DETAILED CORRESPONDENCE
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 .
This action is in response to the papers filed December 1, 2025. Currently, claims 1, 3, 6-12, 14-18, 20-21 are pending.
All arguments have been thoroughly reviewed but are deemed non-persuasive for the reasons which follow.
This action is FINAL.
Any objections and rejections not reiterated below are hereby withdrawn.
Priority
This application claims priority to
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Drawings
The drawings are acceptable.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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, 3, 6-12, 14-18, 20-21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Fan (US Publication 2015/0299784) in view of Sims et al. (US 11,788,120, October 17, 2023, provisional November 2017) in view of Cai et al. (US 2016/0369329, December 22, 2016) and further in view of Ramsey (WO 2013/176767).
Fan teaches multiplex nucleic acid analysis of single cells (abstract). Figure 30 illustrates a flowchart for Example 13.
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With respect to Claims 1, 12 and 17, Fan teaches capturing cells in microwells and loading beads into the microwells such that the cells are in proximity with the beads, i.e. particles. Fan teaches cell lysis of the cells which allows binding of the bead targets with the mRNA from single cell. Fan then teaches performing reverse transcription, i.e. RT, to form cDNA. Multiplex PCR is then performed to obtain a second strand synthesis. Fan teaches using the molecular barcodes to decode the cell type, the sample index region, and different label regions, for example. Each of these regions may be decoded with a probe (see para 252, 253, 244). Fan teaches an oligonucleotide may comprise a universal label, a cellular label, a molecular label, and a sample label. These labels may be used to distinguish target nucleic acids between samples, different target nucleic acids, different cells (see para 244). Fan teaches one or more detectable labels to the molecular barcode, molecular identifier label, sample tag and the labeled nucleic acid may be used (para 457). Fan teaches contacting samples with first-labeled and second labeled nucleic acid sequences sequentially. Sequentially is one after the other such that the first probe was removed (para 115)(limitations of Claim 3). Fan teaches each microwell array may be imaged in its entirety within a single image or a series of images may be “tiled” to create a high resolution image of the entire array (para 221).
Fan does not teach hybridizing a first probe including a first fluorophore to products corresponding to a first subset of target and a second probe including a second fluorophore corresponding to the second subset or performing the reverse transcription, second strand synthesis operation or the two target detection at a sample processing substrate.
However, Sims teaches methods for trapping single-cell lysates in microwells capable of capturing RNA on beads (abstract). Sims teaches mRNA is immobilized on a glass surface, and enzymatic processing steps can take place on chip (col. 14, lines 23-25). Specifically, Sims teaches introducing, i.e. capturing a cell and a capture bead in a microwell. The particle beads are functionalized and comprise oligonucleotides sequences attached thereto. Sims teaches adding a lysis buffer to the microwells and capturing RNA from the cell onto the capture bead and reverse transcribing (see Claim 1 of Sims, step b). Sims further teaches a cDNA library may be generated (col. 19, lines 22-25). Sims teaches optically demultiplexing the capture bead barcodes using fluorescence hybridization wherein sequential fluorescence hybridization comprises sequentially introducing mixtures of labeled probes into the microwell, i.e. detecting a first probe, dehybridizing and hybridizing and detecting a second sequence. Sims teaches 12 rounds of sequential FISH were used to read out the 12-digit binary optical barcode sequence. Each round included background scan, probe hybridization, probe scan and probe stripping (i.e. dehybridization).
Sims teaches 12 sequential rounds that each require probe stripping, i.e. dehybridization (col 20, lines 15-20). Sims teaches representative images from optical phenotyping of cells and from optical demultiplexing of beads in Figure 16A (col. 20, lines 30-35). An automated scanning system was used (see col. 20, lines 35-32). With respect to Claims 6, 11, 15 Sims teaches a barcoded capture oligonucleotide that contain oligo (dT) for capturing mRNA from individual eukaryotic cells (col. 12, lines 65-67 thru Col. 13, lines 1-5). With respect to Claim 7, Sims teaches the bead may be functionalized with primer that are specific to targeted DNA loci or RNA transcripts (col. 13, lines 6-9). With respect to Claims 8, 14, 16, 18, Sims teaches aligning molecules to the human transcriptome for a mixed species analysis (see Figure 18A, for example). Sims also teaches a fluorescence image of single cell transcriptome “prints” arrayed on a glass coverslip (see Figure 4B). With respect to Claim 20, Sims teaches a histogram showing that the number of genes (i.e targets) detected per individual cell ranges from 1,000 to 17,000 (see Figure 12). With respect to Claim 21, Sims teaches genes can be quantified from sequencing data based on the number of unique molecular identifiers associated with each gene.
Further, Cai teaches multiplex labeling of molecules by sequential hybridization barcoding using probes (abstract). As see in Figure 2, Cai teaches analysis of target mRNA sequences using at least two probes that are differentially labeled with different dyes (i.e. purple, blue and green). Cai teaches probes can be stripped and rehybridizes to the same mRNA in multiple cycles of hybridization (para 350).
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Figure 29 also provides hybridization chain reaction of targets using readout probes that are differentially labeled. The sequential barcoding to multiplex different targets can profile large number of targets (para 182). Each target, after rounds of contacting and imaging, has its own unique combination of labels (sequential barcoding) so that information such as quantitative and/or spatial information, can be obtained for a target (para 184).
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Further, Ramsey teaches a method that provides beads in a plurality of wells and performs PCR, RT-PCR or LAMP reactions in the wells (see para 54).
Therefore, it would have been prima facie obvious prior to the effective filing date of the instant application to have modified the nucleic acid detection assay of Fan to perform the enzymatic processing on the substrate, as taught by Sims. Fan specifically teaches wells may be sealed during cell lysis, for example to prevent cross hybridization of target nucleic acid between adjacent microwells (para 310). Sims teaches lysis, reverse transcription and hybridization analysis may all be performed in the same well on-site. Figures 4-5 of Sims illustrates on-chip reverse transcription in single wells for single cells. The ordinary artisan would have recognized the reverse transcription and second-strand synthesis steps on-chip because Sims teaches enzymatic processing steps can take place on-chip. The ordinary artisan would have recognized this was an alternative means for performing enzymatic processing steps without the need to transfer of capture beads from the microwell array to a tube.
The prior art further teaches it would have been obvious to use two or more different probes with two or more different fluorophores to detect targets with a dehybridization step, as taught by Cai. Cai teaches sequential hybridization may be performed using several probes with several different labels to detect and profile large numbers of targets.
Further, it would have been prima facie obvious prior to the effective filing date of the instant application to have modified the nucleic acid detection assay of Fan to perform the RT-PCR and probe detection steps in the microwell, as taught by Ramsey. Ramsey illustrates RT-PCR and probe detection steps may be performed following bead analysis in the well. The ordinary artisan would have recognized additional collection of beads and transfer of the beads to a tube would not be required. Further, the wells ensure one cell is analysis at a time.
With respect to Claim 6, 15 Fan teaches target binding region may comprise a random multimer sequence or an oligo dT sequence (e.g., a stretch of thymidine nucleotides that may hybridize to a poly-adenylation tail on mRNAs)(para 248).
With respect to Claim 7, Fan teaches the target binding nucleic acid may be a gene specific sequence that may attach to a specific location of a specific target nucleic acid (para 248).
With respect to Claims 8, 14, 16, Fan teaches the method allows for identification of transcriptome, such as from a diseased or non-diseased subjected (Para 302). The method may represent from 0.01%-100% of the transcripts of an organism’s transcriptome (para 365).
With respect to Claim 9, Fan teaches analysis of B cells expressing IgM and IgD, for example (para 588).
With respect to Claim 10-11, Fan teaches analysis may be of mRNA or cDNA targets (see para 262, 337, 494).
With respect to Claim 18, Fan teaches a flow cell comprises a plurality of microarray chambers that interface with a plurality of microwell arrays such that one or more samples may be processed in parallel (para 764).
With respect to Claim 20, Fan teaches target specific regions may comprise a panel from 2-50,000 biomarkers (para 17).
With respect to Claim 21, Fan teaches a method for quantitation of RNA molecules in a sample (para 117, see Figure 56, 330).
Response to Arguments
The response traverses the rejection. The claims have been amended to require generating an image dataset from the optical detection of the first and second probe. This was the limitation of previously presented Claim 4 and 5. Claim 4 and 5 were rejected. Fan teaches each microwell array may be imaged in its entirety within a single image or a series of images may be “tiled” to create a high resolution image of the entire array (para 221). The response cites para 252 and Figure 30 from Fan to support a barcode is used to determine the location of the bead. This argument has been reviewed but is not persuasive. Fan may be used for all it teaches. Fan teaches serial images tiled to determine the target location of cells (para 221).
Sims teaches representative images from optical phenotyping of cells and from optical demultiplexing of beads in Figure 16A (col. 20, lines 30-35). Figure 17A and B are further directed to probe hybridization and stripping images. Figure 17 does not process maps of synthetic beads. The figure illustrates probe analysis. Sims teaches the targets may be analyzed using FISH followed by stripping the probe and adding a new probe. This is characterization using probes and does not require sequencing.
Each of these references supports generating an image dataset for the probes and spatially characterizing the biological targets. The ordinary artisan would be motivated to have used different probe detection methods as taught by Cai, for example, for differential detection of targets. Cai teaches sequential hybridization may be performed using several probes with several different labels to detect and profile large numbers of targets. Applying the sequential hybridization method of Cai in wells would have been prima facie obvious in light of the teachings of Fan and Sims that probes may be detected in wells.
Even more, the barcodes on the beads are surrogates for the targets. Therefore, determining positions of beads is indicative of target location.
The response asserts the ordinary artisan would not have combined Fan’s open microwell system with Cai’s method because it would destroy the integrity of the very sample Fan seeks to preserve, rendering the process inoperable. This argument has been reviewed but is not persuasive. Fan specifically teaches methods to seal wells to protect and prevent cross contamination. Sims specifically teaches enzymatic process can take place on-chip. Thus, the ordinary artisan would have recognized the enzymatic processing would eliminate the requirement to transfer the beads to a tube for processing.
The response argues Fan teaches away from an integrated process. This argument has been reviewed but is not persuasive. The MPEP 2145(X)(D)(1) provides “the prior art’s mere disclosure of more than one alternative does not constitute a teaching away from any of these alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed….” The teachings of Fan do not discourage a skilled artisan from performing the enzymatic process on-chip. Sims specially teaches the enzymatic process may be performed on-chip. The mere teaching of a set of method steps is not a teaching away if it does not criticize, discredit or otherwise discourage investigation.
The response argues the combination of Fan, Cai and Ramsy is impermissible hindsight because modifying Fan’s “protective ex situ method” would be contrary to its teachings and would destroy the sample integrity Fan seeks to preserve. As provided by MPEP 2145, "[a]ny judgment on obviousness is in a sense necessarily a reconstruction based on hindsight reasoning, but so long as it takes into account only knowledge which was within the level of ordinary skill in the art at the time the claimed invention was made and does not include knowledge gleaned only from applicant’s disclosure, such a reconstruction is proper." In reMcLaughlin, 443 F.2d 1392, 1395, 170 USPQ 209, 212 (CCPA 1971). Here, the prior art teaches each limitation of the instant claims.
Conclusion
No claims allowable over the art.
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 JEANINE ANNE GOLDBERG whose telephone number is (571)272-0743. The examiner can normally be reached Monday-Friday 6am-3:30pm.
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/JEANINE A GOLDBERG/Primary Examiner, Art Unit 1682
February 6, 2026