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 .
Claim status and Formal Matters
The instant action is in response to papers filed 7/28/2026.
Claims 1, 3-5, 9, 21, 23, 51, 58-60, 62, 65, 78, 80-81, 94 have been amended.
Claims 1, 3-5, 9, 20-21, 23, 51, 58-60, 62, 65, 78, 80-81, 94 are pending and being examined.
Priority
The instant application was filed 02/27/2024 and is a national stage entry of PCT/US2022/075774 with an international filing date: 08/31/2022 and claims priority from provisional application 63239897 , filed 09/01/2021.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 7/28/2026 is 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.
It is noted that the IDS contain office actions related to foreign applications, but do not provide a context or specific claims or references that are discussed in the actions. These have been reviewed for what is provided within them, but the relevant application material such as specification, claims and cited references have not been used for the review.
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 1-5, 9, 20-21, 23, 51, 56, 58-60, 62, 65, 78, 80-81, 94 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 is indefinite because it lacks a positive active step relating back to the preamble. The preamble recites a method for determining the spatial location and copy number of a nucleic acid target in a sample, however the last positive active step is drawn to obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded cellular component-binding reagent specific oligonucleotides, or products thereof. Therefore it is unclear as to whether the method is drawn to determining the spatial location and copy number of a nucleic acid target in a sample or obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded cellular component-binding reagent specific oligonucleotides, or products thereof.
Claim 1 has been amended to recites, “target-binding region configured to hybridize to a nucleic acid target.” The recitation of configured to hybridize suggests there is also not configured to hybridize. The specification and claims provide no limiting definition or standard to differentiated configured to hybridize from not configured to hybridize. Further it is unclear what configured to hybridize requires. Thus the metes and bounds are unclear.
Claim 1 has been to amended to recite, “wherein the contacting comprises contacting the plurality of the spatial regions with the sample such that each
Claim 1 recites, “obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid molecules, or amplification products thereof, wherein each of the plurality of sequencing reads comprises a spatial label sequence, a molecular label sequence, and a subsequence of the nucleic acid target;.” The metes and bounds are unclear what is unclear if the wherein clause, “wherein each of the plurality of sequencing reads comprises a spatial label sequence, a molecular label sequence, and a subsequence of the nucleic acid target” limits the amplification products thereof and if so how.
Claim 1 also recites, “or each spatial label sequence that identifies a respective spatial region of the substrate and thereby a respective spatial location of the sample,
Claim 1 further recites, “unique identifier sequence.” The recitation of, “unique identifier sequence” suggest “non- unique identifier sequence.” The specification and claims provide no specific definition or standard to differentiate the two. Thus the metes and bounds are unclear.
Claim 1 also recites, “configured to specifically binding .” The recitation of “configured” suggest not configured. Further the recitation of “specifically” suggests “non-specifically.” The specification does not provide a definition or standard to differentiate configured from not configured or specifically from non-specifically. Thus the metes and bounds are unclear.
Claim 1 has been amended to recite, “a cellular component-binding reagent specific oligonucleotide comprising a unique identifier sequence for the cellular component-binding reagent.” The recitation of “reagent specific oligonucleotide” suggests there is reagent non-specific oligonucleotide. The specification and claims do not provide a standard to differentiate the two. Further the claim recites, “a unique identifier sequence for the cellular component-binding reagent.” The recitation of a unique identifier sequence for the cellular component-binding reagent suggests a non- unique identifier sequence for the cellular component-binding reagent. Thus the metes and bounds of the claim are unclear.
Claim 1 recites, “for each spatial label sequence, which is associated a respective spatial region of the substrate and thereby a respective spatial location of the sample, counting the number of molecular labels with respective sequences associated with a unique identifier sequence to determine the number of copies of at least one cellular component target of the plurality of cellular component targets at each spatial location of the sample.” The recitation of, “associated.” It is unclear how or what is required of how association is done and/or what is encompassed. The metes and bounds are unclear what respective spatial region is respective to. Further it is unclear what respective spatial location of the sample is respective to. Further it is unclear how or if the spatial region relates to the spatial location.
Claim 23 recites, “further comprising correlating the imaging data and sequencing data for one or more spatial locations of the sample..” Correlating is a relative term. The specification and claims provide no specific definition or standard on how to correlating the imaging data with sequencing data. Thus the metes and bounds are unclear.
Claim 59 recites, “defines respective spatial region.” Defines respective is a relative term and suggest there is non- respective as well as respective. The specification and claims provide no limiting definition to differentiate respective from non- respective.
Claim 62 recites, “wherein the blocking reagent comprises a plurality of decoy oligonucleotides configured to hybridize to at least one of the one or more nucleic acids that are not the nucleic acid target
Claim 65 recites, “random sequence.” Random is a relative term which suggests non-random. The specification and claims fail to provide a standard to differentiate a random sequences from a non-random sequence.
Claim 81recites, “specifically binds.” The metes and bounds are unclear as “specifically binds” which suggest “non-specifically binds.” The specification and claims do not provide a standard to differentiate “specifically binds” from “non-specifically binds.”
Claim 94 recites, “configured to hybridize.” The recitation of configured to hybridize suggests there is configured to not hybridize. However the claims and specification do not provide a standard to differentiate configured to hybridize from configured to not hybridize. Further it is unclear if associated is a covalent attachment, correlated is some way or anything else.
Response to Arguments
The response traverses the rejection of the claims in view of the amendments. This argument has been thoroughly reviewed but is not considered persuasive as the amendment has introduced new issues and/or has not addressed all the issues previously identified.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1, 3-5, 9, 20, 21, 23, 51, 56, 58-60, 94 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Stahl (Science Mag (2016) volume 353 pages 78-82 and supplemental)
With regards to claim 1, Stahl teaches in figure 2A
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Stahl teaches permeabilization and reverse transcription, cDNA library preparation for sequencing, Fluorescent imaging and image alignment, Sequence alignment and generation of gene expression database. (supplemental material and Figure 2)
Claim 1 recites, “cellular component-binding reagent.” The specification does not provide a limiting definition of “cellular component-binding reagent.” Thus the art cited anticipates claim 1.
With regards to claim 3, Stahl teaches permeabilization and reverse transcription, thus the contacting is in the presence of the extension reagents.
With regards to claim 4, Stahl teaches “ RT and dNTPs in permeabilization and reverse transcription . (supplemental material)
With regards to claim 5, Stahl teaches the RNase H treatment of the sample following first strand synthesis. Thus this is a denaturing step. (supplemental material)
With regards to claim 9, Stahl teaches biopsies were sectioned and every 4th section is used for spatial barded array (supplemental material).
With regards 20 Stahl teaches Fluorescent imaging and image alignment. (supplemental material)
With regards to claim 21, Stahl teaches fluorescent imagining, bright field images and visualization of structures. (supplemental material)
With regards to claim 23, Stahl teaches Fluorescent imaging and image alignment, Sequence alignment and generation of gene expression database. (supplemental material and Figure 2)
With regards to claim 51, 56, 58 Stahl teaches oligonucleotide barcodes immobilized on planar substrate (figure 2).
With regards to claim 59 Stahl teaches microwell array collection and preparation of breast cancer biopsy.(figure 2, (supplementary content)
With regards to claim 60, Stahl teaches BSA which can be considered a blocking agent.
With regards to claim 94, Stahl teaches in figure 2A
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Stahl teaches permeabilization and reverse transcription, cDNA library preparation for sequencing, Fluorescent imaging and image alignment, Sequence alignment and generation of gene expression database. (supplemental material and Figure 2) Thus Stahl anticipates claim 94.
Response to Arguments
The response traverses the rejection by asserting that the claim has been amended to provide the limitations of claim 2. This argument has been thoroughly reviewed but is not considered persuasive as claim 2 was previously rejected as anticipated. The response further alleges the art does not teach all the limitations of the claims. This argument has been thoroughly reviewed but is not considered persuasive as the rejection addresses all the limitations of the claims. The response has not specifically identified a limitation not taught by the prior art. Thus the rejection is maintained.
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.
Claim(s) 1, 3-5, 9, 20-21, 23, 51, 58-60, 62, 65, 78, 80-81, 94 is/are rejected under 35 U.S.C. 103 as being unpatentable over Stahl (Science Mag (2016) volume 353 pages 78-82 and supplemental) and Ho (Nucleic Acids Research, 2015, Vol. 43, No. 1 e4)
With regards to claim 1, Stahl teaches in figure 2A
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Stahl teaches permeabilization and reverse transcription, cDNA library preparation for sequencing, Fluorescent imaging and image alignment, Sequence alignment and generation of gene expression database. (supplemental material and Figure 2) Thus Stahl anticipates claim 1.
Stahl does not specifically teach blocking probe or decoy probe (62, 65, 78, 80, 81.)
However, Ho teaches, “Here we provide the first strategy to use a competitive Extendable Blocking Probe (ExBP) for allele specific priming with superior selectivity at the stage of reverse transcription. In order to analyze highly similar RNA variants, a reverse-transcriptase primer whose sequence matches a specific variant selectively primes only that variant, whereas mismatch priming to the alternative variant is suppressed by virtue of hybridization and subsequent extension of the perfectly matched ExBP on that alternative variant template to form a cDNA–RNA hybrid. This hybrid will render the alternative RNA template unavailable for mismatch priming initiated by the specific primer in a hot-start protocol of reverse transcription when the temperature decreases to a level where such mismatch priming could occur. The ExBP-based reverse transcription assay detected BRAF and KRAS mutations in at least 1000-fold excess of wild-type RNA and detection was linear over a 4-log dynamic range. This novel strategy not only reveals the presence or absence of rare mutations with an exceptionally high selectivity, but also provides a convenient tool for accurate determination of RNA variants in different settings, such as quantification of allele-specific ex pression.
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 blocking probes of Ho in the method of Stahl. The artisan would be motivated to bind and reduce non-desirable or off target nucleic acids. The artisan would have a reasonable expectation of success as the artisan is merely applying known methods to reverse transcription reaction.
Claim 2 recites, “cellular component-binding reagent.” The specification does not provide a limiting definition of “cellular component-binding reagent.” Thus the art cited with claim 1 anticipates claim 2.
With regards to claim 3, Stahl teaches permeabilization and reverse transcription, thus the contacting is in the presence of the extension reagents.
With regards to claim 4, Stahl teaches “ RT and dNTPs in permeabilization and reverse transcription . (supplemental material)
With regards to claim 5, Stahl teaches the RNaseH treatment of the sample following first strand synthesis. Thus this is a denaturing step. (supplemental material)
With regards to claim 9, Stahl teaches biopsies were sectioned and every 4th section is used for spatial barded array (supplemental material).
With regards 20 Stahl teaches Fluorescent imaging and image alignment. (supplemental material)
With regards to claim 21, Stahl teaches fluorescent imagining, bright field images and visualization of structures. (supplemental material)
With regards to claim 23, Stahl teaches Fluorescent imaging and image alignment, Sequence alignment and generation of gene expression database. (supplemental material and Figure 2)
With regards to claim 51, 56, 58 Stahl teaches oligonucleotide barcodes immobilized on planar substrate (figure 2).
With regards to claim 59 Stahl teaches microwell array collection and preparation of breast cancer biopsy.(figure 2, (supplementary content)
With regards to claim 60, Stahl teaches BSA which can be considered a blocking agent.
With regards to claim 94, Stahl teaches in figure 2A
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Stahl teaches permeabilization and reverse transcription, cDNA library preparation for sequencing, Fluorescent imaging and image alignment, Sequence alignment and generation of gene expression database. (supplemental material and Figure 2) Thus Stahl anticipates claim 4.
Response to Arguments
The response traverses the rejection by asserting that the claim has been amended to provide the limitations of claim 2. This argument has been thoroughly reviewed but is not considered persuasive as claim 2 was previously rejected as anticipated. The response further alleges the art does not teach all the limitations of the claims. This argument has been thoroughly reviewed but is not considered persuasive as the rejection addresses all the limitations of the claims. The response has not specifically identified a limitation not taught by the prior art. Thus the rejection is maintained.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claim 1, 3-5, 9, 20-21, 23, 51, 58-60, 62, 65, 78, 80-81, 94 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1-6 of US Patent 12,606,859. Although the claims at issue are not identical, they are not patentably distinct from each other because coextensive in scope
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
The instant claims are drawn to a method for determining the spatial location and copy number of a nucleic acid target in a sample, comprising: providing a substrate comprising a plurality of spatial regions, wherein a plurality of oligonucleotide barcodes are associated with each of the spatial regions, wherein each of the oligonucleotide barcodes comprises a universal sequence, a molecular label, a target-binding region configured to hybridize to a nucleic acid target, and a predetermined spatial label, wherein oligonucleotide barcodes of the same spatial region comprise the same spatial label, and wherein oligonucleotide barcodes of the different spatial regions comprise different spatial labels; contacting the substrate with a sample comprising copies of a nucleic acid target and a plurality of cellular component targets, wherein the cellular component targets comprise protein targets, wherein the contacting comprises contacting the plurality of the spatial regions with the sample such that each respective spatial region contacts arespective spatial location of the sample; extending the plurality of oligonucleotide barcodes hybridized to the copies of a nucleic acid target to generate a plurality of barcoded nucleic acid molecules each comprising a sequence complementary to at least a portion of the nucleic acid target; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid molecules, or amplification products thereof, wherein each of the plurality of sequencing reads comprises a spatial label sequence, a molecular label sequence, and a subsequence of the nucleic acid target; for each spatial label sequence that identifies a respective spatial region of the substrate and thereby a respective spatial location of the sample, contacting a plurality of cellular component-binding reagents with the sample, wherein each of the plurality of cellular component-binding reagents comprises a cellular component-binding reagent specific oligonucleotide comprising a unique identifier sequence for the cellular component-binding reagent, and wherein the cellular component-binding reagent is configured to specifically bind to at least one of the plurality of cellular component targets; extending the plurality of oligonucleotide barcodes hybridized to the cellular component-binding reagent specific oligonucleotides to generate a plurality of barcoded cellular component-binding reagent specific oligonucleotides each comprising a sequence complementary to at least a portion of the unique identifier sequence; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded cellular component-binding reagent specific oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a spatial label sequence, a molecular label sequence, and at least a portion of the unique identifier sequence; and for each spatial label sequence that identifies a respective spatial region of the substrate and thereby a respective spatial location of the sample, counting the number of molecular labels with distinct sequences associated with a unique identifier sequence to determine the number of copies of at least one cellular component target of the plurality of cellular component targets at each spatial location of the sample..
The claims of 859 are drawn to A method for simultaneous measurement of protein and gene expressions in cells, comprising: contacting a plurality of cellular component-binding reagents with a plurality of cells comprising a plurality of cellular component targets and copies of a nucleic acid target, wherein the nucleic acid target comprises mRNA, wherein each cellular component-binding reagent of the plurality of cellular component-binding reagents comprises a cellular component-binding reagent specific oligonucleotide comprising a unique identifier for the cellular component-binding reagent and a poly(A) sequence 18-30 nucleotides in length, wherein the cellular component-binding reagent specific oligonucleotide comprises DNA, and wherein the cellular component-binding reagent is capable of specifically binding to at least one of the plurality of cellular component targets; partitioning the plurality of cells associated with the cellular component-binding reagents to a plurality of partitions, wherein a partition of the plurality of partitions comprises a single cell from the plurality of cells associated with the cellular component-binding reagents; in the partition comprising the single cell, contacting a solid support comprising a plurality of oligonucleotide barcodes with the cellular component-binding reagent specific oligonucleotides and the copies of the nucleic acid target for hybridization, wherein the oligonucleotide barcodes each comprise a poly(T) sequence, a first universal sequence, a first molecular label, and a cleavage region, wherein the cleavage region is 5′ of the first universal sequence and comprises one or more cleavage sites; extending the cellular component-binding reagent specific oligonucleotides hybridized to the plurality of oligonucleotide barcodes to generate a plurality of extended cellular component-binding reagent specific oligonucleotides each comprising a complement of the first molecular label, a complement of the cleavage region, and a complement of the first universal sequence; extending the plurality of oligonucleotide barcodes hybridized to the copies of a nucleic acid target to generate a plurality of barcoded nucleic acid molecules each comprising a poly(T) sequence, a first universal sequence, a cleavage region, a sequence complementary to the at least a portion of the nucleic acid target and the first molecular label, wherein the extended cellular component-binding reagent specific oligonucleotides each comprise a double-stranded cleavage region and wherein the plurality of barcoded nucleic acid molecules each comprise a single-stranded cleavage region; contacting the plurality of barcoded nucleic acid molecules and the plurality of extended cellular component-binding reagent specific oligonucleotides with one or more cleaving agents to generate: (i) a plurality of cleaved barcoded nucleic acid molecules not associated with the solid support, and (ii) a plurality of cleaved extended cellular component-binding reagent specific oligonucleotides associated with the solid support separating the plurality of cleaved barcoded nucleic acid molecules from the solid support; denaturing the cleaved extended cellular component-binding reagent specific oligonucleotides associated with the solid support to generate a plurality of denatured cleaved extended cellular component-binding reagent specific oligonucleotides; separating the plurality of denatured cleaved extended cellular component-binding reagent specific oligonucleotides from the solid support obtaining sequence information of the plurality of separated extended cellular component-binding reagent specific oligonucleotides, or products thereof, to determine the number of copies of at least one cellular component target of the plurality of cellular component targets in one or more of the plurality of cells; and obtaining sequence information of the plurality of separated barcoded nucleic acid molecules, or products thereof, to determine the copy number of the nucleic acid target in one or more of the plurality of cells.
Thus it is prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims, the claims of 859 are a species encompassed by the claims and thus obvious variants.
Dependent claims are rejected as they are commensurate in scope.
Claim 1-5, 9, 20, 21, 23, 51, 56, 58-60, 62, 65, 78, 80, 81,94 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 2-8, 14-16, 20-21, 24-25, 32, 34 -35, 38, 49, 65 of co-pending application 18/836303. Although the claims at issue are not identical, they are not patentably distinct from each other because coextensive in scope
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
The instant claims are drawn to method for determining the spatial location and copy number of a nucleic acid target in a sample, comprising: providing a substrate comprising a plurality of spatial regions, wherein a plurality of oligonucleotide barcodes are associated with each of the spatial regions, wherein each of the oligonucleotide barcodes comprises a universal sequence, a molecular label, a target-binding region configured to hybridize to a nucleic acid target, and a predetermined spatial label, wherein oligonucleotide barcodes of the same spatial region comprise the same spatial label, and wherein oligonucleotide barcodes of the different spatial regions comprise different spatial labels; contacting the substrate with a sample comprising copies of a nucleic acid target and a plurality of cellular component targets, wherein the cellular component targets comprise protein targets, wherein the contacting comprises contacting the plurality of the spatial regions with the sample such that each respective spatial region contacts arespective spatial location of the sample; extending the plurality of oligonucleotide barcodes hybridized to the copies of a nucleic acid target to generate a plurality of barcoded nucleic acid molecules each comprising a sequence complementary to at least a portion of the nucleic acid target; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid molecules, or amplification products thereof, wherein each of the plurality of sequencing reads comprises a spatial label sequence, a molecular label sequence, and a subsequence of the nucleic acid target; for each spatial label sequence that identifies a respective spatial region of the substrate and thereby a respective spatial location of the sample, contacting a plurality of cellular component-binding reagents with the sample, wherein each of the plurality of cellular component-binding reagents comprises a cellular component-binding reagent specific oligonucleotide comprising a unique identifier sequence for the cellular component-binding reagent, and wherein the cellular component-binding reagent is configured to specifically bind to at least one of the plurality of cellular component targets; extending the plurality of oligonucleotide barcodes hybridized to the cellular component-binding reagent specific oligonucleotides to generate a plurality of barcoded cellular component-binding reagent specific oligonucleotides each comprising a sequence complementary to at least a portion of the unique identifier sequence; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded cellular component-binding reagent specific oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a spatial label sequence, a molecular label sequence, and at least a portion of the unique identifier sequence; and for each spatial label sequence that identifies a respective spatial region of the substrate and thereby a respective spatial location of the sample, counting the number of molecular labels with distinct sequences associated with a unique identifier sequence to determine the number of copies of at least one cellular component target of the plurality of cellular component targets at each spatial location of the sample.
The claims of 303 are drawn to A method for measuring cellular component target expression in cells, comprising: contacting a plurality of first cellular component-binding reagents with a plurality of first detectable conjugates, wherein each of the plurality of first cellular component-binding reagents comprises a cellular component-binding reagent specific oligonucleotide comprising a unique identifier sequence for the first cellular component-binding reagent, and wherein the first cellular component-binding reagent is capable of specifically binding to at least one of a plurality of cellular component targets, and wherein each of the plurality of first detectable conjugates comprises a detectable moiety, or precursor thereof, and a unique identifier specific oligonucleotide comprising a sequence configured to bind a unique identifier sequence, wherein first detectable conjugates capable of binding the same unique identifier sequence comprise the same detectable moiety, or a precursor thereof, and wherein first detectable conjugates capable of binding different unique identifier sequences comprise different detectable moieties, or precursors thereof; contacting the plurality of first cellular component-binding reagents associated with the plurality of first detectable conjugates with a first plurality of cells comprising a plurality of cellular component targets; and measuring emissions of the detectable moiety of each first detectable conjugate with an instrument as an indication of the amount each of first cellular component- binding reagent bound to a cellular component target and a first detectable conjugate..
Thus it is prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims, the claims of 3039 are a species encompassed by the claims and thus obvious variants.
Dependent claims are rejected as they are commensurate in scope.
Claim 1-5, 9, 20, 21, 23, 51, 56, 58-60, 62, 65, 78, 80, 81,94 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 2-8, 14-16, 20-21, 24-25, 32, 34 -35, 38, 49, 65 of co-pending application 18730,043. Although the claims at issue are not identical, they are not patentably distinct from each other because coextensive in scope
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
method for determining the spatial location and copy number of a nucleic acid target in a sample, comprising: providing a substrate comprising a plurality of spatial regions, wherein a plurality of oligonucleotide barcodes are associated with each of the spatial regions, wherein each of the oligonucleotide barcodes comprises a universal sequence, a molecular label, a target-binding region configured to hybridize to a nucleic acid target, and a predetermined spatial label, wherein oligonucleotide barcodes of the same spatial region comprise the same spatial label, and wherein oligonucleotide barcodes of the different spatial regions comprise different spatial labels; contacting the substrate with a sample comprising copies of a nucleic acid target and a plurality of cellular component targets, wherein the cellular component targets comprise protein targets, wherein the contacting comprises contacting the plurality of the spatial regions with the sample such that each respective spatial region contacts acellular component-binding reagents with the sample, wherein each of the plurality of cellular component-binding reagents comprises a cellular component-binding reagent specific oligonucleotide comprising a unique identifier sequence for the cellular component-binding reagent, and wherein the cellular component-binding reagent is configured to specifically bind to at least one of the plurality of cellular component targets; extending the plurality of oligonucleotide barcodes hybridized to the cellular component-binding reagent specific oligonucleotides to generate a plurality of barcoded cellular component-binding reagent specific oligonucleotides each comprising a sequence complementary to at least a portion of the unique identifier sequence; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded cellular component-binding reagent specific oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a spatial label sequence, a molecular label sequence, and at least a portion of the unique identifier sequence; and for each spatial label sequence that identifies a respective spatial region of the substrate and thereby a respective spatial location of the sample, counting the number of molecular labels with distinct sequences associated with a unique identifier sequence to determine the number of copies of at least one cellular component target of the plurality of cellular component targets at each spatial location of the sample.
The claims of 043 are drawn to A method for labeling nucleic acid targets in a sample, comprising: contacting copies of a nucleic acid target with a first plurality of oligonucleotide barcodes, wherein each oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a first universal sequence, a first molecular label, and a first target- binding region capable of hybridizing to the nucleic acid target; extending the first plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target to generate a plurality of barcoded nucleic acid molecules each comprising the first universal sequence, the first molecular label, and a sequence complementary to at least a portion of the nucleic acid target; contacting the barcoded nucleic acid molecules with a second plurality of oligonucleotide barcodes for hybridization, wherein each oligonucleotide barcode of the second plurality of oligonucleotide barcodes comprises a second universal sequence, a cleavage domain, and a blocking group, wherein the blocking group is capable of preventing extension of the oligonucleotide barcode, wherein the cleavage domain is positioned 5' of the blocking group, and wherein a cleaving enzyme is capable of cleaving the oligonucleotide barcode at a point within or adjacent to the cleavage domain when the cleavage domain is hybridized to a barcoded nucleic acid molecule; contacting the second plurality of oligonucleotide barcodes hybridized to the barcoded nucleic acid molecules with the cleaving enzyme, thereby removing the blocking group from said oligonucleotide barcodes; extending the 3' ends of oligonucleotide barcodes of the second plurality of oligonucleotide barcodes hybridized to the barcoded nucleic acid molecules to generate a plurality of extended barcoded nucleic acid molecules...
Thus it is prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims, the claims of 043 are a species encompassed by the claims and thus obvious variants.
Dependent claims are rejected as they are commensurate in scope.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
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/Steven Pohnert/Primary Examiner, Art Unit 1683
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