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 .
DETAILED ACTION
Status of Claims
Claims 183-197 are pending and the subject of this NON-FINAL Office Action. This is the first office action on the merits.
Claim Rejections - 35 USC § 112- Indefiniteness
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.
Claim 194-197 are 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 pre-AIA the applicant regards as the invention.
In claims 195-196, the following lack antecedent basis: “the . . . unique molecular
labels“; “the . . . mRNAs”; and “the . . . cellular component-binding reagent oligonucleotides.”
Claim 194 is confusing when it recites “the number of occurrences of the target nucleic acid molecule in the single cells indicates identifies the sample origin of the cell.”
Claims 195-197 are confusing because “comparable flowcell methods” is vague. Specifically, comparable in what ways? This is never defined. Although the phrase that follows is “performed using a single fluid injection,” yet it is unclear if this is the definition of “comparable,” or if “comparable” modifies other aspects of the “single fluid injection” technique. Moreover, no specific “single fluid injection” technique is disclosed, much less a “comparable” one. In sum, it is impossible to benchmark or compare the claimed method to another method without clear metes and bounds of the “comparable flowcell methods.”
Claim Rejections - 35 USC § 102
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)Novelty; Prior Art.—A person shall be entitled to a patent unless—
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; or
(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.
Claims 183-197 are rejected under 35 U.S.C. § 102(a)(2) as being anticipated by SIMS (US20210254143, effective filing 11/27/2017).
As to claim 183, SIMS teaches a method a method for determining a number of occurrences of a target nucleic acid molecule in single cells, the method comprising:
(a) providing a flowcell comprising a fluidic channel (Fig. 2A),
wherein the fluidic channel comprises a ceiling, a first sidewall, and a bottom, and
wherein the bottom comprises a substrate which comprises a plurality of microwells (Fig. 2A);
(b) capturing single cells and single beads in the plurality of microwells, wherein a single bead comprises a plurality of tethered barcodes, and wherein the plurality of tethered barcodes further comprises (Figs. 2A, 3B & 5; claim 1):
i) a bead-specific cellular label (paras. 0037-41- “(b) a barcode attached to the PCR handle, wherein the barcode is configured for identification of the attached capture bead and associated cell, wherein the barcode comprises N blocks, wherein each block is an oligonucleotide sequence of length 8 nt to 30 nt chosen from one of N sets of M oligonucleotides, wherein N is at least 2 and M is at least 30”; see also paras. 0028-36 & 0160, claims 41 & 51, Figs. 15 & 22-23);
ii) a diverse set of molecular labels (paras. 0037-41- “(c) a unique molecular identifier (UMI) of length 6 to 16 nucleotides (nt), wherein the UMI is either (i) attached to the barcode or (ii) segments of the UMI appear before or between or after the blocks of oligonucleotides, wherein the UMI may differ between oligonucleotide sequences on the capture bead, and wherein the UMI may differ between different capture beads”; see also paras. 0028-36 & 0160, claims 41 & 51, Figs. 15 & 22-23); and
iii) a plurality of target binding regions capable of hybridizing with target nucleic acid molecules (paras. 0037-41- “(d) an oligo(dT) attached to the UMI”; see also paras. 0028-36 & 0160, claims 41 & 51, Figs. 15 & 22-23),
(c) co-injecting a first fluid and a second fluid into the fluidic channel,
wherein the first fluid is introduced into the fluidic channel immediately before the second fluid,
wherein the first fluid interfaces with a surface of the content of the microwell for a duration,
wherein the first fluid comprises a lysis buffer,
wherein one or more components of the first fluid enter the microwell by diffusion and initiate cellular lysis, and
wherein the second fluid seals the content of the microwell (Figs. 2A and 5, showing “Cells are first deposited in the microwell array by gravity followed by beads (while circles) covalently functionalized with oligo(dT) primers (orange circular outlines). A lysis buffer is introduced followed by rapid displacement of fluid in the channel with oil, which conformally seals the array” as explained in para. 0046, and para. 0049; see also claim 46);
(d) hybridizing target nucleic acid molecules released from single cells following cellular lysis with the plurality of target binding regions tethered to single beads in a stochastic manner (id.);
(e) performing an extension reaction to create a plurality of molecular conjugates each comprising a barcode and a portion of a complementary sequence of one of the target nucleic acid molecule (id.);
(f) amplifying and sequencing the molecular conjugates (e.g. single cell RNA-seq; id.); and
(g) determining the number of occurrences of the target nucleic acid molecule in the single cells (id.).
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As to claim 184, SIMS teaches the density of the first fluid is greater than the density of the second fluid, and wherein the first fluid and the second fluid are immiscible (oil vs aqueous; Fig. 2A).
As to claim 185, SIMS teaches step (b) comprises priming the flow cell, loading the cells, and then loading the beads (paras. 0046 & 0049).
As to claim 186, SIMS teaches step (b) comprises priming the flow cell, displacing the priming buffer with an air injection, loading a cell suspension, displacing the cell suspension with an air injection, and loading the beads (id.)
As to claim 187, SIMS teaches the plurality of tethered barcodes further comprise a universal primer sequence (e.g. “PCR handle”; Figs. 15 & 22-23).
As to claim 188, SIMS teaches the plurality of target binding regions of the plurality of barcodes tethered to a bead comprise a mixture of sequences selected from the group consisting of gene-specific sequences, oligo-dT sequences, random multimer sequences, or any combination thereof. (id.)
As to claim 189, SIMS teaches the wherein the target nucleic acid molecules comprise RNA molecules (id.)
As to claim 190, SIMS teaches the target nucleic acid molecules comprise mRNA molecules (id.)
As to claim 191, SIMS teaches the target nucleic acid molecules comprise cellular component-binding reagent oligonucleotides (para. 0116, 0120). As best the Examiner can determine from Applicants’ disclosure, “cellular component-binding reagent oligonucleotides” means anything that binds a protein (which is anything, such as nucleic acids such as mRNA) (spec., paras. 0204ff).
As to claim 192, SIMS teaches the cellular component-binding reagent oligonucleotides comprise sample indexing oligonucleotides (para. 0116, 0120). Again, the “sample indexing oligonucleotide” is never defined (spec., paras. 0213-15). It is any sequence such as attached DNA (SIMS, para. 0116, 0120).
As to claim 193, SIMS teaches the target nucleic acid molecules comprises cellular component-binding reagent oligonucleotides, and wherein determining the number of occurrences of the target nucleic acid molecule in the single cells indicates the number of copies of a cellular component target in the single cell (id.)
As to claim 194, SIMS teaches the target nucleic acid molecules comprise sample indexing oligonucleotides, and wherein determining the number of occurrences of the target nucleic acid molecule in the single cells indicates identifies the sample origin of the cell (id.)
As to claim 195, SIMS teaches the second fluid sealing the content of the microwell yields an increase in the number of mRNAs and/or cellular component-binding reagent oligonucleotides captured by the barcodes as compared to comparable flowcell methods performed using a single fluid injection (super-Poisson loading; paras. 0127). In addition, this claim merely recited a result of the steps performed in claim 183, which cannot be separated from each other. In other words, same procedure, same results.
As to claim 196, SIMS teaches the second fluid sealing the content of the microwell yields an increase in the number of occurrences of unique molecular labels associated with each of the mRNAs and/or cellular component-binding reagent oligonucleotides determined as compared to comparable flowcell methods performed using a single fluid injection flowcell (id.) In addition, this claim merely recited a result of the steps performed in claim 183, which cannot be separated from each other. In other words, same procedure, same results.
As to claim 197, SIMS teaches the second fluid sealing the content of the microwell yields an increase in the signal-to-noise ratio as compared to comparable flowcell methods performed using a single fluid injection (id.) In addition, this claim merely recited a result of the steps performed in claim 183, which cannot be separated from each other. In other words, same procedure, same results.
Double Patenting- Obvious Type
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 obviousness-type 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); and 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 a nonstatutory double patenting ground provided the conflicting application or patent either is shown to be commonly owned with this application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement.
Effective January 1, 1994, a registered attorney or agent of record may sign a terminal disclaimer. A terminal disclaimer signed by the assignee must fully comply with 37 CFR 3.73(b).
Instant claims 183-197 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over conflicting claims 1-22 of US11976269, in view of SIMS (US20210254143, effective filing 11/27/2017).
The instant claims are obvious over the conflicting claims because the conflicting claims teach the same flowcell loading scheme for microwell loading of biological materials; and the prior art clearly teaches that such loading was directly applicable to . More specifically, the conflicting claims teach:
1. A method for introducing one or more components to contents of microwells comprising:
(a) introducing a first fluid into a fluidic channel comprising a ceiling, a first sidewall, and a bottom,
wherein the bottom of the fluidic channel comprises a plurality of microwells,
whereby the fluidic channel and each microwell of the plurality of microwells comprise the first fluid;
(b) introducing a first displacement fluid into the fluidic channel to displace the first fluid from the fluidic channel at a first flow rate;
(c) introducing a second fluid into the fluidic channel at a second flow rate while in contact with a second displacement fluid that is immiscible to the second fluid such that the second fluid is immediately followed by and in contact with the second displacement fluid and the second fluid and the second displacement fluid have the same flow rate,
wherein one or more components of the second fluid enters the content in the microwell when the second fluid comes into contact with the content in the microwell for a first duration, and
wherein the second displacement fluid displaces the second fluid from the fluidic channel and/or seals the content of the microwell.
2. The method of claim 1, comprising:
(d) introducing a third fluid into the fluidic channel at a third flow rate while in contact with a third displacement fluid that is immiscible to the third fluid such that the third fluid is immediately followed by and in contact with the third displacement fluid and the third fluid and the third displacement fluid have the same flow rate,
wherein one or more components of the third fluid enters the content in the microwell when the third fluid comes into contact with the content in the microwell for a second duration, and
wherein the third displacement fluid displaces the third fluid from the fluidic channel and/or seals the content of the microwell.
3. The method of claim 1, comprising introducing a third displacement fluid immediately prior to introducing a second fluid of the plurality of second fluids.
4. The method of claim 1, comprising introducing a third displacement fluid immediately after introducing the second displacement fluid.
5. The method of claim 1, comprising introducing a third displacement fluid immediately prior to introducing the second displacement fluid.
6. The method of claim 1, comprising introducing a third fluid immediately prior to introducing a second fluid of the plurality of second fluids.
7. The method of claim 1, comprising introducing a third fluid immediately after introducing the second displacement fluid.
8. The method of claim 1, comprising introducing a third fluid immediately prior to introducing the second displacement fluid.
9. The method of claim 1, wherein the second fluid and/or the third fluid enters the content of the microwell by diffusion.
10. The method of claim 1, wherein the flow rate of the first fluid ranges from 0.001 ml/sec to 100 ml/sec.
11. The method of claim 10,
wherein the fluidic channel further comprises an anterior position and a posterior position along a longitudinal path of the fluidic channel, the anterior position being located nearer to an inlet wherein the first fluid is introduced into the fluidic channel, and
wherein the ratio of the flow rate of the first fluid at the anterior position relative the posterior position ranges from 1:1 to 1:10000.
12. The method of claim 10,
wherein the first duration of the contact depends on the first speed of the second fluid in the flow channel and the longitudinal length of the second fluid in the flow channel, and/or
wherein the second duration of the contact depends on the second speed of the third fluid in the flow channel and the longitudinal length of the third fluid in the flow channel.
13. The method of claim 12,
wherein the longitudinal length of the second fluid in the flow channel depends on the volume of the second fluid introduced, the volume of the fluidic channel, the volume of the flowcell, or a combination thereof, and/or
wherein the longitudinal length of the third fluid in the flow channel depends on the volume of the third fluid introduced, the volume of the fluidic channel, the volume of the flowcell, or a combination thereof.
14. The method of claim 1, wherein the first flow rate is a fixed flow rate, wherein the second flow rate is a fixed flow rate, and/or wherein the third flow rate is a fixed flow rate.
15. The method of claim 1, wherein the first flow rate is a variable flow rate, wherein the second flow rate is a variable flow rate, and/or wherein the third flow rate is a variable flow rate.
16. The method of claim 1, wherein the first flow rate is an increasing flow rate, wherein the second flow rate is an increasing flow rate, and/or the third flow rate is an increasing flow rate.
17. The method of claim 1, wherein the first flow rate is a decreasing flow rate, wherein the second flow rate is a decreasing flow rate, and/or wherein the third flow rate is a decreasing flow rate.
18. The method of claim 1, wherein the second fluid and the second displacement fluid are dispensed together into the fluidic channel from a container comprising both the second fluid and the second displacement fluid.
19. The method of claim 18, wherein the second fluid and the second displacement fluid are dispensed together into the fluidic channel from the container using a pump.
20. The method of claim 18, wherein the container is a pipette.
21. The method of claim 20, wherein the second fluid and the second displacement fluid are aspirated sequentially into the pipette prior to being dispensed together.
22. The method of claim 1, wherein the fluidic channel further comprises an inlet and an outlet, and
wherein the second displacement fluid begins displacing the second fluid from the fluidic channel and/or sealing the contents of the microwell immediately adjacent to the inlet before the second fluid has reached the outlet.
The conflicting claims do not explicitly teach (b) capturing single cells and single beads in the plurality of microwells, wherein a single bead comprises a plurality of tethered barcodes, and wherein the plurality of tethered barcodes further comprises: i) a bead-specific cellular label; ii) a diverse set of molecular labels; and iii) a plurality of target binding regions capable of hybridizing with target nucleic acid molecules; (d) hybridizing target nucleic acid molecules released from single cells following cellular lysis with the plurality of target binding regions tethered to single beads in a stochastic manner; (e) performing an extension reaction to create a plurality of molecular conjugates each comprising a barcode and a portion of a complementary sequence of one of the target nucleic acid molecule; (f) amplifying and sequencing the molecular conjugates; and (g) determining the number of occurrences of the target nucleic acid molecule in the single cells. However, SIMS, as explained above, very clearly teaches to use the same flowcell loading scheme to load the same barcoded beads for RNA-seq. Thus, the prior art clearly, and explicitly teaches that the claimed loading scheme was directly applicable to RNA-seq using barcoded beads.
Prior Art
The following prior art is also pertinent: US 20220064717; US2018/0088112; US2018/0346970; WO 2016145409 A1; US11156611; US 20180340939; US 20180305681; US 20180024139; US 20180179591.
The line of Applications and patents stemming from WO 2018058073 A2 may also become the basis for obvious-type double patenting rejections depending on future amendments.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Aaron Priest whose telephone number is (571)270-1095. The examiner can normally be reached 8am-6pm.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Gary Benzion can be reached at (571) 272-0782. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/AARON A PRIEST/Primary Examiner, Art Unit 1681
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