DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election without traverse of Group I, claims 22-28, in the reply filed on 5/26/2026 is acknowledged. Claims 1-21 and 29-63 have been cancelled.
Priority
This application is a CON of 16/766,928 filed 05/26/2020 now PAT 11,788,120 which is a 371 of PCT/US2018/062650 filed 11/27/2018 which claims benefit of 62/590,889 filed 11/27/2017.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 1/22/2025 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
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.
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.
Claim(s) 22-28 is/are rejected under 35 U.S.C. 103 as being unpatentable over Fan et al (WO 2015031691, March 2015).
Regarding claims 22-28, Fan et al teach teaches methods of capturing mRNA from a cell and sequencing the captured mRNA, wherein the method comprises distributing a single substrate into a microwell array, (e.g., para [0177] and [0579]), wherein each substrate is attached to an oligonucleotide having an oligo(dT) at its 3’ end, which hybridizes to a poly(dA) region of mRNA (e.g., para [0579-0580]), and having at it’s 5’ end a universal priming site (which serves as a “sequencing adapter”), a cell label and a molecular label (e.g., para [0579]). Fan teaches that the methods disclosed therein include performing a step of imaging microwells to detect labeled nucleic acids and/or labeled amplicons (e.g., para [0213] and [0396-0388]). It is disclosed that the molecular labels – i.e., barcodes – are unique to each individual cell and bead (e.g., para [0003], [0237], [0241]). It is stated “The oligonucleotide may be referred to as a molecular bar code. The oligonucleotide may be referred to as a label (e.g., molecular label, cellular label) or tag (e.g., sample tag)” (para [0237]). It is disclosed that an oligonucleotide may comprise 5 or 8 or more molecular labels / barcodes (e.g., para [0248] and [0270]). Fan teaches detecting mRNAs by hybridization with a labeled probe (e.g., para [0376]), including a fluorescently-labeled probe (e.g., para [0455]). Fan (para [0152]) states: “In some instances, the plurality of probes comprises a sequence that is complementary to at least a portion of the sample tag, molecular identifier label, nucleic acid, or a combination thereof. In other instances, the plurality of probes comprises a sequence that is complementary to the junction formed by the attachment of the sample tag or molecular identifier label to the nucleic acid.”
Fan teaches wherein the substrate is selected from the group consisting of a polydimethylsiloxane (PDMS) solid support, a polystyrene solid support, a glass solid support, a polypropylene solid support, an agarose solid support, a gelatin solid support, a magnetic solid support, a pluronic solid support, and any combination thereof. In some embodiments, the plurality of oligonucleotides comprise a linker comprising a linker functional group, and the solid support comprises a solid support functional group; wherein the solid support functional group and linker functional group connect to each other. In some embodiments, the linker functional group and the solid support functional group are individually selected from the group consisting of C6, biotin, streptavidin, primary amine(s), aldehyde(s), ketone(s), and any combination thereof. In some embodiments, molecular labels of the plurality of oligonucleotides comprise at least 15 nucleotides ([0004]). Fan teaches wherein the microwell array of the substrate may comprise of a first and second or more substrate surfaces which may be clamped together with a seal (see e.g., [00100] – [0103] and Figures 70-73). Fan explains that the at paragraphs [00137] – [0138]: [00137] The terms "support", "solid support", "semi-solid support", and "substrate" may be used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. A support may refer to any surface that is transferable from solution to solution or forms a structure for conducting oligonucleotide-based assays. The support or substrate may be a solid support. Alternatively, the support is a non-solid support. A support may refer to an insoluble, semi-soluble, or insoluble material. A support may be referred to as "functionalized" when it includes a linker, a scaffold, a building block, or other reactive moiety attached thereto, whereas a solid support may be "nonfunctionalized" when it lacks such a reactive moiety attached thereto. The support may be employed free in solution, such as in a microtiter well format; in a flow-through format, such as in a column; or in a dipstick.
[00138] The support or substrate may comprise a membrane, paper, plastic, coated surface, flat surface, glass, slide, chip, or any combination thereof. In many embodiments, at least one surface of the support may be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like. According to other embodiments, the solid support(s) may take the form of resins, gels, microspheres, or other geometric configurations. Alternatively, the solid support(s) comprises silica chips, microparticles, nanoparticles, plates, and arrays. Solid supports may include beads (e.g., silica gel, controlled pore glass, magnetic beads, Dynabeads, Wang resin; Merrifield resin, Sephadex/Sepharose beads, cellulose beads, polystyrene beads etc.), capillaries, flat supports such as glass fiber filters, glass surfaces, metal surfaces (steel, gold silver, aluminum, silicon and copper), glass supports, plastic supports, silicon supports, chips, filters, membranes, microwell plates, slides, or the like, plastic materials including multiwell plates or membranes (e.g., formed of polyethylene, polypropylene, polyamide, polyvinylidenedifluoride), wafers, combs, pins or needles (e.g., arrays of pins suitable for combinatorial synthesis or analysis) or beads in an array of pits or nanoliter wells of flat surfaces such as wafers (e.g., silicon wafers), wafers with pits with or without filter bottoms. See also paragraphs [00147]- [00159, [00164 and [00168]. Fan teaches that linkage of molecular reagents/components or nucleic acid to the solid support may be covalent or non-covalent ([00141], [00146] and [00173]). Fan also discuss oligonucleotide attachment or immobilized on functionalized solid supports ([00169] – [00171]. See also para. [00186]. Fan et al discuss adding at least one buffer, wherein the buffer may encompass a lysis buffer into the microwell ([0007], [0051] and [0054]). Finally, Fan teaches analyzing the substrate using fluorescence-based methodologies (see [0054, [0082], [0092], [00127] and [00167].
Fan does not expressly teach which of the substrate surfaces comprises a siloxane material or glass or which of the substrate surfaces comprise of the oligo-dT primer as claimed. However, the Fan teaches multiple embodiments including wherein a first surface may be functionalized to comprise of an affinity resin and a second surface functionalized to comprise of a component ([0010]- [0011]). The reference teaches where one surface may be functionalized with a nuclei acid or an antigen binding protein and a second functionalized with a second components or reagent ([0011] – [0014] and [0018). MPEP 2144.04 states that the selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results); In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930).
Thus, it would have been prima facie obvious to one of ordinary skill in the art at the time of the effective filing date of the claimed invention to have select which substrate surface to immobilize the nucleic acid or other reagents based on the practitioner’s desired results. Such modifications of the methodology of the Fan are within the ordinary artisan capabilities and would not negatively alter or modify the results of capturing and sequencing RNA as taught by Fan. Accordingly, the ordinary artisan could expect a reasonable expectation of success carrying out the invention of Fan to achieve the results of capturing RNA.
Prior Art
9. The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Gerahn et al (WO 2017124101) [0010] The present application provides a high-throughput parallel single cell biochemical analysis in an array of wells or containers comprising or characterized by providing a first functionalized surface of each well or container, wherein the functionalized surface comprises an affinity resin; and providing a second functionalized surface of an array material to a top surface, wherein the functionalized surface provides accessible ionic functional groups. In an aspect of the method, the method further comprises at least one or more additional functionalized surface of each well or container, wherein the additional functionalized surfaces provides for multiplexing reactions. In a further aspect, the array material comprises polydimethylsiloxane, polycarbonate, polystyrene, polymethylmethacrylate, polyvinylidene difluoride, polyvinylchloride, polypropylene, cyclic olefin co-polymer, a glass, or silicon. In another aspect of the method, the array material is doped with an ionic functional group anywhere from 1% to 30% (by molar basis). In an embodiment of the method, the array material is doped with a functional group which can undergo further molecular bonding or modification via covalent bonding anywhere from 1% to 30% (by molar basis). In an embodiment, the ionic functional group is a negatively charged functional group. In a further embodiment, the negatively charged functional group is a carboxylic acid. In an aspect of the method, the top surface comprises an array functionalized to an organosilane. In a further aspect, the organosilane functionalized array surface provides free alcohols upon treatment. In an embodiment, the treatment comprises air plasma under mild vacuum. In an embodiment, the free alcohols are reacted with an amino- silate providing an amine. In a further embodiment, the amine is activated with an isothiocyanate. In an embodiment, the isothiocyante is conjugated to a polysaccharide. In an embodiment, the ionic functional group is a positively charged functional group. In a further embodiment, the positively charged functional group is an amine. In an embodiment, the amine is a polyamine. In an aspect of the method, the amine is selected from a group consisting of chitosan, poly(lysine), polyglucosamine and poly(acetyl)glucosamine. In another aspect of the method, the affinity resin comprises a negatively charged surface, wherein the negatively charged surface repels mRNA, and wherein the negatively charged surface enables mRNA to be captured by poly(dT) beads. In an aspect, the affinity resin comprises a carboxylate. In a further aspect, the carboxylate is poly(glutamate) glutamate or aspartate. In an embodiment, the wells or containers comprise micro-sized wells, nano-sized wells, or pico-sized wells. In an aspect of the method, the wells or containers are sealed with a membrane. In an embodiment, the membrane. In an embodiment, the membrane is an ultrafiltration membrane. In an embodiment, the membrane comprises a weak cationic exchange surface. In an embodiment, the ultrafiltration membrane comprises a polymeric surface functionalized with chitosan.
[0011] The present application provides a microwell comprising or characterized by a first functionalized surface of each well or container, wherein the functionalized surface comprises an affinity resin; and a second functionalized surface, wherein the functionalized surface provides accessible ionic functional groups. In an embodiment of the microwell, the second functionalized surface is configured for attachment of an ion exchange membrane. In an embodiment of the microwell, the top surface comprises an array functionalized to an organosilane. In a further embodiment, the organosilane functionalized array surface provides free alcohols upon treatment. In another embodiment, the treatment comprises air plasma under mild vacuum. In an embodiment, the free alcohols are reacted with an amino-silane providing an amine. In another embodiment, the amine is activated with an isothiocyanate. In an embodiment, the isothiocyante is conjugated to a polysaccharide. In another embodiment, the ionic functional group is a positively charged functional group. In an embodiment, the positively charged functional group is an amine. In a further embodiment, the negatively charged functional group is a polyamine. In an embodiment, the amine is selected from a group consisting of chitosan, poly(lysine), polyglucosamine and poly(acetyl)glucosamine. In another embodiment, the affinity resin comprises a negatively charged surface, wherein the negatively charged surface repels mRNA, and wherein the negatively charged surface enables mRNA to be captured by poly(dT) beads. In an embodiment of the microwell, the affinity resin comprises a carboxylate. In a further aspect, the carboxylate is poly(glutamate)glutamate or aspartate. In an embodiment, the wells or containers comprise micro-sized wells, nano-sized wells, or pico-sized wells. In an embodiment, the first functionalized surface comprises a nucleic acid. In an embodiment, the first functionalized surface comprises an antigen binding protein. In an embodiment, the microwell is configured to contain a barcoded bead. The present application also provides an array comprising two or more microwells of the aforementioned microwells.
[0012] In a related aspect, the present application provides method for high-throughput parallel single cell biochemical analysis in an array of wells or containers comprising or characterized by (a) a first functionalized surface of an open well or container, wherein the functionalized surface comprises an affinity resin or antigen binding protein; (b) loading each functionalized surface well or container with anywhere from 1 to 5 cells; (c) further loading each functionalized surface well or container with a barcoded poly (dT) bead, wherein the barcoded bead; (d) sealing the first functionalized surface well or container with a second functionalized surface; wherein the second functionalized surface is an ultrafiltration membrane; wherein the second functionalized surface is transfixed to the edges of the well or container providing a closed vessel; wherein the functionalized surface provides accessible ionic functional groups; (e) performing successive buffer exchanges to lyse the cells in solution, wherein the lyse cells provide protein and nucleic acids; and, (f) removing the ultrafiltration membrane. In another aspect, the present application provides a method for high-throughput parallel single cell biochemical analysis in an array of wells or containers comprising: (a) delivering a first reagent to a container comprising a functionalized inner surface; (b) attaching a membrane to a functionalized top surface; and (c) delivering a second reagent to the container by transfer through the membrane. In an embodiment, the method further comprises analyzing the barcoded beads, protein, and nucleic acids. In an embodiment, the method further comprises analyzing the reaction products.
[0014] The present application provides an improved high-throughput parallel single cell biochemical analysis in an array of wells or containers comprising or characterized by providing a first functionalized surface of each well or container, wherein the functionalized surface comprises an affinity resin; and providing a second functionalized surface of an array material to a top surface, wherein the functionalized surface provides accessible ionic functional groups.
Conclusion
10. No clams are allowed. Any inquiry concerning this communication or earlier communications from the examiner should be directed to CYNTHIA B WILDER whose telephone number is (571)272-0791. The examiner can normally be reached Flexible.
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/CYNTHIA B WILDER/Primary Examiner, Art Unit 1681
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