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 .
Office Action: Notice
This action is a second NON-FINAL, in response to Applicant’s Arguments (3/31/2026).
Claim Status
Claims 25-48 (6/16/2022) are under examination.
Priority
Claims 25-48 receive a priority date of 12/16/2019, the filing date of Italian Republic Provisional IT102019000024159.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
The Information Disclosure Statement from 12/23/2025 is considered.
Objections Withdrawn
Specification:
The objections to the specification due to the use of a trademark or tradenames are withdrawn in view of Applicant’s amendments.
The objections to the specification due to the use of hyperlinks are withdrawn in view of Applicant’s amendments.
Drawings:
The specific deficiency regarding nucleotide and/or amino acid sequence disclosures is withdrawn in view of Applicant’s amendments to Drawings (3/31/2026).
Rejections Withdrawn
Claim Rejections - 35 USC § 112(b)
The rejections of claims 25-48 under 35 U.S.C. 112(b) or pre-AIA 35 U.S.C. 112, 2nd paragraph, are withdrawn in view of Applicant’s amendments of claims 25, 27, 37 and 40, to address antecedent basis issues.
Claim Rejections – 35 USC § 102
The 102 (a) (1) and 102 (a) (2) rejections of claims 25-42 and 44 are withdrawn in view of Applicant’s arguments (3/31/2026). Specifically, under further consideration of Applicant’s arguments, they are found to be persuasive that the portions of Belgrader relied upon in the rejection disclose whole genome amplification generally, but do not specifically teach whole genome amplification by DRS-WGA or MALBAC in the specified workflow as recited in independent claim 25.
New Rejections
Claim Rejections - 35 USC § 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 (i.e., changing from AIA to pre-AIA ) 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 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 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.
Claims 25-48 are rejected under 35 U.S.C. 103 as being unpatentable over Belgrader et al. (US PGPub 2018/0179591 A1; published 6/28/2018) and Huo et al. (“Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing”, GIGA SCIENCE, published 2015), in view of Ritter et al. (US PG Pub 2015/0337368 A1; published 11/26/2015); Huang et al. (US PG Pub 2015/0322492 A1; published 11/12/2015); and Fontana et al. (US Patent No. 9938574 B2; issued 4/10/2018).
Regarding claim 25, Belgrader teaches the need for methods, compositions and systems for analyzing genomic and proteomic information from individual cells or a small population of cells including, but not limited to, cancer cells, fetal cells, and immune cells involved in immune responses (Paragraph 5, lines 1-5). Further Belgrader teaches the amplification of the cell's nucleic acids is carried out until the barcoded overlapping fragments within the partition constitute at least 1× coverage of the particular portion or all of the cell's genome, at least 2×, at least 3×, at least 4×, at least 5×, at least 10×, at least 20×, at least 40× or more coverage of the genome or its relevant portion of interest and once the barcoded fragments are produced, they may be directly sequenced on an appropriate sequencing system, e.g., an Illumina Hiseq®, Miseq® or X10 system, or they may be subjected to additional processing, such as further amplification, attachment of other functional sequences, e.g., second sequencing primers, for reverse reads, sample index sequences, and the like (Paragraph 226, lines 1-10). Specifically, Belgrader teaches that the labelling agents may be coupled, through the coupling approaches as described herein, to a reporter oligonucleotide comprising a nucleic acid barcode sequence that permits identification of the labelling agent, as described herein and in some embodiments, the nucleic acid barcode sequence coupled to the labelling agent may comprise a unique molecular identifier (UMI) sequence segment (Paragraph 192, lines 1-20). Further, Belgrader teaches the inclusion of an identifier which may be an oligonucleotide comprising a nucleic acid barcode sequence or binding agent barcode sequence (Paragraph 332, lines 1-5). Belgrader also teaches that prior to co-partitioning, the cells may be incubated with the library of labelling agents, that may represent antibodies to a broad panel of different cell surface features, e.g., receptors, proteins, etc., and which include their associated reporter oligonucleotides where unbound labelling agents may be washed from the cells, and the cells may then be co-partitioned along with the barcode oligonucleotides described above and as a result, the partitions may include the cell or cells, as well as the bound labelling agents and their known, associated reporter oligonucleotides (Paragraph 232, lines 15-25). Belgrader additionally teaches that in some cases, the methods and compositions may be used for sequencing the genome and transcriptome from a single cell in parallel (Paragraph 348, lines 1-5).
Regarding claim 26, Belgrader teaches that the previously described method can be applied to the first amplification products and/or second amplification products, which may be subject to sequencing for sequence analysis (Paragraph 296, lines 15-20).
Regarding claim 27, Belgrader teaches that the previously described method includes a UMI identifying sequence that in some instances, in which the cell may be bound to at least about 5 different labelling agents, at least about 10 different labelling agents, at least about 50 different labelling agents or nucleotides (Paragraph 316m lines 10-15).
Regarding claims 28-31, Belgrader teaches that the previously described method includes a method for RNA expression analysis in individual cells using the methods described, where shown, at operation 602 a cell containing sample is sorted for viable cells, which are quantified and diluted for subsequent partitioning and the individual cells separately co-partitioned with gel beads or droplets bearing the barcoding oligonucleotides as described herein (Figure 6; Paragraph 257, lines 1-5).
Regarding claim 32, Belgrader teaches that the previously described method includes following the generation of amplification products, subsequent operations may include purification (e.g., via solid phase reversible immobilization (SPRI)), further processing (e.g., shearing, ligation of functional sequences, and subsequent amplification (e.g., via PCR)). These operations may occur in bulk (e.g., outside the partition) (Paragraph 296, lines 1-5).
Regarding claims 33-34, Belgrader teaches that the previously described method includes methods, compositions and systems for analyzing individual cells or a small population of cells, including the analysis and attribution of nucleic acids and proteins from and to these individual cells or cell populations (Paragraph 5, lines 1-5).
Regarding claims 35-39, Belgrader teaches that the previously described method includes a labelling agent (e.g., antibody) and 5221 is indirectly (e.g., via hybridization) coupled to an oligonucleotide 5222 comprising a barcode sequence 5223 that identifies the label agent 5221 and the labelling agent 5221 is directly (e.g., covalently bound, bound via a protein-protein interaction, such as with Protein G) coupled to a hybridization oligonucleotide 5232 that hybridizes with sequence 5231 of oligonucleotide 5222, where hybridization of oligonucleotide 5232 to oligonucleotide 5231 couples label agent 5221 to oligonucleotide 5222 (Figure 52B; Paragraph 367, lines 1-10). Further Belgrader teaches that the oligonucleotide 5222 also includes additional sequences (sequence 5224 comprising a reverse complement of a template switch oligo and sequence 5225 comprising a PCR handle) suitable for downstream reactions and FIG. 52B (panel II) also shows an additional oligonucleotide 5226 (e.g., which may have been released from a bead as described elsewhere herein) comprising a barcode sequence 5228, a UMI sequence 5229 and additional sequences (sequence 5227 comprising a sequencing read primer binding site ‘pR1’ and sequence 5220 comprising a template switch oligo) suitable for downstream reactions (Paragraph 367, lines 10-20).
Further, Belgrader teaches that the first individual barcode molecule or the second individual barcode molecule may be capable of coupling to the labelling agent via a third nucleic acid molecule coupled to the labelling agent and the third nucleic acid molecule can be coupled to the labelling agent and comprise a third nucleic acid barcode sequence that identifies the coupled labelling agent (and, thus, a cell surface feature to which the labelling agent is bound), where in a primer extension reaction, the first individual barcode molecule or the second individual barcode molecule can be extended such that a complement of the third barcode sequence is added to the first or second individual barcode molecule and during sequencing, the first or second barcode sequence of these molecules can identify the partition from which the molecules were synthesized and, where a partition comprises a single cell, the third barcode sequence can associate a particular cell surface feature with that single cell (Paragraph 384, lines 1-10).
Regarding claim 40, Belgrader teaches that the previously described method includes and also provided is at least one labelling agent, such as a library of labelling agents, capable of binding to a cell surface feature of interest, where a labelling agent may include, but is not limited to, an antibody, an antibody fragment, a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi-specific antibody, a bi-specific T-cell engager, a T-cell receptor engager, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold, or any combination thereof (Paragraph 317, lines 10-20).
Regarding claims 41-42, 44, Belgrader teaches the need for methods, compositions and systems for analyzing genomic and proteomic information from individual cells or a small population of cells including, but not limited to, cancer cells, fetal cells, and immune cells involved in immune responses (Paragraph 5, lines 1-5). Specifically, Belgrader teaches kits comprising antibody-binding proteins conjugated with reporter oligonucleotides, e.g., in well plates (Paragraph 324, lines 1-5). Further Belgrader teaches the amplification of the cell's nucleic acids is carried out until the barcoded overlapping fragments within the partition constitute at least 1× coverage of the particular portion or all of the cell's genome, at least 2×, at least 3×, at least 4×, at least 5×, at least 10×, at least 20×, at least 40× or more coverage of the genome or its relevant portion of interest and once the barcoded fragments are produced, they may be directly sequenced on an appropriate sequencing system, e.g., an Illumina Hiseq®, Miseq® or X10 system, or they may be subjected to additional processing, such as further amplification, attachment of other functional sequences, e.g., second sequencing primers, for reverse reads, sample index sequences, and the like (Paragraph 226, lines 1-10). Specifically, Belgrader teaches that the labelling agents may be coupled, through the coupling approaches as described herein, to a reporter oligonucleotide comprising a nucleic acid barcode sequence that permits identification of the labelling agent, as described herein and in some embodiments, the nucleic acid barcode sequence coupled to the labelling agent may comprise a unique molecular identifier (UMI) sequence segment (Paragraph 192, lines 1-20). Further, Belgrader teaches the inclusion of an identifier which may be an oligonucleotide comprising a nucleic acid barcode sequence or binding agent barcode sequence (Paragraph 332, lines 1-5). Belgrader also teaches that prior to co-partitioning, the cells may be incubated with the library of labelling agents, that may represent antibodies to a broad panel of different cell surface features, e.g., receptors, proteins, etc., and which include their associated reporter oligonucleotides where unbound labelling agents may be washed from the cells, and the cells may then be co-partitioned along with the barcode oligonucleotides described above and as a result, the partitions may include the cell or cells, as well as the bound labelling agents and their known, associated reporter oligonucleotides (Paragraph 232, lines 15-25). Belgrader additionally teaches that in some cases, the methods and compositions may be used for sequencing the genome and transcriptome from a single cell in parallel (Paragraph 348, lines 1-5).
Belgrader also teaches that the previously described method can be applied to the first amplification products and/or second amplification products, which may be subject to sequencing for sequence analysis (Paragraph 296, lines 15-20).
Belgrader does not teach or suggest performing the recited whole genome amplification using DRS-WGA or MALBAC, as applied to the specific workflow.
Belgrader does not teach or suggest the specific SEQ ID NOs corresponding to the library primers or tagged oligonucleotides, including, SEQ ID NO: 1, 2, 3, 8-15, 16-27, 28-29, 30-37 and/or 38-49.
Huo teaches Multiple Annealing and Looping Based Amplification Cycles (MALBAC) as a commonly used whole genome amplification (WGA) method for single-cell genomic analysis and further teaches that MALBAC provides high genome coverage and effective detection of genomic variations, including single-nucleotide variations (SNVs) and copy number variations (CNVs), making it suitable for downstream sequencing and genomic analysis (Abstract). Specifically, Huo teaches performing whole genome amplification using MALBAC in single-cell samples prior to sequencing and teaches that MALBAC provides efficient genome-wide amplification with sufficient genome coverage and accuracy for detecting genomic variations, thereby demonstrating its suitability for whole genome amplification in genomic sequencing workflows (Figures 1, 4; Background: Paragraphs 1-3; Discussion: Paragraphs 1-3).
Ritter teaches a method for amplifying a target nucleic acid sequence comprising a first amplification using specified first and second primer pairs (Abstract). Specifically, Ritter teaches SEQ ID NO:
PNG
media_image1.png
126
617
media_image1.png
Greyscale
77, a 100% similarity match to SEQ ID NO: 2 of the instant application.
Huang teaches compositions with uniquely designed oligonucleotide primers for identifying a plurality of microorganism in a sample (Abstract). Huang also teaches SEQ ID NO: 44, a 100% similarity match to SEQ ID NO: 8 and 30 of the instant application.
PNG
media_image2.png
193
652
media_image2.png
Greyscale
PNG
media_image3.png
182
619
media_image3.png
Greyscale
Huang also teaches SEQ ID NO: 52, a 100% similarity match to SEQ ID NO: 16 of the instant application.
PNG
media_image4.png
132
615
media_image4.png
Greyscale
Fontana teaches a method for detecting a first and or a second target DNA sequence from a DNA library, differing in that a mutation generates/eliminates a restriction site for a restriction endonuclease (Abstract). Fontana also teaches SEQ ID NO: 1, a 100% similarity match to SEQ ID NO: 29 of the instant application.
It would have been obvious to one of ordinary skill in the art at the time of the invention to employ the MALBAC whole genome amplification technique taught by Huo in the genomic analysis workflow of Belgrader in order to obtain whole genome amplification suitable for downstream sequencing and genomic analysis. One of ordinary skill in the art would have been motivated to do so because Huo teaches that MALBAC provides effective genome-wide amplification with high genome coverage and reliable genomic variation detection in single-cell samples. A person of ordinary skill in the art would have had a reasonable expectation of success because both Belgrader and Hou are directed to single-cell genomic sequencing workflows that rely upon amplified genomic DNA for subsequent sequencing and analysis.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the methods of Belgrader/Huo with the specific primer and oligonucleotide artificial sequences as taught by Ritter, Huang and Fontana because Belgrader expressly teaches that its reporter oligonucleotides may incorporate known primer sequences, amplification handles, index sequences and functional adapters to enable downstream sequencing (Paragraphs 192, 226, 232, 296). Specifically, Ritter, Huang and Fontana each teach primer sequences or oligonucleotide tags that are identical to SEQ ID NOs 2, 8, 16, 29 and 30 of the instant application. One skilled in the art would have been motivated to use these known primers in Belgrader’s modular system in order to facilitate amplification and sequencing with standard sequencing platforms/workflows, as Belgrader specifically instructs the user to select appropriate amplification primers based on the desired sequencing method. Further, substituting the known primer or tagged sequences of Ritter, Huang or Fontana into Belgrader’s reporter oligo design would have represented a routine design choice that predictably yields an amplifiable, sequence-ready construct, and the art provides a reasonable expectation of success because each reference teaches functional primers of conventional length, structure and compatibility with the same sequencing platforms as Belgrader.
Applicant’s Response: The Applicant argues that Belgrader does not teach the recited whole genome amplification methods, namely deterministic restriction-site whole genome amplification (DRS-WGA) or Multiple Annealing and Looping Based Amplification Cycles (MALBAC), and that Belgrader’s generic amplification to achieve genome coverage cannot be equated with these specific amplification techniques. Applicant further argues that Belgrader does not teach simultaneously applying genomic DNA and the tagged oligonucleotide, asserting instead that Belgrader teaches separation of nucleic acid species into different daughter droplets for subsequent analysis.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered and are found partially persuasive, as discussed below.
The rejection under 35 USC 102 has been withdrawn and replaced with the present rejection under 35 USC 103. While the Applicant correctly notes that Belgrader does not disclose performing whole genome amplification using DRS-WGA or MALBAC, Hou teaches MALBAC as a known whole genome amplification technique for single-cell genomic analysis and teaches that MALBAC provides efficient genome-wide amplification and high genome coverage suitable for downstream sequencing applications.
It would have been obvious to one of ordinary skill in the art to employ the MALBAC whole genome amplification method taught by Huo in the genomic analysis workflow of Belgrader to obtain whole genome amplification for downstream sequencing and analysis. As set forth in MPEP 2143, a claimed invention is unpatentable where the prior art would have suggested the claimed subject matter to a person of ordinary skill in the art and provided a reasonable expectation of success. Specifically, Belgrader teaches the claimed barcoded binding-agent and reporter oligonucleotide workflow, while Huo teaches the recited MALBAC amplification method. The combination merely applies a known amplification technique to a known genomic analysis system for its recognized purpose and would have yielded predictable results.
Applicant’s argument regarding simultaneous amplification is likewise unpersuasive because the obviousness rejection does not rely on Belgrader alone. Belgrader teaches co-partitioning cells with associated reporter oligonucleotides and performing parallel genomic analysis workflows, and when considered in combination with Huo, the applied references collectively render the claimed subject matter obvious. See MPEP 2145.
Conclusions
No claim is allowed. As an aside, SEQ ID NOs: 1, 3, 28 and 38-49 are free of the prior art.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ELIZABETH ROSE LAFAVE whose telephone number is (703)756-4747. The examiner can normally be reached Compressed Bi-Week: M-F 7:30-4:30.
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, Heather Calamita can be reached on 571-272-2876. 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.
/ELIZABETH ROSE LAFAVE/
Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684
Prosecution Insights