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 .
DETAILED ACTION
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office Action.
Status of Claims
Claims 1-4, 6-9, 14, 17, 20-25, 27-29, 32-37 and 39-42 are currently pending in the instant application. Claims 1, 2, 4 and 17 have been amended by Applicants’ amendment filed 06-02-2026. Claim 18 has been canceled by Applicants’ amendment filed 06-02-2026. No claims have been added by Applicants’ amendment filed 06-02-2026.
Applicant's election of Group I with traverse of claims 1-9, 14-19, 32, 34 and 35, directed to a method for preparing a sequencing library; and the election of Species without traverse as follows:
Species (A): wherein processing comprising contacting subsets with reverse transcriptase (claim 2);
Species (B): wherein the primer comprises a poly-T nucleotide sequence (claim 3);
Species (C): wherein the predetermined RNA nucleic acids are mRNA (claim 14);
Species (D): wherein the nucleotide label comprises a nucleotide analog, a hapten-labeled nucleotide, etc. (claim 17);
Species (E): wherein the predetermined condition comprises exposure to an agent (claim 21);
Species (F): wherein the agent comprises a protein, a non-ribosomal protein, etc. (claim 22);
Species (G): wherein distributing comprises dilution (claim 28);
Species (H): wherein adding comprises contacting nucleic acid fragments comprising one or more index sequence (claim 31);
Species (I): wherein the compartment comprises a well or a droplet (claim 34);
Species (J): wherein the primer comprises RNA nucleic acids (claim 45);
Species (K): wherein the primer comprises a poly-T nucleotide sequence that anneals to mRNA poly(A) tail (claim 46);
Species (L): wherein the label comprises a nucleotide analog, a hapten-labeled nucleotide, etc. that can be modified (claim 57);
Species (M): adding one or more of the first, second or third compartment comprises contacting nucleic acid fragments with a transposome complex (claim 69); and
Species (N): all compartments are wells or droplets (instant claim 71), in the reply filed on July 13, 2022 was previously acknowledged.
Please Note: Upon reconsideration, the Examiner has rejoined instant claims 2-4 as reading on the elected species of Group I.
Claims 10-13, 20-25, 31 and 36-86 were previously withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on July 13, 2022.
Claims 6-9, 14, 27, 29, 32, 33 and 35 were previously withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected species, there being no allowable generic or linking claim.
The restriction requirement was deemed proper was made FINAL.
The claims will be examined insofar as they read on the elected species.
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 1-4, 17, 28 and 34 are under consideration to which the following grounds of rejection are applicable.
Priority
The present application filed December 20, 2019 is a 35 U.S.C. 371 national stage filing of
International Application No. PCT/US2019/035422, filed June 4, 2019; which claims the benefit of US
Provisional Patent 62/821,678, filed March 21, 2019; and US Provisional Patent 62/680,259, filed June 4,
2018.
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 disclosures of the prior-filed applications including US Provisional Patent Application 62/680,259, filed June 4, 2018; and US Provisional Patent Application 62/821,678, filed March 21, 2019, which 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: “incorporating individual labeled nucleotides into RNA as RNA is synthesized in the subsets of nucleic or cells to result in RNA comprising the labeled RNA nucleic acids and unlabeled pre-existing RNA nucleic acids” in lines 5-7.
Therefore, the priority date for the presently claimed invention is June 4, 2019, the filing date of PCT/US2019/035422.
Response to Arguments
Applicant’s arguments filed June 2, 2026 have been fully considered but they are not persuasive. Applicants essentially assert that: (a) US Provisional Patent Applications 62/680,259 and 62/821,678 provide adequate support or enablement for the phrase "incorporating a label into RNA as it is synthesized in the subsets of nuclei or cells to result in labeled RNA nucleic acids and unlabeled pre-existing RNA nucleic acids" as recited in claim 1, such that in 62/821,678 support can be found at pg. 2, lines 8-11; pg. 7, lines 3-7 and 17-18; pg. 113, penultimate paragraph), such that the priority date is March 21, 2019 (Applicant Remarks, pg. 1, Priority through pg. pg. 3, first partial paragraph).
Regarding (a), the Examiner disagrees that support for the phrase can be found in the cited portions of US Provisional Patent Application 62/821,678 for the phrase “incorporating individual labeled nucleotides into RNA as RNA is synthesized in the subsets of nucleic or cells to result in RNA comprising the labeled RNA nucleic acids and unlabeled pre-existing RNA nucleic acids” in lines 5-7. It is noted that two of the four paragraphs cited are very broad teachings (e.g., “adding compartment-specific index to the nucleic acid fragments”; “current methods capture a snapshot of cell state…labeling newly synthesized RNA allows capture…or any single-cell indexing method”; “various methods exist for labeling newly synthesized nucleic acid so it can be distinguished from previously existing nucleic acid”; and “by labeling newly synthesized mRNA with 4-thiouridine…its past state (past state memory)”. Applicant has not indicated where support can be found for the complete and specific step of “incorporating individual labeled nucleotides into RNA as it is synthesized in the subsets of nuclei or cells. The Examiner contends that the step as recited in instant claim 1b of labeling nucleotides as RNA is synthesized (incorporating a labeled nucleotide while RNA is being transcribed) is clearly distinct from labeling “newly synthesized RNA” (labeling nucleotides in RNA that has already been synthesized). Moreover, there is no teaching in the provisional patent applications that these steps refer to (or read on) the same process. There is no teaching that a labeled nucleotide is incorporated into RNA as RNA is being synthesized. Thus, the priority date for the presently claimed invention is June 4, 2019.
Withdrawn Objections/Rejections
Applicants’ amendment and arguments filed June 2, 2026 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.
Claim Rejections - 35 USC § 103
The rejection of claims 1, 17, 18, 28 and 34 is withdrawn under 35 U.S.C. 103 as being unpatentable over unpatentable Seelig et al. (hereinafter “Seelig”) (US Patent No. 10900065, issued January 26, 2021; effective filing date November 13, 2015; of record) in view of New England Biolabs (Instruction Manual, 2016, 1-25); as evidenced by NEBNext (Illumina, 2025, 1-5; of record); and Boone et. al. (hereinafter “Boone”) (Nucleic Acids Research, March 2018, 46(6), 2701-2721; of record).
Seelig does not specifically exemplify incorporating a label as the RNA is synthesized.
In view of the withdrawn rejection, Applicant’s arguments are rendered moot.
The rejection of claims 1, 17, 18, 28 and 34 is withdrawn under 35 U.S.C. 103 as being unpatentable over unpatentable Cao et al. (hereinafter “Cao”) (bioRxiv Preprint, Feb 2017, 1-35; of record) in view of New England Biolabs (Instruction Manual, 2016, 1-25); as evidenced by NEBNext (Illumina, 2025, 1-5); and Boone et. al. (hereinafter “Boone”) (Nucleic Acids Research, March 2018, 46(6), 2701-2721).
The combined references of Cao and NEB do not specifically exemplify incorporating a label as the RNA processing to comprise labeled RNA and unlabeled pre-existing RNA.
In view of the withdrawn rejection, Applicant’s arguments are rendered moot.
Maintained Objections/Rejections
Claim Rejections - 35 USC § 112(b)
The rejection of claim 17 is maintained, and claims 2 and 4 are newly rejected, under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter
which applicant regards as the invention.
Claim 2 is indefinite for the recitation of the term “adding of step (c) comprises:…or the unlabeled pre-existing RNA” such as recited in claim 2, lines 1-8 because claim 1, lines 16-17 already recites what “adding” comprises (e.g., adding comprises ligation, primer extension…or a combination thereof), such that dependent claim 2 cannot recite that “adding” comprises something different than what is recited in the independent claim. Moreover, claim 2 is indefinite for the recitation of the term “step (c)” because claim 2 depends from instant claim 1, wherein claim 1 does not recite a “step” and, thus, the metes and bounds of the claim cannot be determined. The Examiner suggests that Applicant amend the claim 2 to recite, for example, wherein adding in (c) further comprises…”
Claim 2 is indefinite for the recitation of the term “the primer extension” such as recited in claim 2, lines 4-5. There is insufficient antecedent basis for the term “the primer extension” in the claim. Moreover, claim 2 depends from instant claim 1, wherein claim 1 does not recite primer extension and, thus, the metes and bounds of the claim cannot be determined.
Claim 2 is indefinite for the recitation of the term “the other strand” such as recited in claim 2, line 7. There is insufficient antecedent basis for the term “the other strand” in the claim.
Claim 4 is indefinite for the recitation of the term “a predetermined DNA nucleic acid or a predetermined RNA nucleic acid” such as recited in claim 4, line 4 because claim 4 depends from instant claims 1-3, wherein claim 1-3 do not recite the presence of a predetermined DNA nucleic acid or the presence of a predetermined RNA nucleic acid and, thus, the metes and bounds of the claim cannot be determined.
Claim 17 is indefinite for the recitation of the term “click-functionalized nucleotide” such as recited in claim 17, lines 2-3 because it is unclear whether the term refers to nucleotides that have been functionalized via click reactions, and/or whether the term refers to functional groups on the nucleotide that can undergo a click reaction. Moreover, it is unclear what nucleotide functionalizations are encompassed by the term “click-functionalized nucleotide” and whether the term refers to nucleotides functionalized with azides, alkynes, carbonyls, halogens, aromatic rings, triazoles, alcohols, ethers, benzimidazoles, etc. as evidenced by Fantoni (Abstract; and pg. 7125, Figure 2) and Kolb (pg. 1229 through pg. 1230, col 1, first partial paragraph; and Figure 1), and/or functionalized with amines, alcohols, sulfonyls, Ar-OSO2F, SO2F, etc. as evidenced by Guo (Abstract; and pg. 1, Scheme 1) and, thus, the metes and bounds of the claim cannot be determined.
Claim Rejections - 35 USC § 102
The rejection of claims 1, 17, 28 and 34 is maintained, and claims 2-4 are newly rejected ,under 35 U.S.C. 102(a1)/102(a2) as being anticipated by Cao et al. (hereinafter “Cao”) (Seattle Organism Molecular Atlases, 2018, 1-9).
Regarding claims 1-4, 17, 28 and 34, Cao teaches a workflow comprising: Steps (1)-(4): nuclei extraction from tissue, nuclei fixation by paraformaldehyde, permeabilization, sonication and filtering, indexed reverse transcription, pooling, and redistributing:
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(5) Indexed hairpin ligation, pool and redistribute:
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(6) Second strand synthesis, tagmentation, purification, USER treatment, indexed PCR:
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; and
(7) Library purification and sequencing (interpreted as including hairpin ligation duplex, claim 1) (pg. 1, entire page). Cao teaches Step 5: Reverse Transcription, wherein each well of 4 x 96 well plate comprises 80,0000 nuclei in nuclei buffer, dNTP, and indexed oligo-dT, incubate, prepare the reverse transcription reaction mix, start RT reaction, after the reaction add NBB into each well, pool the nuclei from all wells, and pellet the nuclei (interpreted as providing compartments; and incorporating individual labeled nucleotides into RNA as RNA is synthesized in the subset of nuclei; including wells, claims 1, 17, 18 and 34) (pg. 5, Step 5; and pg. 6, Step 5). Cao teaches Step 6: Ligation, wherein cells are resuspended in NSB in the wells of 4 x 96 well plates, indexed ligation primers are added into each well, ligate, pool the nuclei from all wells, add another NBB to the nuclei mix, pellet the nuclei, dump the supernatant, resuspend the cells in NBB, filter nuclei, count the nuclei concentration with hemacytometer, distribute the diluted nuclei (in NBB) into several 96 well plates (interpreted as processing the RNA; first compartment specific index; adding comprising ligation; combining and pooling; distributing subsets of pooled indexed nuclei or cells into second compartments; adding indexed nucleic acids by ligating a hairpin ligation duplex; including wells; combining the dual indexed nuclei or cells; and dilution, claims 1-4 and 28) (pg. 6, Step 6; and pg. 7, Step 6).
Cao meets all the limitations of the claims and, therefore, anticipates the claimed invention.
Response to Arguments
Applicant’s arguments filed June 2, 2026 have been fully considered but they are not persuasive. Applicants essentially assert that: (a) there is nothing in the Cao document that indicates that it was published in 2018 and the links suggest that the Figures were published in 2019, such that it appears that the document was publically available sometime during 2019 (Applicant Remarks, pg. 22-24).
Regarding (a), please note that the link indicates that the Cao reference is version 3. Additionally, the Protocol clearly indicates that the experimental lab protocol was released on September 16, 2018 (See; Release Version: 09/16/2018), while it appears that initial datasets were released in GitHub on 12/05/2018. Please see the Protocol (in part), below:
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Moreover, the GitHub link indicates that the files for the process pipeline were uploaded 8 years ago (e.g., 2018). As shown below (in part):
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Thus, the claims remain rejected.
Double Patenting
The rejection of claims 1, 17, 28 and 34 is maintained, and claims 2-4 are newly rejected, on the ground of nonstatutory double patenting as being unpatentable over: Claims 1-24 of U.S. Patent No. 11981891 for the reasons of record.
Response to Arguments
Applicant’s arguments filed June 2, 2026 have been fully considered but they are not persuasive. Applicants essentially assert that: (a) Applicant traverses the rejection, such that upon an indication of otherwise allowable subject matter and in the event this rejection is maintained, Applicant will provide an appropriate response (Applicant Remarks, pg. 24, last full paragraph).
Regarding (a), Applicant did not specifically indicate how the claims of the copending applications recited supra are patentably distinct from the instant claims as required by 37 CFR 1.111(b). Thus, the claims remain rejected for the reasons already of record.
New Objections/Rejections
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 2 and 4 are rejected 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. This is a new rejection necessitated by amendment of the claims in the response filed 06-02-2026.
Claim 2 recites (in part): “wherein the adding step (c) comprises: contacting each of the subsets of nuclei or cells…or the unlabeled pre-existing RNA” such as recited in claim 2, lines 1-8 because claim 2 depends from instant claim 1, wherein claim 1 does not recite a “step” (c) and/or primer extension. Thus, claim 2 is an improper dependent claim 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 4 recites (in part): “wherein the processing the labeled RNA and the unlabeled preexisting RNA further comprises…or a predetermined RNA nucleic acid” such as recited in claim 4, lines 1-4 because claim 4 depends from instant claims 1-3, wherein claims 1-3 do not recite a predetermined DNA nucleic acid or a predetermined RNA nucleic acid. Thus, claim 4 is an improper dependent claim 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.
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 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 set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-4, 17, 28 and 34 are rejected under 35 U.S.C. 103 as being unpatentable over Seelig et al. (hereinafter “Seelig”) (US Patent No. 10900065, issued January 26, 2021; effective filing date November 13, 2015; of record) in view of Jao et al. (hereinafter “Jao”) (PNAS, 2008, 105(41), 15779-15784); and further in view of Saxonov et al. (hereinafter “Saxonov”) (US Patent No. 10190115, issued January 29, 2019; filed September 22, 2014) as evidenced by New England Biolabs (Instruction Manual, 2016, 1-25; of record). This is a new rejection necessitated by amendment of the claims in the response filed 06-02-2026.
Regarding claim 1 (in part), Seelig teaches methods of uniquely labeling or barcoding molecules within a cell, a plurality of cells, and/or a tissue, wherein the molecules to be labeled can include, but are not limited to, RNAs, DNAs, cDNAs, proteins, peptides and/or antigens, wherein Figure 1 depicts ligation of nucleic acid tags to form a label or a barcode (interpreting nucleotides that make up RNA and that can be modified by chemical reaction as incorporating individual labeled nucleotides into RNA within a cell or nuclei as it is synthesized; including by ligation, claim 1) (Abstract; and col 1, lines 60-61; and Figure 1). Seelig teaches that next generation sequencing (NGS) can be used to identify and/or quantify individual transcripts from a sample of cells (col 1, lines 32-34). Seelig teaches that the plurality of cells can be re-pooled and the method can be repeated any number of times, adding more tags to the cDNAs creating a set of nucleic acid tags that can act as a barcode (interpreted as processing to comprise a first and second compartment-specific barcode; pooling cells to generate pooled indexed nuclei or cells, claim 1) (col 6, lines 42-45). Seelig teaches that in an aspect of the disclosure relates to methods of labeling nucleic acids in a first cell, wherein the method can comprise: (a) generating complementary DNAs (cDNAs) within a plurality of cells comprising the first cell by reverse transcribing RNAs using a reverse transcription primer comprising a 5' overhang sequence; (b) dividing the plurality of cells into a number (n) of aliquots; (c) providing a plurality of nucleic acid tags to each of then aliquots, wherein each labeling sequence of the plurality of nucleic acid tags provided into a given aliquot is the same, and wherein a different labeling sequence is provided into each of the n aliquots; (d) binding at least one of the cDNAs in each of the n aliquots to the nucleic acid tags; (e) combining then aliquots; and (f) repeating steps (b), (c), (d), and (e) with the combined aliquot (interpreting dividing aliquots as distributing comprises dilution; generating indexed nuclei or cells; compartment specific index; the index in other compartments is different; pooling the indexed nuclei or cells in step (e) to carry out steps 1(a)-1(c); repeating combining, providing tags in each aliquot, binding tags, and combining aliquots as encompassing distributing into a second compartment comprising indexed nucleic acids; indexing again; pooling the indexed nuclei nucleic acids in steps 1(d), 1(e) and 1(f); nucleotide labels are different for different compartments; obtaining dual indexed nuclei or cells; and the nucleotide label comprises a nucleotide analog, claim 1) (col 3, lines 11-26). Seelig teaches that barcoded cDNA can be mixed together and sequenced (e.g., using NGS), such that the methods can be useful in assessing, analyzing, or studying the transcriptome (i.e., the different RNA species transcribed from the genome of a given cell) of one or more individual cells (col 4, lines 4-12). Seelig teaches that data can be gathered regarding RNA expression at the level of a single cell, such that an aliquot or group of cells can be separated into different reaction vessels or containers and a first set of nucleic acid tags can be added to the plurality of cDNA transcripts, wherein the aliquots of cells can then be re-grouped (interpreted as pooling), mixed, and separated again and a second set of nucleic acid tags can be added to the first set of nucleic acid tags (interpreted as cells pooled and separated into different compartments; a first compartment-specific index sequence; a second compartment-specific index sequence; subsets of cells; and distributing subsets of cells, claim 1) (col 4, lines 4-7 and 13-19). Seelig teaches in Examples 7 and 8, 3’ adapter ligation and producing Illumina compatible sequencing products by ligating a 3’ adapter oligo to barcoded cDNA, wherein the cDNA can be amplified using PCR as shown in Figures 15 & 16 (col 18, lines 5-19, 30-32 and 48-50; and Figures 15 & 16). Seelig teaches that the products can be sequenced on an Illumina MiSeq using paired end sequencing and multiplex sequencing primers (col 19, lines 44-46).
Regarding claims 2 (in part) and 4 (in part), Seelig teaches that the reverse transcription primer can be configured to reverse transcribe all, or substantially all, RNA in a cell (e.g., a random hexamer with a 5' overhang), wherein the reverse transcription primer can be configured to reverse transcribe RNA having a poly(A) tail (e.g., a poly(dT) primer, such as a dT(15) primer, with a 5' overhang), such that the reverse transcription primer can be configured to reverse transcribe predetermined RNAs (e.g., a transcript-specific primer) (interpreted as compartment-specific indexes; poly-T nucleotide sequence and reverse transcriptase; and predetermined RNA sequences, claims 2-4) (col 6, lines 54-63). Seelig teaches that Figure 2 show the formation of cDNA by in situ reverse transcription, wherein Panel A depicts a cell that is fixed and permeabilized; Panel B depicts addition of a poly(T) primer, as discussed above, which can template the reverse transcription of polyadenylated transcripts; and that reverse transcription can be conducted or performed on the plurality of cells including on a fixed and/or permeabilized plurality of cells, wherein M-MuLV reverse transcriptase (ENZYMATIC) can be used in the reverse transcription (interpreted as poly-T nucleotide sequence; and reverse transcriptase, claims 2 and 3) (col 7, lines 1-5 and 13-18).
Regarding claim 3, Seelig teaches that the reverse transcription primer can be configured to reverse transcribe all, or substantially all, RNA in a cell (e.g., a random hexamer with a 5' overhang), wherein the reverse transcription primer can be configured to reverse transcribe RNA having a poly(A) tail (e.g., a poly(dT) primer, such as a dT(15) primer, with a 5' overhang), such that the reverse transcription primer can be configured to reverse transcribe predetermined RNAs (e.g., a transcript-specific primer) (interpreted as compartment-specific indexes; poly-T nucleotide sequence and reverse transcriptase; and predetermined RNA sequences, claim 3) (col 6, lines 54-63).
Regarding claim 17, Seelig teaches that the present disclosure can be useful in assessing, analyzing, or studying the transcriptome such as the different RNA species transcribed from the genome of a given cell of one or more individual cells (interpreting individual nucleotides being transcribed to encompass generating a nucleotide analog or a mutagenic nucleotide, claim 17) (col 4, lines 7-11).
Regarding claim 28, Seelig teaches that an aliquot or group of cells can be separated into different reaction vessels or containers and a first set of nucleic acid tags can be added to the plurality of cDNA transcripts (interpreting transferring into vessels or containers as diluting, claim 28) (col 4, lines 12-16). Seelig teaches that each cell line can be transferred into its own 15 ml conical centrifuge tube, wherein 2 ml of Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) can be added to each tube, wherein the number of cells in each tube can be calculated (e.g., with a hemocytometer or on a flow cytometer), such that 200 μl of the sample can be transferred from each tube into separate 1.7 ml microcentrifuge tubes and 100 μl of the sample can be run on an ACCURI Flow Cytometer to calculate the cell concentration (interpreting transferring to tubes using DMEM medium as diluting, claim 28) (col 13, lines 31-40). Seelig teaches that each tube of cells can be resuspended in PBS and the cells can be counted such as on a flow cytometer (interpreted as diluting and distributing, claim 28) (col 17, lines 15-17).
Regarding claim 34, Seelig teaches that after repeated rounds of separating, tagging, and re-pooling, the cDNAs of each cell can be bound to a unique combination or sequence of nucleic acid tags that form a barcode, wherein cells in a single sample can be separated into a number of different reaction vessels including four 1.5 ml microcentrifuge tubes, a plurality of wells of a 96-well plate, or another suitable number and type of reaction vessels (interpreted as compartments comprising wells, claim 34) (col 4, lines 21-29).
Seelig does not specifically exemplify labeling nucleotides as RNA is synthesized; labeled RNA nucleotides; unlabeled pre-existing RNA; and barcoded hairpin adapter (claims 1, 2 and 4, all in part).
Regarding claims 1, 2 and 4 (all in part), Jao teaches a chemical method to detect RNA synthesis in cells, based on the biosynthetic incorporation of the uridine analog 5-ethynyluridine (EU) into newly transcribed RNA, on average once every 35 uridine residues in total RNA, wherein EU-labeled cellular RNA is detected quickly and with high sensitivity by using a copper (I)-catalyzed cycloaddition reaction (often referred to as “click” chemistry) with fluorescent azides, followed by microscopic imaging (interpreted as labeling nucleotides with 5-EU into RNA as RNA is synthesized in the subsets of nuclei or cells; and click-functionalized nucleotides, claims 1 and 17) (Abstract, lines 1-8). Jao teaches a chemical method to assay DNA synthesis in vivo by using 5-ethynyl-2-deoxyuridine (EdU), a thymidine analog that incorporates efficiently into DNA, wherein EdU can be detected rapidly and with great sensitivity with fluorescent azides by means of a Sharpless–Meldal copper (I)-catalyzed Huisgen cycloaddition reaction, a highly efficient and selective reaction often referred to as a ‘‘click’’ reaction, such that the methodology extends to a chemical method to assay RNA synthesis in vivo, wherein 5-ethynyluridine (EU) is incorporated into RNA transcripts generated by RNA polymerases I, II, and III in cells, such that EU-labeled cellular RNA can be detected quickly and with high sensitivity with fluorescent azides; and detection of EU is much faster than an anti-BrU immune-stain and allows whole-mount staining of large organ and tissue fragments, wherein EU does not significantly label cellular DNA, thus making it a specific transcriptional label (interpreted as labeling nucleotides with 5-EU into RNA as RNA is synthesized in the subsets of nuclei or cells; nucleotide analogs; and click-functionalized nucleotides, claims 1, 4 and 17) (pg. 15779, col 1, last full paragraph; and last partial paragraph; and col 2, first partial paragraph). Jao teaches that EU (Figure 1A) was incorporated into cellular RNA, wherein EU-labeled RNA could then be detected by reaction with fluorescent azides, by means of a copper (I)-catalyzed alkyne-azide cycloaddition, where EU was synthesized from 5-iodo-uridine; and the fluorescent azides used in this study were Alexa594-azide, Alexa568-azide, and tetramethyl-rhodamine (TMR)-azide, such that NIH 3T3 cells were incubated overnight with increasing concentrations of EU, fixed and stained with a fluorescent azide; and as shown in Figure 1B, the cells showed staining that increased in intensity with increasing EU concentration, wherein unlabeled cells showed very low background staining (Figure 1Bi), such that the staining was very strong in cell nuclei, where the nucleoli, sites of abundant ribosomal RNA transcription stained very intensely, while cytoplasmic staining was detectable but weaker than the nuclear signal; and importantly, all cells showed comparable levels of EU staining, such that the click staining reaction for the EU label was fast and proceeded smoothly with all three fluorescent azides used in this study (interpreted as labeling nucleotides with 5-EU into RNA as RNA is synthesized in the subsets of nuclei or cells; click-functionalized nucleotides; labeled RNA nucleotides; and unlabeled pre-existing RNA, claims 1 and 17) (pg. 15780, col 1).
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 for uniquely labeling and/or barcoding nucleic acid molecules including RNA as disclosed by Seelig to include the method of incorporating a uridine analog into RNA during transcription that can be detected with fluorescent azides as disclosed by Jao with a reasonable expectation of success in fluorescently labeling and uniquely barcoding nucleic acids within a biological sample including a plurality of cells and/or a tissue; and/or in detecting, analyzing, identifying and imaging uniquely barcoded and fluorescently detectable nucleic acids within a plurality of cells including within a tissue.
The combined references of Seelig and Jao do not specifically teach hairpin adaptors (claim 1, in part).
Regarding claim 1 (in part), Saxonov teaches methods, compositions, and assays, many of which involve amplification reactions such as digital PCR or droplet digital PCR, wherein the assays can be used for such applications as sequencing, copy number variation analysis; as well as, subdividing a sample into multiple partitions (e.g., droplets) and merging the partitions with other partitions that comprise adaptors with barcodes (Abstract). Saxonov teaches that barcodes can be present on adaptors, and an adaptor with a barcode can be attached to a polynucleotide by ligation, wherein a variety of types of adaptors can be used in the methods, compositions, systems, and kits described herein such that an adaptor can include a double-stranded sequence (interpreted as a barcoded ligation duplex adapter, claim 1) (col 7, lines 38-43). Saxonov teaches that an adaptor can comprise a single-stranded nucleic acid or a double-stranded nucleic acid including RNA, DNA and a DNA/RNA hybrid duplex (interpreted as an adaptor; a duplex adapter, claim 1) (col 8, lines 19-28). Saxonov teaches that an adaptor can comprise a hairpin (or hairpin loop), wherein a hairpin can comprise DNA and/or RNA; and the number of non-base paired bases in a loop of a hairpin can be about, more than about, or at least about 4, 5, 6, 7, 8…28, 29, or 30 bases; and the number of non-base-paired bases in a loop of a hairpin can be about 4 to about 8, about 4 to about 10, about 4 to about 14, about 4 to about 16, about 4 to about 20, about 4 to about 24, about 4 to about 26, or about 4 to about 30 bases, wherein the length of the stem (base-paired portion) of the adaptor can be about, more than about, or at least about 4, 5, 6, 7, 8…28, 29, or 30 base-pairs, such that a hairpin adaptor is ligated to only one end of a polynucleotide; a first hairpin adaptor is ligated to one end of a polynucleotide and a second hairpin adaptor is ligated to the other end of the polynucleotide, such that hairpin adaptors that are ligated to each end of a polynucleotide can comprise the same nucleic acid sequence or different nucleic acid sequences, wherein each of the adaptors can include barcodes that are the same or barcodes that are different (interpreted as a barcoded hairpin ligation duplex adapter, claim 1) (col 8, lines 40-61). Saxonov teaches commercially available kits comprising adaptors with barcodes can be used in the methods described herein including the ENCORE 384 Multiplex System (NUGEN), which can comprise 384 molecularly barcoded library adaptors; the ENCOR NGS Multiplex Library Systems for ION TORRENT can comprise adaptors with barcodes that can be ligated to fragments; the ENCORE Complete RNA Seq IL Multiplex System 1-8 (NUGEN); and ENCOR Complete RNA-Seq IL Multiplex System 9-16 (NUGEN) can provide barcoded adaptors for multiplex sequencing; the ENCOR Complete RNA-Seq DR Multiplex system 1-8 (NUGEN) and ENCORE Complete RNA-Seq DR Multiplex system 9-16 (NUGEN) can provide a dedicated read (DR) barcode design; as well as, kits comprising adaptors with barcodes from LIFE TECHNOLOGIES include 5500 SOLiD Fragment Library Barcode Adaptors; SOLiD RNA Barcoding Kit, Module 17-32, SOLiD RNA Barcoding Kit, Module 33-48, SOLiD RNA Barcoding Kit, Module 49-64, SOLiD RNA Barcoding Kit, Module 49-96, SOLiD RNA Barcoding Kit, Module 65-80, or SOLiD RNA Barcoding Kit, Module 81-96; SureSelect AB Barcode Adaptor Kit (AGILENT TECHONOLOGIES), Bico Scientific's AIR Barcoded Adapters, NEXTFLEX DNA Barcodes, ILLUMINA TRUSEQ RNA and DNA Sample Preparation Kits, RAINDANCE Technologies DEEPSEQ FFPE solution, NEBNEXT Multiplex Oligos for ILLUMNIA (Index Primers 1-12), or NEBNEXT Multiplex Small RNA Library Prep set for ILLUMNIA (Index Primers 1-12); and/or next generation sequencing techniques that comprise real-time SMRT technology by Pacific Biosciences (interpreted as including barcoded hairpin duplex adaptors, claim 1) (col 9, lines 32-67; col 10, lines 1-15; and col 29, lines 1-3), where it is known that NEBNext adaptors comprise a loop comprising a uracil (U) are designed for use in library prep, wherein the adaptors enable high-efficiency adaptor ligation, high library yields, and minimizes adaptor-dimer formation as evidenced by New England Biolabs (pg. 2, Workflow Overview, last full paragraph).
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 barcoding nucleic acids and processing them to comprise adapters including hairpin adapters and adapters obtained using commercial adapter kits as exemplified by Saxonov, 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 incorporating labeled nucleotides into RNA during transcription with molecules that can be detected and imaged using fluorescent azides as disclosed by Jao, and the method of uniquely labeling and/or uniquely barcoding nucleic acids within a cell as taught by Seelig to include the method of preparing polynucleotides in adapter-filled partitions including using partitions comprising barcoded hairpin adapters as taught by Saxonov with a reasonable expectation of success in detecting and/or imaging labeled nucleic acids that bind fluorescent azides in a sample including a tissue sample, selecting a tissue area or a compartment of interest, and analyzing and/or identifying by sequencing, the uniquely barcoded nucleic acids within a plurality of cells including within a tissue; in conducting multiplex sample preparation for next-generation sequencing including sequencing including on the Illumina platform, wherein barcoded hairpin adaptors can ligate with high-efficiency and high library yields, while minimizing adaptor-dimer formation including; and/or in combinatorially labeling and barcoding nucleic acids within cell samples that have been distributed into separate compartments, while reducing the cost of sequencing per sample.
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(a) as obvious over the art.
Conclusion
Claims 1-4, 17, 28 and 34 remain rejected.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, 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.
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/AMY M BUNKER/Primary Examiner, Art Unit 1684
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