Adey, Kaper, Cusanovich, Rio, and Vaidyanathan references are evidentiary references as indicated in the rejection of record after the phrase “as evidenced by”. Thus, the claims remain rejected.
Regarding (b), although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26USPQ2d 1057 (Fed. Cir. 1993). As noted in MPEP 2112.01(I),
where the claimed and prior art products are identical or substantially identical in structure or composition, or are produced by identical or substantially identical processes, a prima facie case of either anticipation or obviousness has been established. In re Best, 562 F.2d 1252, 1255, 195 USPQ 430, 433 (CCPA 1977). "When the PTO shows a sound basis for believing that the products of the applicant and the prior art are the same, the applicant has the burden of showing that they are not." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990).
MPEP 2112.01(II) states:
"Products of identical chemical composition cannot have mutually exclusive properties." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990). A chemical composition and its properties are inseparable. Therefore, if the prior art teaches the identical chemical structure, the properties applicant discloses and/or claims are necessarily present. Id. (Applicant argued that the claimed composition was a pressure sensitive adhesive containing a tacky polymer while the product of the reference was hard and abrasion resistant. "The Board correctly found that the virtual identity of monomers and procedures sufficed to support a prima facie case of unpatentability of Spada’s polymer latexes for lack of novelty") (underline added).
Moreover, it is noted that none of the references has to teach each and every claim limitation. If they did, this would have been anticipation and not an obviousness-type rejection. One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Applicant’s assertion that regions of genomic DNA present in closed chromatin are not converted into library molecules, are not present in a sequencing library, and are not the result of "genome-wide uniform incorporation...that is not restricted to sites of chromatin accessibility" (claim 1, step d); and modifying Giresi in view of Arrigoni does not result in the claimed invention, is not found persuasive. As an initial matter, the limitation “genome-wide uniform incorporation…chromatin accessibility” is not taught in the as-filed Specification, and it has been rejected as being indefinite under 35 USC 112(b). Moreover, although instant claim 1 recites steps (a) – (k), claim 1 does not recite much of what Applicant asserts including: converting the gDNA present in closed chromatin into library molecules, and/or limitations directed to opening regions of closed chromatin. Chromatin is not recited in claim 1, and there is only single mention of ‘chromatin accessibility’. There are no active steps recited with regard to closed chromatin conversion into library molecules. To that end –
Giresi teaches:
ATAC-Seq provides accurate and sensitive measure of chromatin accessibility genome-wide (interpreted as genome-wide incorporation, claim 1) (paragraph [0174]).
ATAC-seq is an information rich assay, allowing simultaneous interrogation of factor occupancy, nucleosome positions in regulatory sites, and chromatin accessibility genome-wide (interpreted as genome-wide incorporation, claim 1) (paragraph [0181], lines 8-11).
ATAC-seq protocol wherein nuclei are prepared from 50,000 cells, cells are permeabilized, and chromosomal DNA transposed with TN5 transposase.
ATAC-seq is a sensitive, accurate probe of open chromatin state. (a) ATAC-seq reaction schematic, wherein transposase (green), loaded with sequencing adapters (red and blue), inserts only in regions of open chromatin (nucleosomes in grey) and generates sequencing library fragments that can be PCR amplified.
ChIP-Seq and FAIRE-Seq methods for assaying chromatin accessibility.
Arrigoni teaches:
A method of nuclei extraction by sonication (NEXSON) is highly effective across various organisms, cells types, and cell numbers, wherein the method has the potential to replace complex cell-type-specific, but largely ineffective, nuclei isolation protocols, such that this fully standardized ChIP-seq allows even small cell numbers (~10,000 cells per ChIP) can be easily processed without application of modified chromatin or library preparation protocols.
NEXSON produces efficient nuclei isolation using a simple and reproducible procedure to generate high quality genome-wide chromatin maps across many different cell types (pg. 2, col 1, first full paragraph; lines 10-14).
All cells types were fixed in 1% methanol-free formaldehyde in D-MEM at room temperature; and that to enhance nuclei extraction, wherein other nuclei extraction buffer with different detergent compositions were tested including in shearing buffer (10 mM Tris-HCl pH 8; 0.1% SDS; 1 mM EDTA).
The BLUEPRINT protocol uses high SDS concentration (1% SDS) in the shearing buffer.
The combined references of Giresi and Arrigoni teach all of the limitations of the claims including nucleosome-depletion and the generation of nucleosome-depleted nuclei. As a result, there is genome-wide uniform incorporation of the index sequences that are not restricted to sites of chromatin accessibility. Thus, the claims remain rejected.
Regarding (c), Applicant’s assertion that even if the skilled person modified Giresi or Arrigoni to produce libraries that were not limited to open chromatin, such a modification would render Giresi and Arrigoni inoperative for its intended purpose, is not found persuasive. Applicant has provided no analysis and/or reasoning regarding how or why modifying Giresi or Arrigoni would render the references inoperative for their intended purpose. Thus, the claims remain rejected.
Conclusion
Claims 1, 7, 13-16, 18, 19, 21 and 26-28 are rejected.
THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to AMY M BUNKER whose telephone number is (313) 446-4833. The examiner can normally be reached on Monday-Friday (6am-2:30pm).
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/AMY M BUNKER/Primary Examiner, Art Unit 1684