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 8-17 and 19, drawn to a microfluidic process, in the reply filed on 8/7/2025 is acknowledged. Accordingly, the claim 18 is withdrawn from consideration as being drawn to non-elected invention.
Priority
This application is a DIV of 16/494,537 filed 09/16/2019 now US PAT 11,427,853 which is a 371 of PCT/EP2018/056551 filed 03/15/2018.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 11/22/2022 are 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.
Drawings
The drawings were received on 11/22/2022. These drawings are found acceptable by the Examiner.
Specification
The disclosure is objected to because of the following informalities:
(a) The use of the term “Illumina” page 9, “Triton X”, “Tween-20”, “IGEPAL CA 630”, “Superscriptase”, “MultiScribe”, “SMartScribe”, at pages 31-33, which are a trade names or marks used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore, the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM, or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claim 17 is objected to because of the following informalities:
(a) Claim 17 recites the limitation “reagents-droplets”. No other recitation of the limitation with a dash line (-) between the limitations “reagents” and “droplets” has been noted in the claims or specification. It cannot be determined if the limitation is intended to represent a single terminology or multiple limitations. Appropriate correction is required.
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) is/are rejected under 35 U.S.C. 103 as being unpatentable over Nicol et al {Nicol, used interchangeably herein} (WO 2016145409), September 2016) in view of Labanieh et al {Labanieh, used interchangeably herein} (Micromachines (Basel), 6(10): 1469-1482, October 2015).
The claims of the instant invention are directed to a microfluidic method for barcoding nucleic acids from a cell comprising primarily: providing a microfluidic device, a droplet of a first type and its migration step, a droplet of a second type and its migration step, two droplet fusion steps and two barcode introduction techniques in the fused droplets.
Regarding claim 8, Nicol discloses a method of assigning a binding phenotype of an expressed polypeptide to a potential genotype, comprising: partitioning cells into individual compartments, wherein the individual compartments comprise an origin-specific barcode; the individual compartments comprise a single cell; and the individual compartments comprise individual droplets generated on a microfluidic device,
(see claims 1, 2, 34 beginning at page 108 which are equivalent to claim 8 herein, "a microfluidic method for barcoding cell nucleic acids, comprising providing a microfluidic device comprising a chip comprising at least one microfluidic channel and a plurality of reservoirs"); forming the single droplet by merging a first droplet comprising the cell with a second droplet comprising the barcode; and further comprising merging the single microdroplet with a third microdroplet comprising an additional reagent; and the third droplet may comprise reverse transcription reagents, cell lysis reagents, dNTPs, and DNA polymerase, among others (see also e.g., para. [0023], [0026]-[0027], [0030], [0113]-[0120], [0207], [0212], [0216], [0226] and [0238]);
(see claims 35, 37-38 at page 112-113,which are equivalent to the steps of "-injecting a carrier fluid into the inlet of said microfluidic channel, as described in the instant claim 8 herein, the carrier fluid comprises a plurality of droplets of a first type dispersed in the carrier fluid, wherein the droplets of the first type are single cell droplets or RT droplets, wherein at least some of the RT droplets comprise a reverse transcriptase and at least one oligonucleotide, and wherein at least some of the single cell droplets comprise one single cell, wherein the single cell comprises a nucleic acid from a single cell for a plurality of reservoirs (compartments), a first migration step wherein at least one droplet of the first type in the plurality of droplets is moved into one of the plurality of reservoirs by buoyant force; -injecting a carrier fluid into the inlet of the microfluidic channel, the carrier fluid comprises a plurality of droplets of the second type dispersed in the carrier fluid, wherein the droplets of the second type are single cell droplets or RT droplets, and wherein the droplets of the second type are RT droplets when the droplets of the first type are single cell droplets, or the droplets of the second type are single cell droplets when the droplets of the first type are RT droplets, for a plurality of reservoirs, a second migration step wherein at least a portion of at least one droplet of the second type enters into one of the plurality of reservoirs; for a plurality of reservoirs, said at least one droplet of said first type fuses with said at least one droplet of said second type in the respective reservoir or at an edge thereof, resulting in fused droplets "); tagging the expressed target nucleic acids and the bound polypeptide captor molecules with barcodes to produce barcoded expressed target nucleic acids and barcoded polypeptide captor molecules; the barcode may further comprise primer sequences, sequencing adaptors, restriction sites, enrichment capture sequences; and tagging the expressed target nucleic acid and the captor molecule nucleic acid identifier comprises introducing reagents into the individual compartments, the reagent is sufficient to allow the first and second types of source-specific barcodes to hybridize to captor molecule nucleic acid identifiers of the target nucleic acids and the polypeptide captor molecules, respectively, and serve as a template to generate cDNA copies of all or a portion of the target nucleic acids and captor molecule specific nucleic acid identifiers, the sequence of the oligonucleotide barcode is pooled in each target nucleic acid cDNA product and captor molecule nucleic acid identifier cDNA product (see also [0119] – [0120], [0135] – [0140], [0142] –[0143], [0170], [0207], [0212], [0216], [0226] and [0238]),
(see Nicol claims 1, 8, 11, which are equivalent to the step(s) of "a) a fused droplet as described in the instant claim 8, hybridizing at least some of said nucleic acids from a single cell with said at least one oligonucleotide in said fused droplets, b) in each fused droplet, reverse transcribing at least some of the nucleic acids from the single cell present in the fused droplets, thereby generating single cell cDNA, and c) attaching at least one barcode sequence to the single cell cDNA obtained in step b), wherein the at least one barcode sequence encodes the identity of the single cell; or a) for each fused droplet, hybridizing at least some of the nucleic acids from the single cell from the at least one cell with the at least one oligonucleotide in the fused droplets, wherein the at least one oligonucleotide comprises at least one barcode sequence, b) in each fused droplet, reverse transcribing at least some of said nucleic acids from the single cell present in said fused droplet, thereby generating barcoded single cell cDNA, wherein said at least one barcode sequence encodes the identity of said single cell") (see also para. [0026] – [0027], [0030], [0155], [0170], [0207], [0212], [0216], [0226] and [0238]).
With regards to the limitations associated with specific details of microfluidic chip device as instantly claimed, Nicol teaches various aspects of a microfluidic chip device comprising a plurality of compartments (reservoirs) and steps of fusing reagent hydrogel beads into emulsion droplets wherein the drops are fused using an electric field generated with electrodes such that when applied two droplets merge (see e.g., Figures 11 and 14), Nicol does not expressly discuss inlets and plurality of microfluidic channels associated with the microfluidic chip disclosed therein or the specific migration step. However, such microfluidic device structures are well-known in the art as evidence by Labanieh et al.
Labanieh et al teach the design, fabrication and use of a dual-layered microfluidic device for the ultrahigh- throughput droplet trapping, analysis, and recovery using droplet buoyancy. Labanieh teach wherein the device comprises of a floating droplet chip, wherein the device consists of two layers of PDMS, one for droplet generation and assembly and the other for droplet trapping. The top layer is designed with a microwell array whose well dimension can be varied according to the desired droplet size to be trapped. The bottom PDMS layer was fabricated with a height of 50 micromolar and contains two aqueous inlets and a single oil inlet whereby respective fluids are directed to a flow focusing structure for droplet generation (Section 3.1). Labanieh teach after the flow-focusing structure, a widening winding channel which reduces the velocity of the droplets and aides in droplet visualization was included in the structure. Labanieh also teaches that the bottom layer also contains a chamber which is oriented below the well array (section 3.1)
Labanieh teach clustering multiple droplets of differing contents (i.e., cells, reagents, or samples) and merging them using a chemical reagent or externally applied electric field, wherein said steps are achieved in channels (page 5-6) based on colocalization of droplets in a highly parallel and controlled fashioned (page 9, first full paragraph). Labanieh teach using the device, they were able to quantify the number of droplets, which contain an enzyme-conjugated bead among a cluster of immobilized droplets, and demonstrated that the device is useful for droplet clustering and real-time analysis by clustering several droplets together into microwells and monitoring diffusion of fluorescein, a product of the enzymatic reaction and its substrate between droplets (abstract and conclusion).
It would have been obvious to one of ordinary skill in the art at the time of the effective filing date of the claimed invention to have combined the floating droplet array of Labanieh to the droplet barcoding cell nucleic acid microfluidic process of Nicol, since the microfluidic chip device used in the droplet barcoding cell nucleic acid assay of Nicol comprise of similar structural and functional features of the device as taught by Labanieh. Such combinations are within the ordinary artisan capabilities and would not negatively alter or modify results of carrying out the droplet barcoding assay. The ordinary artisan would have been motivated to do so based on the advantages taught by Labanieh that the floating droplet array technology is advantageous because it combines compartmentalizing samples by droplet microfluidics with the ultrahigh-throughput analytics and parallelization capabilities of microarray formats and have the capabilities to be used in applications of single-cell or molecule analysis, genetic sequencing, biochemical profiling, cell culture, pathogen detection and drug discovery (page 10, beginning at line 7).
Regarding claim 9, Nicol teaches wherein at least some of the reagent droplets comprise a reverse transcriptase, a nuclease, a polymerase, or a ligase enzyme (e.g., [0026], [0034], and [0041]).
Regarding claim 10, Nicol teaches wherein at least some of the reagent droplets further comprise a polymer [e.g., [0079] which teaches exemplary discrete volumes or spaces useful in the disclosed methods include droplets (for example, microfluidic droplets and/or emulsion droplets), hydrogel beads or other polymer structures (for example poly-ethylene glycol di- acrylate beads or agarose beads).
Regarding claims 11-12, Nicol teaches wherein the plurality of droplets of the first or second type may comprise of picoliter volumes of from 1 to 750 pL or up to 1000 ml ([0154] and [0155]; see also [0196] at ~100 pL droplet). MPEP states “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Regarding claims 13-15 and 19, Nicol teaches that fusion is accomplished using an electric field generated with electrodes such that when applied the two droplets merge in microchannels [0026] [0216]. Nicol discusses fusing droplets containing reagents with other droplets containing components (see e.g., claims 35, 37). Nicol does not expressly teach the fusion occupancy efficiency or capturing efficiency or total volume of one droplet compared to another droplet. However, these percentages merely recite a plethora of conventional nucleic acid manipulation of reagents and methodologies, as well as well as routine optimization of reaction components, concentrations, and parameters and could easily been obtained based on the teaching provided by the ordinary artisan based on desired results. MPEP states “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). MPEP also states "The discovery of a previously unappreciated property of a prior art composition, or of a scientific explanation for the prior art’s functioning, does not render the old composition patentably new to the discoverer.” Atlas Powder Co. v. Ireco Inc., 190 F.3d 1342, 1347, 51 USPQ2d 1943, 1947 (Fed. Cir. 1999). Thus, the claiming of a new use, new function or unknown property which is inherently present in the prior art does not necessarily make the claim patentable. In re Best, 562 F.2d 1252, 1254, 195 USPQ 430, 433 (CCPA 1977). >In In re Crish, 393 F.3d 1253, 1258, 73 USPQ2d 1364, 1368 (Fed. Cir. 2004)". MPEP 2112.
Regarding claim 16, Nicol teaches wherein the method incorporated phenotypic coupling along with the droplet barcoding assay ([001], [003], [0017] , [0019], [0108] – [0109], [0174], [0179]).
Regarding claim 17, Nicol teaches wherein at least some of the reagents-droplets comprise at least one particle of a first type to which the at least one oligonucleotide is bound ([0030], [0034], [0041], [0192]).
Prior art
12. The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. Rotem et al (WO 2013134261, September 2013) teach embodiments related to microfluidics and epigenetic sequencing. In one set of embodiments, cells contained within a plurality of microfluidic droplets are lysed and the DNA (e.g., from nucleosomes) within the droplets are labeled, e.g., with adapters containing an identification sequence. The adapters may also contain other sequences, e.g., restriction sites, primer sites, etc., to assist with later analysis. After labeling with adapters, the DNA from the different cells may be combined and analyzed, e.g., to determine epigenetic information about the cells. For example, the DNA may be separated on the basis of certain modifications (e.g., methylation), and the DNA from the separated nucleosomes may be sequenced using techniques such as chromatin immunoprecipitation ("ChIP"). In some cases, the DNA sequences may also be aligned with genomes, e.g., to determine which portions of the genome were epigenetically modified, e.g., via methylation (abstract).
Conclusion
13. No claims 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