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 .
Election/Restrictions
Applicant’s election without traverse of Applicant elects: i. fixed biological particle (claim 1), ii. cross linked nucleic acid molecule (claim 2), iii. polyethylene glycol (claim 5), iv. fixed single cell (claim 6) in the reply filed on 7/29/2025 is acknowledged.
Claims 45-47, 52 and 53 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 7/29/2025.
Claims 1-6, 9, 13, 15-16, 24, 27, 30-31 and 35, are being examined.
Priority
The instant application was filed 10/13/2022 and is a continuation of PCT/US2021/027784 , filed 04/16/2021 which claims priority from provisional application 63010768 , filed 04/16/2020 and claims priority from provisional application 63132278 , filed 12/30/2020.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 7/11/2023, 10/20/2023, 1/25/2024, 4/8/2024 are being considered by the examiner.
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.
Drawings
The drawings fired 10/13/2023 are in color. Color photographs and color drawings are not accepted in utility applications unless a petition filed under 37 CFR 1.84(a)(2) is granted. Any such petition must be accompanied by the appropriate fee set forth in 37 CFR 1.17(h), one set of color drawings or color photographs, as appropriate, if submitted via the USPTO patent electronic filing system or three sets of color drawings or color photographs, as appropriate, if not submitted via the via USPTO patent electronic filing system, and, unless already present, an amendment to include the following language as the first paragraph of the brief description of the drawings section of the specification:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2).
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-6, 9, 13, 15-16, 24, 27, 30-31 and 35, rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 b) recites, “subjecting said partition to a condition sufficient to generate a nucleic acid molecule comprising said nucleic acid sequence coupled to said nucleic acid barcode molecule.” The recitation of, “condition sufficient to generate” suggests there are condition not sufficient to generate. The specification provides no standard to differentiate condition sufficient to generate” from condition not sufficient to generate. Thus, the metes and bounds are unclear as to what is required of “condition sufficient to generate.
Claim 1 d) recites, “subjecting said released nucleic acid molecule comprising said nucleic acid sequence coupled to said nucleic acid barcode molecule to a condition sufficient to extend said nucleic acid barcode molecule.” The recitation of, “condition sufficient to extend” suggests there are condition not sufficient to extend. The specification provides no standard to differentiate condition sufficient to extend” from condition not sufficient to extend. Thus, the metes and bounds are unclear as to what is required of “condition sufficient to extend.”
Claim 2 recites, “cross- linked nucleic acid molecule, a cross-linked ribonucleic acid (RNA) molecule, or a cross-linked messenger RNA (mRNA) molecule.” The metes and bounds are unclear what is required of cross- linked nucleic acid molecule, a cross-linked ribonucleic acid (RNA) molecule, or a cross-linked messenger RNA (mRNA) molecule. While the specification teaches, “[000104] Herein, "crosslinking" means connecting or attaching two or more separate substances to each other. The connecting or attaching is due to formation of crosslinks. In some examples, crosslinking refers to formation of chemical bonds between two or more atoms in a molecule or in different molecules. In some examples, nucleic acid molecules may be crosslinked. A nucleic acid molecule that is not crosslinked maybe said to be "unlinked."” It is unclear what the recited nucleic acids are crosslinked to.
Claim 15 recites, “a capture sequence configured to anneal to a nucleic acid molecule.” The recitation of “configured to anneal to a nucleic acid molecule” suggest there are capture sequence which are not configured to anneal to a nucleic acid molecule. The specification and claims provide a standard to differentiate capture sequence which are not configured to anneal to a nucleic acid molecule from capture sequence which are configured to anneal to a nucleic acid molecule. Thus, the metes and bounds are unclear.
Claim 16 recites, “subjecting said partition to a condition sufficient to hybridize a template switching oligonucleotide (TSO).” The recitation of subjecting said partition to a condition sufficient to hybridize a template switching oligonucleotide (TSO) suggests there are subjecting said partition to a condition not sufficient to hybridize a template switching oligonucleotide (TSO). The specification and claims provide no standard to differentiate the two. Thus, the metes and bounds are unclear.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 1-6, 9, 13, 15-16, 24, 27, 30-31 and 35is/are rejected under 35 U.S.C. 103 as being unpatentable over Hindson (US20150376609), Abate(WO2015/200717), Kim (ACS Appl. Mater. Interfaces 2016, 8, 33521−33528)
The specification teaches, “[00088] Herein, "barcode" generally refers to a label, or identifier, that conveys or is capable of conveying information about an analyte. A barcode can be part of an analyte. A barcode can be independent of an analyte. A barcode can be a tag attached to an analyte (e.g., nucleic acid molecule) or a combination of the tag in addition to an endogenous characteristic of the analyte (e.g., size of the analyte or end sequence(s)). A barcode may be unique. Barcodes can have a variety of different formats. For example, barcodes can include polynucleotide barcodes; random nucleic acid and/or amino acid sequences; and synthetic nucleic acid and/or amino acid sequences. A barcode can be attached to an analyte in a reversible or irreversible manner. A barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before, during, and/or after sequencing of the sample. Barcodes can allow for identification and/or quantification of individual sequencing-reads.” Thus, the broadest reasonable interpretation of a barcode encompasses nucleic acid sequences specific for target nucleic acid, such as a primer sequence.
With regards to claim 1, Hindson teaches, “[0006] An aspect of the disclosure provides a method of analyzing nucleic acids from cells that includes providing nucleic acids derived from an individual cell into a discrete partition; generating one or more first nucleic acid sequences derived from the nucleic acids within the discrete partition, which one or more first nucleic acid sequences have attached thereto oligonucleotides that comprise a common nucleic acid barcode sequence; generating a characterization of the one or more first nucleic acid sequences or one or more second nucleic acid sequences derived from the one or more first nucleic acid sequences, which one or more second nucleic acid sequences comprise the common barcode sequence; and identifying the one or more first nucleic acid sequences or one or more second nucleic acid sequences as being derived from the individual cell based, at least in part, upon a presence of the common nucleic acid barcode sequence in the generated characterization.” Hindson teaches, “ 0065] In other aspects, in addition to or as an alternative to droplet-based partitioning, cells may be encapsulated within a microcapsule that comprises an outer shell or layer or porous matrix in which is entrained one or more individual cells or small groups of cells, and may include other reagents. Encapsulation of cells may be carried out by a variety of processes. In general, such processes combine an aqueous fluid containing the cells to be analyzed with a polymeric precursor material that may be capable of being formed into a gel or other solid or semi-solid matrix upon application of a particular stimulus to the polymer precursor. Such stimuli include, e.g., thermal stimuli (either heating or cooling), photo-stimuli (e.g., through photo-curing), chemical stimuli (e.g., through crosslinking, polymerization initiation of the precursor (e.g., through added initiators), or the like.” Hindson teaches, “[0067] For example, in the case where the polymer precursor material comprises a linear polymer material, e.g., a linear polyacrylamide, PEG, or other linear polymeric material, the activation agent may comprise a cross-linking agent, or a chemical that activates a cross-linking agent within the formed droplets.”
Hindson does not specifically teach the cells are fixed.
However, Abate teaches, “[00187] RT-PCR carried out on a fixed cell encapsulated in a microdroplet can be carried out by first diluting the microdroplet and performing the RT-PCR reaction on a sample of the diluted microdroplet. Such dilution of the cellular sample can help to limit any cellular compounds that would interfere with RT-PCR. In other embodiments, the RT-PCR reagents are added directly to the microdroplet containing the fixed cell in a "one pot" reaction without any dilution of sample. In certain embodiments, fixed cells are solubilized prior to the RT-PCR using proteases and detergents.”
Kim teaches, “First, in the initial stage of PCR, PEG-nGO decreases primer dimerization and nonspecific annealing between primers and template DNA by adsorbing excess primers. Second, in the later stage of PCR, when amplified PCR products have accumulated, PEG-nGO attenuates the reannealing of amplified dsDNA by preferential binding to denatured ssDNA, thus facilitating the annealing of primers to template strands. Third, owing to its preferential interaction with ssDNA, PEG-nGO can shorten the time required for denaturation by facilitating the melting of amplified dsDNA, which serves as a template for subsequent rounds of PCR. Hence, PEG-nGO enhances the specificity and efficiency of PCR by preferential binding to ssDNA, which is analogous to the role of SSBs in DNA replication in vivo.”
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to use the encapsulated fixed cells in the method of Hindson. The artisan would be motivated as Abate suggests the use of fixed cells and fix cells allow for examination of samples preserved from surgery or other surgical samples. The artisan would be motivated to provide PEG in the sample as Kim teaches it decreases primer dimerization and nonspecific annealing between primers and template DNA by adsorbing excess primers. Second, in the later stage of PCR, when amplified PCR products have accumulated, PEG-nGO attenuates the reannealing of amplified dsDNA by preferential binding to denatured ssDNA, thus facilitating the annealing of primers to template strands. Third, owing to its preferential interaction with ssDNA, PEG-nGO can shorten the time required for denaturation by facilitating the melting of amplified dsDNA, which serves as a template for subsequent rounds of PCR. Hence, PEG-nGO enhances the specificity and efficiency of PCR by preferential binding to ssDNA, which is analogous to the role of SSBs in DNA replication in vivo.” The artisan would have a reasonable expectation of success as the artisan is merely using known techniques.
With regards to claim 2, Abate teaches fixed cells contain crosslinked RNA.
With regards to claim 3-4, Abate teaches the use of protease which will remove crosslinks.
With regards to claims 5, Hindson and Kim teach use of PEG.
With regards to claim 6, Hindson teaches single cell analysis (I. 0044-0048) Abate teaches, “[0053] FIG. 32 shows an embodiment of a single cell RT-PCR microfluidic device as described herein.”
With regards to claim 9, Abate teaches , “In some embodiments, the encapsulated viruses are subjected to one or more virus lysing techniques, such as proteinase k digestion or thermal lysis.” (0010)
With regards to claim 13, Hindson teaches, “[0072] Additional reagents may also be co-partitioned with the cells, such as endonucleases to fragment the cell's DNA, DNA polymerase enzymes and dNTPs used to amplify the cell's nucleic acid fragments and to attach the barcode oligonucleotides to the amplified fragments. Additional reagents may also include reverse transcriptase enzymes, including enzymes with terminal transferase activity, primers and oligonucleotides, and switch oligonucleotides (also referred to herein as “switch oligos”) which can be used for template switching. In some cases, template switching can be used to increase the length of a cDNA. In one example of template switching, cDNA can be generated from reverse transcription of a template, e.g., cellular mRNA, where a reverse transcriptase with terminal transferase activity can add additional nucleotides, e.g., polyC, to the cDNA that are not encoded by the template, such, as at an end of the cDNA. Switch oligos can include sequences complementary to the additional nucleotides, e.g. polyG. The additional nucleotides (e.g., polyC) on the cDNA can hybridize to the sequences complementary to the additional nucleotides (e.g., polyG) on the switch oligo, whereby the switch oligo can be used by the reverse transcriptase as template to further extend the cDNA. Switch oligos may comprise deoxyribonucleic acids, ribonucleic acids, modified nucleic acids including locked nucleic acids (LNA), or any combination.”
With regards to claim 15, Hindson teaches the use of a capture oligonucleotide (0121).
With regards to claim 16, Hindson teaches, “0072] Additional reagents may also be co-partitioned with the cells, such as endonucleases to fragment the cell's DNA, DNA polymerase enzymes and dNTPs used to amplify the cell's nucleic acid fragments and to attach the barcode oligonucleotides to the amplified fragments. Additional reagents may also include reverse transcriptase enzymes, including enzymes with terminal transferase activity, primers and oligonucleotides, and switch oligonucleotides (also referred to herein as “switch oligos”) which can be used for template switching. In some cases, template switching can be used to increase the length of a cDNA. In one example of template switching, cDNA can be generated from reverse transcription of a template, e.g., cellular mRNA, where a reverse transcriptase with terminal transferase activity can add additional nucleotides, e.g., polyC, to the cDNA that are not encoded by the template, such, as at an end of the cDNA. Switch oligos can include sequences complementary to the additional nucleotides, e.g. polyG. The additional nucleotides (e.g., polyC) on the cDNA can hybridize to the sequences complementary to the additional nucleotides (e.g., polyG) on the switch oligo, whereby the switch oligo can be used by the reverse transcriptase as template to further extend the cDNA. Switch oligos may comprise deoxyribonucleic acids, ribonucleic acids, modified nucleic acids including locked nucleic acids (LNA), or any combination.”
With regards to claim 24, Hindson teaches photo-labile linkages.
With regards to claim 27, Hindson teaches, “[0052] As used herein, in some aspects, the partitions refer to containers or vessels (such as wells, microwells, tubes, through ports in nanoarray substrates, e.g., BioTrove nanoarrays, or other containers). In many some aspects, however, the compartments or partitions comprise partitions that are flowable within fluid streams. These partitions may be comprised of, e.g., microcapsules or micro-vesicles that have an outer barrier surrounding an inner fluid center or core, or they may be a porous matrix that is capable of entraining and/or retaining materials within its matrix. In some aspects, however, these partitions comprise droplets of aqueous fluid within a non-aqueous continuous phase, e.g., an oil phase.”
With regards to claim 30, Hindson teaches, “sequencing the oligonucleotide reporter groups and attached barcodes.” (0020)
With regards to claim 31, Hindson teaches, “[0007] In some embodiments, the discrete partition is a discrete droplet. In some embodiments, the oligonucleotides are co-partitioned with the nucleic acids derived from the individual cell into the discrete partition. In some embodiments, at least 10,000, at least 100,000 or at least 500,000 of the oligonucleotides are co-partitioned with the nucleic acids derived from the individual cell into the discrete partition.”
The courts have haled that rearrangement of steps is obvious in the absence of unexpected results. Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959) (Prior art reference disclosing a process of making a laminated sheet wherein a base sheet is first coated with a metallic film and thereafter impregnated with a thermosetting material was held to render prima facie obvious claims directed to a process of making a laminated sheet by reversing the order of the prior art process steps.). See also In re Burhans, 154 F.2d 690, 69 USPQ 330 (CCPA 1946) (selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results); In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930) (Selection of any order of mixing ingredients is prima facie obvious.).
With regards to claim 35, Abate teaches, “The use of fixed and/or permeabilized cells (as discussed in greater detail below) also allows for increased levels of multiplexing.”
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to fix cells prior to the method. The artisan would be motivated as Abate suggests the use of “fixed cells.” The artisan would have a reasonable expectation of success as the artisan is merely using known methods in a logical order.
Summary
No claims are allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Chakrabarti (Nucleic Acids Research (2001) volume 29, pages 2377-2381)
Chomczynsk (BioTechniques 40:454-458 (April 2006) doi 10.2144/000112149)
Any inquiry concerning this communication or earlier communications from the examiner should be directed to STEVEN C POHNERT PhD whose telephone number is (571)272-3803. The examiner can normally be reached Monday- Friday about 6:00 AM-5:00 PM, every second Friday off.
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, Anne Gussow can be reached at (571)272-6047. 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.
/Steven Pohnert/ Primary Examiner, Art Unit 1683