CTFR 18/000,222 CTFR 100667 DETAILED ACTION Notice of Pre-AIA or AIA Status 07-03-aia AIA 15-10-aia The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA. 07-103 AIA Please note: The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action. Interview Summary Applicant’s summary of the Interview conducted on March 20, 2026 is acknowledged. Claim Status Claims 1-5, 7-10, 12-13, and 25-33 are pending and being examined on the merits. Information Disclosure Statement 06-49-06 AIA The listing of references in the specification is not a proper information disclosure statement (e.g., pg 44, citation 17; pg 6, paragraph 2). 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. Applicant’s submission of an IDS on 1/30/2026 is acknowledged. However, this IDS did not contain the reference of Han et al. 2000, as noted above, or other additional references throughout the specification. As noted above, these references have not been considered by the examiner unless cited on the PTO-892 form. Additionally, Feldman et al., 2019 (citation 13 on IDS of 1/30/2026) has been struck through given that this is a duplicate of a reference already on the record (see IDS of 11/4/2025, citation 1). It is also noted that the date for citation 23 on 1/30/2026 is incorrect (the reference is dated as 4/20/2024 when it should be 4/20/2014). Abstract The objection to the Abstract for containing more than 150 words and a typo is withdrawn in light of Applicant’s amendments to the Abstract. Claim Rejections - 35 USC § 112b - Indefiniteness T rejection of claims 6-7, 13, and 25 under 35 U.S.C. 112(b) are withdrawn in light of Applicant’s cancellation of claim 6 and amendments of claims 7, 13, and 25. Claim Rejections - 35 USC § 103 Withdrawn: The rejection of claims 1-6, 8-9, 12, and 26-32 under 35 U.S.C. 103 as being obvious over Chen (Chen et al., US 2019/0360044 A1; cited on IDS of 11/4/2025) is withdrawn in light of Applicant’s amendments to the claims. The rejection of claim 10 under 35 U.S.C. 103 as being unpatentable over Chen (Chen et al., US 2019/0360044 A1; cited on IDS of 11/4/2025) as applied to claims 1-6, 8-9, 12, and 26-32 above, and further in view of Samusik (Samusik et al., US 2019/0055594 A1) is withdrawn in light of Applicant’s amendments to the claims. New (Necessitated by Amendments): 07-21-aia AIA Claim s 1-2, 4-5, 8-10, 12-13, 26-28, and 33 are rejected under 35 U.S.C. 103 as being unpatentable over Nilsson (Nilsson et al., US 2020/0224244 A1, EFD of 10/9/2017) in view of Bava (Bava et al., US 2019/0093156 A1) . Claims 1 and 26: Nilsson teaches a method of target nucleic acid detection (Abstract). Nilsson teaches that this may be performed upon cells of a tissue sample (paragraph [0127]), and that detection comprises sequencing of the nucleic acid (paragraph [0089, 0130]). Nilsson teaches permeabilizing the cells of the tissue sample that contains the target nucleic acid (paragraph [0197]). Nilsson teaches contacting the cells with a padlock probe that comprises a sequence complementary to the target nucleic acid sequence which produces a padlock probe bound to the target nucleic acid sequence (paragraph [0008, 0026-0027, 0029-0034]). Nilsson teaches contacting the padlock probe bound to the target nucleic acid sequence with a reverse transcriptase when the template is RNA and gap-filling is performed on the padlock probe (paragraphs [0034 and 0093]). Nilsson teaches contacting the target nucleic acid sequence on the padlock probe with a ligase to form a circularized padlock probe having the target nucleic acid sequence (paragraph [0029, 0034, 0140-0141]). Nilsson teaches performing rolling circle amplification (RCA) on the circularized padlock probe which creates a linear repeating sequence comprising multiple copies of the target nucleic acid sequence (paragraph [0006,0029]). Nilsson then teaches that the target nucleic acid sequence is detected, for example by sequencing (paragraph [0130-131]). Nilsson teaches, after RCA, digesting the RACA products into monomers, each containing the repeat sequence, and then amplifying said monomers with primers comprising Illumina index adapter sequences, thus obtaining multiple copies of the target nucleic acid sequence comprising the sequence of the extended primer and the index adapter sequence to facilitate sequencing (paragraph [0252]). Nilsson does not teach, after performing RCA, annealing multiple copies of a primer to the linear repeating sequences, extending the multiple copies of the primer to generate amplicons comprising the target nucleic acid sequence, or performing combinatorial indexing on the amplicons to generate extended products comprising the target nucleic acid sequence and a barcode sequence capable of identifying a cell of origin of the extended products. Additionally, Nilsson does not teach obtaining this single cell nucleic acid sequencing data for extended products from millions of cells in a single run. However, this methodology, applied to millions of cells in a single run, is known in the art, as taught by Bava. Bava teaches a method for the detection of target molecules (Abstract). Bava teaches that said method comprises the use of a padlock probe amplified via RCA to generate a template comprising multiple copies of a target nucleic acid sequence (paragraph [0006] and Figure 12). Bava teaches, after amplification, annealing multiple copies of a primer to said template and forming an extension product which is a copy of the UBA (UBA = unique binding agent, which corresponds to the target nucleic acid sequence, paragraph [0006] and Figure 12). Bava teaches performing combinatorial indexing on the amplicons to generate extended producs which comprise the target nucleic acid sequence and a barcode sequence which is capable of identifying a cell of origin of the extended products (cell originating barcode, COB; paragraph [0006] and Figure 12). Bava teaches obtaining single cell nucleic acid sequencing data by identifying the polynucleotide sequence of the extended products and obtaining single cell nucleic acid sequencing data from the sample (paragraph [0007-0008]). Bava teaches that this methodology is performed on 10 6 cells (reads on about 1,000,000 and about 1x10 12 cells in a single run ; paragraph [0345]). Bava teaches that each cell originating barcode is composed of subcodes (SCs) that make up the barcode (paragraph [0345-0347]). Therefore, the barcode is actually a combination of barcode sequences (relevant to claim 26). It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Nilsson to perform combinatorial indexing on the primers annealed to multiple copies of the target nucleic acid within the RCA product as taught by Bava. One would be motivated to do so given the assertion by Bava that this enables the generation of millions of unique cellular barcodes with a high confidence of tag uniqueness (paragraph [0200-0208, 0345]) and enables the detection of target molecules on a single cell basis within samples with complex cell populations (paragraph [0090]). One would have a reasonable expectation of success given that Bava teaches that this methodology can be applied to cells within permeabilized tissue samples (paragraph [0148, 0187]), as is employed in the methodology of Nilsson. As taught by Nilsson, the primers may contain index adapters for sequencing, thus with the incorporation of the methodology of Bava, the primers annealed to the multimeric RCA product may include an index adaptor sequence, thus facilitating downstream sequencing and incorporating the index adaptor sequence into the extended product containing the target nucleic acid sequence and the barcode sequence identifying the cell of origin. Claim 2, 5, and 27: Nilsson teaches that the target nucleic acid sequence comprises a target RNA sequence (paragraph [0001]). Nilsson teaches that the RNA is mRNA (paragraph [0124]). Claim 4: Bava teaches a cell originating barcodes comprised of two 7-mer subcodes added via split pool assembly (SC1 + SC2; paragraph [0345-0347]). This generates a barcode of 14 nucleotides (which reads on between 6 and 20 nucleotides in length ). Claim 8: Bava teaches that the combinatorial indexing is applied in combination with cell splitting (paragraph [0006, 0345]). Claim 9: Bava teaches that the combinatorial indexing comprises use of a microfluidic chamber (paragraph [0035]). Claim 10: Nilson teaches the RCA is performed by a DNA polymerase (paragraph 0029]). Claim 12 and 28: Bava teaches the target sequence is present at 5 copies in a single cell (paragraph 0219]). Claim 13: Bava teaches that extending the multiple copies of the primers is performed with a non-strand displacing polymerase (T4 polymerase, Figure 12). Claim 33: Nilsson teaches using a non-strand displacing reverse transcriptase (paragraph [0093]) . 07-22-aia AIA Claim (s) 3 is rejected under 35 U.S.C. 103 as being unpatentable over Nilsson (Nilsson et al., US 2020/0224244 A1, EFD of 10/9/2017) in view of Bava (Bava et al., US 2019/0093156 A1) as applied to claim s 1-2, 4-5, 8-10, 12-13, 26-28, and 33 above, and further in view of Chen (Chen et al., US 2019/0360044 A1; cited on IDS of 11/4/2025) . The teachings of Nilsson in view of Bava are detailed above. Relevant to the instantly rejected claims, Nilsson in view of Bava teach detection of a target nucleic acid at single cell resolution comprising hybridization of a padlock probe to the target analyte, ligation of the padlock probe, amplification of the padlock probe via RCA, annealing and extension of multiple primers to the RCA product, combinatorial indexing of the extended primers and sequencing of said extended indexed amplicons for single cell target nucleic acid analysis. Nilsson in view of Bava do not teach that the padlock probe comprises a UMI sequence. However, inclusion of UMI sequences in padlock probes is known in the art, as taught by Chen. Chen teaches a method of single-cell analysis comprising quantifying target nucleic acids in individual cells based on sequencing amplicons of said target nucleic acids (Abstract). Chen teaches that amplification of the target nucleic acid can be achieved through hybridization of padlock probes (also called “MIPs”) to the target analyte and amplification through RCA or PCR (paragraphs [0038, 0042], Figure 5, Figure 7). Chen teaches that the padlock probes comprise a UMI (paragraph [0042]). It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Nilsson in view of Bava to incorporate UMIs into the padlock probe. One would be motivated to do so given the assertion by Chen that incorporation of a UMI allows for determination of the number of transcripts that given rise to a particular amplification product (paragraph [00257]). One would have a reasonable expectation of success given that Chen teaches that this is particular advantageous when aiming to detect and quantify “unique amplified products” and the preferred mode of amplification is isothermal (which RCA is; paragraph [0257]) . 07-22-aia AIA Claim s 7 and 25 are rejected under 35 U.S.C. 103 as being unpatentable over Nilsson (Nilsson et al., US 2020/0224244 A1, EFD of 10/9/2017) in view of Bava (Bava et al., US 2019/0093156 A1) as applied to claim s 1-2, 4-5, 8-10, 12-13, 26-28, and 33 above, and further in view of Russel (Russel et al., WO 2018/109206 A1) . The teachings of Nilsson in view of Bava are detailed above. Relevant to the instantly rejected claims, Nilsson in view of Bava teach detection of a target nucleic acid at single cell resolution comprising hybridization of a padlock probe to the target analyte, ligation of the padlock probe, amplification of the padlock probe via RCA, annealing and extension of multiple primers to the RCA product, combinatorial indexing of the extended primers and sequencing of said extended indexed amplicons for single cell target nucleic acid analysis. Nilsson in view of Bava do not teach that the target nucleic acid is a DNA barcode sequence attached to an antibody which has been applied to the sample wherein detection of the barcode sequence identifies the antibody and its abundance or the levels of abundance of a protein bound by the antibody. However, detection of DNA barcode sequences attached to antibodies to determine the abundance of a target protein bound by said antibody is known in the art, as taught by Russel. Russel teaches detecting an analyte in a sample using padlock probes specific to a target sequence which are them amplified by RCA (Abstract and pg 1, ln 1-5). Russel teaches that RCA is used to detect a nucleic acid molecule which can be detected as a “proxy” for the presence of a target analyte, such as a protein (pg 2, ln 21-24, 26). Russel teaches that the target nucleic acid sequence may be attached to an antibody (antibody:nucleic acid conjugate) that is contacted with the sample and which binds to the analyte (pg 14, ln 17-19; pg 19, ln 20-30). Given that the target nucleic acid sequence is provided to the antibody, a target-specific reporter sequence that is the proxy for the analyte constitutes a “barcode” sequence which identifies said antibody or its bound protein (pg 18, ln 28-30). It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Nilsson in view of Bava with that of Russel to use antibody-oligonucleotide conjugated targets. One would be motivated to do so given the assertion by Russel that providing a “proxy” for detection of an analyte allows for RCA amplification of targets that may not have their own nucleic acid to be targeted (pg 2, ln 21-24, 26; pg 14, ln 11-24). One would have a reasonable expectation of success given that Russel is also using RCA to amplify the signal of the target nucleic acid and applies this to a cell or tissue sample of an organism (pg 21, ln 20). Additionally, Nilsson teaches that their methodology can be applied to DNA targets as well (paragraph [0013]) . 07-21-aia AIA Claim s 29-32 are rejected under 35 U.S.C. 103 as being unpatentable over Nilsson (Nilsson et al., US 2020/0224244 A1, EFD of 10/9/2017) in view of Bava (Bava et al., US 2019/0093156 A1) and Chen (Chen et al., US 2019/0360044 A1; cited on IDS of 11/4/2025) . Claim 29: Nilsson teaches a method of target nucleic acid detection (Abstract). Nilsson teaches that this may be performed upon cells of a tissue sample (paragraph [0127]), and that detection comprises sequencing of the nucleic acid (paragraph [0089, 0130]). Nilsson teaches permeabilizing the cells of the tissue sample that contains the target nucleic acid (paragraph [0197]). Nilsson teaches contacting the cells with a padlock probe that comprises a sequence complementary to the target nucleic acid sequence which produces a padlock probe bound to the target nucleic acid sequence (paragraph [0008, 0026-0027, 0029-0034]). Nilsson teaches contacting the padlock probe bound to the target nucleic acid sequence with a reverse transcriptase when the template is RNA and gap-filling is performed on the padlock probe (paragraphs [0034 and 0093]). Nilsson teaches contacting the target nucleic acid sequence on the padlock probe with a ligase to form a circularized padlock probe having the target nucleic acid sequence (paragraph [0029, 0034, 0140-0141]). Nilsson teaches performing rolling circle amplification (RCA) on the circularized padlock probe which creates a linear repeating sequence comprising multiple copies of the target nucleic acid sequence (paragraph [0006,0029]). Nilsson then teaches that the target nucleic acid sequence is detected, for example by sequencing (paragraph [0130-131]). Nilsson teaches, after RCA, digesting the RACA products into monomers, each containing the repeat sequence, and then amplifying said monomers with primers comprising Illumina index adapter sequences, thus obtaining multiple copies of the target nucleic acid sequence comprising the sequence of the extended primer and the index adapter sequence to facilitate sequencing (paragraph [0252]). Nilsson does not teach, after performing RCA, annealing multiple copies of a primer to the linear repeating sequences, extending the multiple copies of the primer to generate amplicons comprising the target nucleic acid sequence, or performing combinatorial indexing on the amplicons to generate extended products comprising the target nucleic acid sequence and a barcode sequence capable of identifying a cell of origin of the extended products. Additionally, Nilsson does not teach obtaining this single cell nucleic acid sequencing data for extended products from millions of cells in a single run. However, this methodology, applied to millions of cells in a single run, is known in the art, as taught by Bava. Bava teaches a method for the detection of target molecules (Abstract). Bava teaches that said method comprises the use of a padlock probe amplified via RCA to generate a template comprising multiple copies of a target nucleic acid sequence (paragraph [0006] and Figure 12). Bava teaches, after amplification, annealing multiple copies of a primer to said template and forming an extension product which is a copy of the UBA (UBA = unique binding agent, which corresponds to the target nucleic acid sequence, paragraph [0006] and Figure 12). Bava teaches performing combinatorial indexing on the amplicons to generate extended producs which comprise the target nucleic acid sequence and a barcode sequence which is capable of identifying a cell of origin of the extended products (cell originating barcode, COB; paragraph [0006] and Figure 12). Bava teaches obtaining single cell nucleic acid sequencing data by identifying the polynucleotide sequence of the extended products and obtaining single cell nucleic acid sequencing data from the sample (paragraph [0007-0008]). Bava teaches that this methodology is performed on 10 6 cells (reads on about 1,000,000 and about 1x10 12 cells in a single run ; paragraph [0345]). Bava teaches that each cell originating barcode is composed of subcodes (SCs) that make up the barcode (paragraph [0345-0347]). Therefore, the barcode is actually a combination of barcode sequences (relevant to claim 26). It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Nilsson to perform combinatorial indexing on the primers annealed to multiple copies of the target nucleic acid within the RCA product as taught by Bava. One would be motivated to do so given the assertion by Bava that this enables the generation of millions of unique cellular barcodes with a high confidence of tag uniqueness (paragraph [0200-0208, 0345]) and enables the detection of target molecules on a single cell basis within samples with complex cell populations (paragraph [0090]). One would have a reasonable expectation of success given that Bava teaches that this methodology can be applied to cells within permeabilized tissue samples (paragraph [0148, 0187]), as is employed in the methodology of Nilsson. As taught by Nilsson, the primers may contain index adapters for sequencing, thus with the incorporation of the methodology of Bava, the primers annealed to the multimeric RCA product may include an index adaptor sequence, thus facilitating downstream sequencing and incorporating the index adaptor sequence into the extended product containing the target nucleic acid sequence and the barcode sequence identifying the cell of origin. Nilsson in view of Bava do not teach that the padlock probe comprises a UMI sequence. However, inclusion of UMI sequences in padlock probes is known in the art, as taught by Chen. Chen teaches a method of single-cell analysis comprising quantifying target nucleic acids in individual cells based on sequencing amplicons of said target nucleic acids (Abstract). Chen teaches that amplification of the target nucleic acid can be achieved through hybridization of padlock probes (also called “MIPs”) to the target analyte and amplification through RCA or PCR (paragraphs [0038, 0042], Figure 5, Figure 7). Chen teaches that the padlock probes comprise a UMI (paragraph [0042]). It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Nilsson in view of Bava to incorporate UMIs into the padlock probe. One would be motivated to do so given the assertion by Chen that incorporation of a UMI allows for determination of the number of transcripts that given rise to a particular amplification product (paragraph [00257]). One would have a reasonable expectation of success given that Chen teaches that this is particular advantageous when aiming to detect and quantify “unique amplified products” and the preferred mode of amplification is isothermal (which RCA is; paragraph [0257]). Claim 30: Nilsson teaches that the target nucleic acid sequence comprises a target RNA sequence (paragraph [0001]). Claim 31: Bava teaches that the combinatorial indexing comprises use of a microfluidic chamber (paragraph [0035]). Claim 32: Bava teaches that the combinatorial indexing is performed in the permeabilized cells and applied with 2 iterations of cell splitting (paragraphs [0341-0347]). Response to Remarks In light of Applicant’s amendments to the claims, all previous 103 rejections were withdrawn. Therefore, Applicant’s arguments (Remarks of 3/30/2026) regarding the previous rejections are moot. New 103 rejections have been made above. Conclusion No claims are allowed. 07-40 AIA 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. Any inquiry concerning this communication or earlier communications from the examiner should be directed to KAILEY E CASH whose telephone number is (571)272-0971. The examiner can normally be reached Monday-Friday 8:30am-6pm ET. 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. 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If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000. /KAILEY ELIZABETH CASH/Examiner, Art Unit 1683 /STEPHEN T KAPUSHOC/Primary Examiner, Art Unit 1683 Application/Control Number: 18/000,222 Page 2 Art Unit: 1683 Application/Control Number: 18/000,222 Page 3 Art Unit: 1683 Application/Control Number: 18/000,222 Page 4 Art Unit: 1683 Application/Control Number: 18/000,222 Page 5 Art Unit: 1683 Application/Control Number: 18/000,222 Page 6 Art Unit: 1683 Application/Control Number: 18/000,222 Page 7 Art Unit: 1683 Application/Control Number: 18/000,222 Page 8 Art Unit: 1683 Application/Control Number: 18/000,222 Page 9 Art Unit: 1683 Application/Control Number: 18/000,222 Page 10 Art Unit: 1683 Application/Control Number: 18/000,222 Page 11 Art Unit: 1683 Application/Control Number: 18/000,222 Page 12 Art Unit: 1683 Application/Control Number: 18/000,222 Page 13 Art Unit: 1683 Application/Control Number: 18/000,222 Page 14 Art Unit: 1683 Application/Control Number: 18/000,222 Page 15 Art Unit: 1683 Application/Control Number: 18/000,222 Page 16 Art Unit: 1683
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