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 the Claims
Claims 27-40, 44 and 46 are pending and the subject of this NON-FINAL Office Action.
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 03/15/2026 has been entered.
Previous Election/Restrictions
Applicant’s election without traverse of species of beads in microwells in the Reply filed 10/28/2024 is acknowledged.
Thus, claims 41-43 and 45 are withdrawn pursuant to 37 CFR 1.142(b), as being drawn to a nonelected inventions, there being no allowable generic or linking claim.
Thus, the requirement for election of species is hereby made FINAL.
Large IDS With Multiple Irrelevant References
On 03/29/2024, Applicants filed a 77-page IDS with 1700 listed references. Then, on 09/05/2025, Applicants filed yet another large IDS of 30 pages with almost 700 more references. First, it is impossible to search in detail through all 2400 references in any reasonable amount of time. To this end, after a random examination of the references within thew time allotted to the Examiner, the Examiner determines that many of the references are irrelevant to the claimed subject matter, having nothing to do with the barcoded nucleic acid beads claimed, and many that are not prior art (e.g. IDS 09/05/2025 ref # 1-16, 195, 246, 263, 404, 417, 443, 450, 510 and 680). Which leads to the larger problem: which references are relevant, and why were these seemingly irrelevant references filed? Before filing an IDS, Applicants have a duty to examine the references themselves and determine which references are reasonably pertinent to the claimed invention. If any particular preference(s) is/are directly pertinent to the claimed invention, then Applicants are encouraged to point this out in the form of a new IDS and/or the next reply.
Claim Interpretations
A “cellular label sequence” is a barcode (AKA index, or tag) sequence that labels a cell source.
A “molecular label sequence” is a barcode (AKA index, or tag) sequence that labels a molecule (e.g. mRNA 1).
A “sample label sequence” is a barcode (AKA index, or tag) sequence that labels a sample source.
A “universal label sequence” is any sequence as broadly defined in the specification. Based on the specification examples, and for compact prosecution, the Examiner applies prior art that uses a sequencing primer or amplification primer sequence as the “universal primer.”
The amended “compartment” claims do not require an “apparatus comprising an optical imager.” In fact, a “compartment” comprising an optical imager would be impossible here (e.g. due to written description issues). Instead, at best, claim 27 merely states the following intended use of the claimed “compartment” (microwell on an array cartridge) with an optical imager: “the apparatus being configured to process images of the microwell array to identify cells and particles and to identify microwells containing more than one cell or more than one particle.” Thus, to fulfill the claim limitations, the Examiner need only show any prior art that teaches compartment is a microwell of a microwell array packaged within a cartridge having an optically transparent window for optical imaging of the microwell array (the array must also be configures to interface with an optical imager, which minimally requires the simple microwell array with transparent window).
The “optically transparent window” can be a cover or a bottom of the array.
Claim Priority
The claims receive a priority date of 08/28/2014 because the United States non-provisional application (14/472,363) filed on that date is the first priority document to fully disclose microwell arrays having optically transparent window, and configured to interface with an optical imager. United States provisional application 61/952,036 only teaches the use of microwells instead of emulsions, and the enabling technique of microwell sizing specifically configured for bead and cell loading along with compartmentalized lysis and nucleic acid amplification reactions. United States provisional application 61/871,232 filed on 08/28/2013 merely states “[t]he methods and kits disclosed herein may further comprise distributing one or more beads in the microwell plate,” which can be “at least about 6 wells, 12 wells, 48 wells, 96 wells, 384 wells, 960 wells or 1000 wells” (paras. 0066-67). At best, “the microwell plate may comprise at least one well in which no more than one bead is distributed” (para. 0279). “The solid support may comprise the use of beads that self assemble in microwells” such as “Illumina's BeadArray Technology. Alternatively, the solid support comprises Abbott Molecular' s Bead Array technology, and Applied Microarray's FlexiPlexTM system” (para. 0296). However, none of the BeadArray, Bead Array or FlexiPlex are designed/configured for compartmentalizing both cells and barcoded oligo beads for individual reactions similar to emulsions, much less microwell arrays having optically transparent window, and configured to interface with an optical imager. Thus, the priority date of the claimed microwell arrays having optically transparent window, and configured to interface with an optical imager is 08/28/2014.
New Grounds of 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 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.
The factual inquiries set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 27-40, 44 and 46-47 are rejected under 35 U.S.C. 103 as being unpatentable over HINDSON (US20140378350, effective filing 08/14/2012), in view of VIGNEAULT (US20140357500, effective filing 03/15/2013) as evidenced by IPR2023-00876, in further view of LINNARSSON (US20160244742, effective filing 09/30/2013), in further view of LEAMON (US20050130173).
It would have been prima facie obvious to a skilled artisan at the time of filing to apply familiar single-cell barcodes and microwell sizing to the barcoded single-cell techniques of the prior art to yield familiar single-cell and single-bead loading on microwells with a reasonable expectation of success.
As to claims 27-38, 44 and 46, HINDSON teaches a microwell (paras. 0096, 0390, 0413) comprising a gel, silica or other material bead (para. 0105, for example) comprising 100,000 or more oligos (para. 0290, claim 23), each oligo comprises an mRNA target-binding region N-mer or Poly-T (paras. 0364-65, Fig. 4, for example), a sample label sequence 408 (Fig. 4) and a universal label sequence 403 or 415 (Fig. 4).
HINDSON does not explicitly teach molecular label sequence wherein the plurality of oligonucleotides comprise 10,000 different molecular label sequences, cellular label sequence wherein the cellular label sequence of each of the plurality of oligonucleotides is the same, or the diameter of the microwell is from 1-fold to 2-fold the diameter of said single particle, is about equal to the sum of the diameter of said single particle and the diameter of said single cell, and/or is at least 10 micrometers; the depth of the microwell is about equal to the sum of the diameter of said single particle and the diameter of said single cell, and/or is at least 1-fold the diameter of said single particle.
However, first as to the combination of molecular barcodes and sample barcodes, this was a well-known option. For example, VIGNEAULT utilizes both molecular barcodes (UID) beads and sample barcodes (SBC) to allow counting of unique molecules (paras. 0314, 0328, 0359) and pooling samples (para. 0363). VIGNEAULT utilizes 100,000 or more different UID on each bead (“each reverse transcription primer comprises a different UID. This can allow for uniquely barcoding each of the RNA molecules being reverse transcribed”; para. 0328).
Cellular barcodes were also a well-known option. For example, LINNARSSON teaches beads with RT barcoded oligos with cellular barcodes (e.g. Fig. 7, Cell ID). Each cell ID is the same on each bead (para. 0058). A sample barcode can also be included on the bead-attached oligos (para. 0058). This allows one to scale single-cell analyses to “approach a million single-cell transcriptomes” (para. 0011).
Finally, the combination of cell barcodes and molecular barcodes has been determined to have been obvious before 08/20/2009. See IPR2023-00876.
Thus, as to the combination of molecular barcodes, sample barcodes and cell barcodes, these were familiar barcode options in the art regularly combined with success to achieve familiar tracking and barcoding results.
As to utilizing microwells instead of emulsions, the prior art is replete with statements that microwells were a familiar options that allow cheaper and more efficient processing than emulsions. To this end, LINNARSSON teaches that they are in fact substitutes: “The plurality of compartments may, for example, be wells of a microwell array, or be droplets formed by an emulsifying or droplet microfluidics apparatus” (para. 0021; see also Example 5). LINNARSSON also explains how bead loading is performed on microwells using microwells with diameter and depth to fit a single bead and cell:
The volume of each compartment should be greater than the volume of the largest single cell to be captured. Preferably, the diameter of each compartment is from about 1 μm to about 1 mm. Therefore, the preferred diameter of each compartment will depend on the nature of the target cells. For example, when the single cells are bacterial cells, the diameter of each compartment is preferably 1-10 μm. When the single cells are typical mammalian cells, the diameter of each compartment is preferably 10-100 μm. When the single cells are large mammalian cells (such as early embryos or Purkinje cells), plant cells or protists, the diameter of each compartment is preferably 100-1000 μm.
The plurality of compartments may, for example, be wells on a microwell array. A suspension of single cells may be pipetted onto the microwell array and the cells allowed to settle into the microwells. The number of cells is adjusted so that there is a low probability of having more than one cell per well, i.e. the average number of cells per compartment is less than one. This means that each well is unlikely to contain more than a single cell. Therefore, some wells will be empty. The cells may settle into the wells by gravity flow, or may be forced by centrifugation. The microfluidic chip may comprise a glass bottom layer suitable for microscope imaging, a silicon layer having etched microwells and/or a plastic enclosure or lid having an inlet and an outlet to allow easy addition and removal of reagents on the microwell array. The thickness of each layer is arbitrary and may be adjusted to fit manufacturing or imaging constraints. The enclosure or lid is not required, but may be eliminated or replaced with a jig or other contraption intended to facilitate liquid flow across the microwell array. Likewise, the bottom layer need not be transparent if the intended application does not require imaging of the captured cells. The well diameters may vary across the full range of cell sizes, from about 1 μm to about 1 mm.
[ . . . ]
The method also includes the step of randomly placing the plurality of solid supports into a plurality of compartments such that the average number of solid supports per compartment, λ2, is less than 1. This means that each compartment is unlikely to contain more than a single solid support. Preferably, λ1 is less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3 or less than 0.2.
As disclosed above, the plurality of compartments may, for example, be wells on a microwell array. In this embodiment, a solution containing a plurality of solid supports is typically flowed over the microwell array. Due to the geometry of the wells (width and aspect ratio), cells do not escape from the wells. The plurality of solid supports are typically added at a density designed to place at most one solid support per well. Solid supports are allowed to settle in the microwells, and will reside above cells in those wells that contain single cells, thereby trapping the cells. The diameter of the solid supports is typically adjusted to prevent passage of cells between the solid support and the well wall. Optionally, the well depth may be adjusted to prevent loading more than one solid support per well; this can allow very high occupancy of the wells without risk of doublets, i.e. two solid supports in a single well
(paras. 0052-53 & 0055-56; emphasis added). A skilled artisan would have recognized that microwell compartments, with their sizes configured for one cell and one bead, were a known substitute for emulsions.
Thus, it would have been prima facie obvious to one having ordinary skill in the art at the time of filing to substitute wells for emulsions to yield familiar benefits.
Previous Response to Arguments
The rejection is maintained at least because LINNARSSON was only relied on exclusively for the bead a well sizing of claims 38-40, which receive a priority date later than the effective filing date of LINNARSSON.
Furthermore, as explained above, in fact, all claims likely receive a priority date of 03/12/2014 because the provisional filed on that date is the first priority document o disclose single bead particle that comprises an oligo with all of cellular label, molecular label and target-binding region, in a specifically-designed microwell plate for loading such beads/particles.
Previous Response to Arguments
The rejection is maintained because Applicants substitute evidence of reasonable expectation of success (RES) with attorney argument; and fail to provide sufficient evidence of unexpected results. Applicants first argue that
One of skill in the art would not have a reasonable expectation of success to add the alleged molecular barcodes of VIGNEAULT to the alleged beads with RT barcoded oligos with cellular barcodes of LINNARSSON, because synthesizing a plurality of oligonucleotides having an identical cellular label sequence, a diverse molecular label sequence, and a target binding region was technically challenging and commercially impractical
(pg. 8). However, Applicants fail to provide evidence to support this assertion. Attorney assertions cannot take the place of evidence. Furthermore, this argument begs the question: what have Applicants done to overcome this supposed lack of RES? The claims merely require a single generic particle associated with a plurality of oligonucleotides, the plurality of oligonucleotides each comprise a generic cellular label sequence, a generic molecular label sequence, and a generic target-binding region, the cellular label sequence of each of the plurality of oligonucleotides is the same, and at least 100 of the plurality of oligonucleotides comprise different molecular label sequences. What has suddenly enabled Applicants to combine cellular label sequences and molecular label sequences, whereas prior artisans could not? The reader is left wondering. And this issue of RES of combining molecular and cellular or samples barcodes was also addressed in IPR2023-00876, where PTAB held the arguments unconvincing (pgs. 40-42).
Applicants also argue that “oligonucleotides comprising a cellular label
sequence and a molecular label sequence into a compartment (particularly one sized for one cell and one particle) achieves superior results that are not envisioned by any of the cited references- for example, the claimed invention enables counting the exact number of hundreds of transcripts in large numbers of single cells in parallel” (pg. 9). However, this was known. For example,
Combining a barcode and a sample index into a primer capable of amplifying regions of a sample nucleic acid (e.g., via PHASE amplification) may enable parallelization of sample indexing. Sets of primers may be used to index nucleic acids from different samples. Each set of primers may be associated with nucleic acid molecules obtained from a particular sample and comprise primers comprising a diversity of barcode sequences and a common sample index sequence
(HINDSON, para. 0315). HINDSON also teaches that thousands to millions of barcodes allows counting thousands of transcripts (paras. 0007, 0012, 0044, 0209, 0239, 0262-67). Similarly, VIGNEAULT describes thousands to millions of barcodes to allow immune repertoire sequencing of thousand to millions of barcoded combinations of VH and VL and thousands to millions of single cells barcoded for thousand to millions of heavy and IgL pairing of antibodies (Abstract; paras. 0247, 0314, 0316, 0328, 0359). LINNARSSON also explains that thousands to millions of barcoded beads in wells allows one “to process thousands or millions of cells in parallel” (para. 0048). In other words, the prior art as a whole clearly suggested to a skilled artisan that using oligonucleotides comprising a cellular label sequence and a molecular label sequence in a compartment (particularly one sized for one cell and one particle) would be expected to yield counting the exact number of hundreds of transcripts in large numbers of single cells in parallel.
As to Applicants’ assertions that “the presently claimed invention solved a long-felt but unresolved needs in the field of high throughput single-cell analysis and received industry praises as a result,” here no evidence of long-felt unresolved need is provided, nor explanation how the praise of others has any nexus with the claimed invention or is reasonably commensurate in scope with the claims. See MPEP § 716. For example, the Fan publication may have been recognized as a “milestone,” but how is this pertinent to the claims. Was it the specific method of Fan that received this praise? Or the oligo-beads? And if the beads, what specific oligo-beads compared to the claimed oligo-beads? What exactly received praise? Until these burdens are met, the assertions of long-felt but unresolved needs and industry praise are merely attorney argument with no probative value.
The rejections are maintained.
As to claim 47, HINDSON teaches fluorescent-labeled probes (paras. 0237, 0241-42).
Microwell Arrays with Optically Transparent Windows Configured to Interface with an Optical Imager
None of the above references explicitly disclose the compartment is a microwell of a microwell array packaged within a cartridge having an optically transparent window for optical imaging of the microwell array, and wherein the cartridge is configured to interface with an apparatus comprising an optical imager. However, numerous of the references disclose microwell arrays used to compartmentalize the single bead-oligos with single cells; and a skilled artisan would have been motivated to apply familiar microwell arrays to detect the single cells and/or beads. For example, LINNARSSON teaches microfluidic chips with microwells to capture single cells and single beads (Fig. 13, for example). “The microfluidic chip may comprise a glass bottom layer suitable for microscope imaging, a silicon layer having etched microwells and/or a plastic enclosure or lid having an inlet and an outlet to allow easy addition and removal of reagents on the microwell array” (0054). Thus, LINNARSSON at least suggests that the microfluidic chip with microwells to capture single cells and single beads may be set up for imaging using optically transparent window.
To this end, another familiar system to allow one to image single cells and single beads from the top through a clear/transparent cover is disclosed in LEAMON. Specifically, LEAMON teaches that the other option to optically detect items in wells, such as single beads, is to use optically transparent covers (Figs. 13-14 and para. 0204 (“An example of the means for delivering reactants to the reaction chamber is the perfusion chamber of the present invention is illustrated in FIG. 13. The perfusion chamber includes a sealed compartment with transparent upper and lower side.”), 0283 (“When the substrate does not provide for fiber optic coupling, a lens system can also be used as described above, in which case either the substrate or the perfusion chamber cover is optically transparent. An exemplary CCD imaging system is described above.”); c.f. paras. 0188, 0190). Thus, an optically transparent cover is the only other option for optical detection of material inside a well.
In sum, a skilled artisan would have found the substitution of an optically transparent cover the obvious other option to optically transparent bottom.
Double Patenting- Obvious Type - Maintained
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 27-40, 44 and 46-47 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over conflicting claims 1-28 of U.S. Patent No. US 9567645.
The conflicting claims anticipate the instant claims because the conflicting claims teach wells with (a) cell and (b) barcoded bead with cellular label, sample label, molecular label and target hybridizing sequence. Specifically, the conflicting claims teach:
1. A method comprising: associating a single bead with a single cell, wherein said single bead comprises a plurality of oligonucleotides, wherein each of said plurality of oligonucleotides comprises an identical cellular label sequence, a molecular label sequence, and a target-binding region, and wherein at least 100 of said plurality of oligonucleotides comprise different molecular label sequences.
2. The method of claim 1, wherein said single cell is selected from the group consisting of a rare cell, a tumor cell, a cell from a human, a cell from a tissue, a cell from a tumor, a cell infected with viral polynucleotides, and any combination thereof.
3. The method of claim 1, further comprising lysing said single cell after associating said single bead with said single cell.
4. The method of claim 1, further comprising lysing said single cell before associating said single bead with said single cell.
5. The method of claim 1, wherein said single bead is magnetic.
6. The method of claim 1, wherein said plurality of oligonucleotides comprises at least 100,000 oligonucleotides.
7. The method of claim 1, wherein said plurality of oligonucleotides each comprise a universal label sequence.
8. The method of claim 1, wherein said target-binding region comprises a sequence selected from the group consisting of an oligo-dT sequence, a gene-specific sequence, and a random multimer sequence.
9. The method of claim 1, further comprising stochastically labelling target nucleic acid molecules from said single cell.
10. The method of claim 9, wherein said labelling comprises hybridizing said target nucleic acid molecules to the plurality of oligonucleotides on said single bead.
11. The method of claim 1, wherein at least 1,000 of said plurality of oligonucleotides comprise different molecular label sequences.
12. The method of claim 1, wherein at least 10,000 of said plurality of oligonucleotides comprise different molecular label sequences.
13. The method of claim 1, wherein said single bead and said single cell are in the same well.
14. The method of claim 1, wherein said single bead and said single cell are in the same droplet.
15. The method of claim 1, wherein said single bead comprises silica gel, controlled pore glass, Dynabead, Wang resin, Merrifield resin, Sephadex/Sepharose bead, cellulose bead, polystyrene bead, or any combination thereof.
16. The method of claim 10, wherein said target nucleic acid molecules comprise mRNA molecules.
17. The method of claim 16, further comprising reverse transcribing said target nucleic acid molecules, thereby generating labelled target cDNAs.
18. The method of claim 17, wherein said reverse transcribing is done while said plurality of oligonucleotides is on said single bead.
19. The method of claim 17, further comprising performing second strand synthesis on said labelled target cDNAs, thereby generating double-stranded labelled target polynucleotides.
20. The method of claim 19, further comprising amplifying said double-stranded labelled target polynucleotides, thereby generating labelled target amplicons.
21. The method of claim 20, wherein said amplifying is performed with a universal primer and a random multimer primer.
22. The method of claim 20, wherein said amplifying is performed with a universal primer or a gene-specific primer.
23. The method of claim 20, wherein said amplifying comprises conducting a nested PCR reaction.
24. The method of claim 20, wherein said amplifying comprises amplifying the whole transcriptome of said single cell.
25. The method of claim 20, further comprising estimating the number of said target nucleic acid molecules.
26. The method of claim 25, wherein said estimating comprises determining the sequence of at least a portion said double-stranded labelled target amplicons.
27. The method of claim 25, wherein said estimating comprises determining the sequence of at least a portion said double-stranded labelled target amplicons and at least a portion of said oligonucleotide.
28. The method of claim 25, wherein said estimating comprises counting the number of unique oligonucleotides associated with a distinct target polynucleotide of said single cell.
Instant claims 27-40, 44 and 46-47 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over conflicting claims 1-30 of U.S. Patent No. US 10131958.
The conflicting claims anticipate the instant claims because the conflicting claims teach wells with (a) cell and (b) barcoded bead with cellular label, sample label, molecular label and target hybridizing sequence. Specifically, the conflicting claims teach:
1. A composition comprising:
a single bead, wherein the bead comprises a plurality of oligonucleotides,
wherein each of the plurality of oligonucleotides comprises a cellular label sequence, a molecular label sequence, and a target-binding region, wherein the cellular label sequence of each of the plurality of oligonucleotides is the same, wherein the cellular label sequence comprises at least 6 nucleotides, wherein the molecular label sequence comprises at least 6 nucleotides, and at least 100 of the plurality of oligonucleotides comprise different molecular label sequences.
2. The composition of claim 1, further comprising a single cell, or a lysate thereof.
3. The composition of claim 2, wherein said plurality of oligonucleotides is capable of labeling individual occurrences of target nucleic acid molecules associated with said single cell.
4. The composition of claim 3, wherein said plurality of oligonucleotides is capable of labeling individual occurrences of target nucleic acid molecules associated with said single cell via a nucleic acid extension reaction.
5. The composition of claim 4, wherein the nucleic acid extension reaction comprises a reverse transcription reaction.
6. The composition of claim 4, wherein the nucleic acid extension reaction is performed using a reverse transcriptase, a DNA polymerase, or a combination thereof.
7. The composition of claim 3, wherein a target nucleic acid molecule of said target nucleic acid molecules comprises a messenger ribonucleic acid (mRNA) molecule.
8. The composition of claim 3, wherein a target nucleic acid molecule of said target nucleic acid molecules comprises a deoxyribonucleic acid (DNA) molecule.
9. The composition of claim 3, wherein a target nucleic acid molecule of said target nucleic acid molecules comprises a sample tag oligonucleotide.
10. The composition of claim 9, wherein the sample tag oligonucleotide is 50-500 nucleotides in length.
11. The composition of claim 1, wherein the molecular label sequence is 6-30 nucleotides in length.
12. The composition of claim 1, wherein the cellular label sequence is 6-30 nucleotides in length.
13. The composition of claim 1, wherein at least 10,000 of said plurality of oligonucleotides comprise different molecular label sequences.
14. The composition of claim 1, wherein at least 700,000 of said plurality of oligonucleotides comprise different molecular label sequences.
15. The composition of claim 1, wherein about 1,000,000 of said plurality of oligonucleotides comprise different molecular label sequences.
16. The composition of claim 3, wherein a target nucleic acid molecule of said target nucleic acid molecules is associated with said single cell via a peptide.
17. The composition of claim 16, wherein the peptide comprises an antibody.
18. The composition of claim 17, wherein the peptide is capable of binding to said single cell.
19. The composition of claim 1, comprising a peptide.
20. The composition of claim 19, wherein the peptide comprises an antibody.
21. The composition of claim 19, wherein the peptide is associated with a sample tag oligonucleotide.
22. The composition of claim 21, wherein the plurality of oligonucleotides is capable of labeling the sample tag oligonucleotide.
23. The method of claim 1, wherein said plurality of oligonucleotides comprises at least 700,000 oligonucleotide.
24. The method of claim 1, wherein said plurality of oligonucleotides comprises about 1,000,000 oligonucleotide.
25. The composition of claim 1, wherein said target-binding region comprises a sequence selected from the group consisting of a sequential sequence of an oligo-dT sequence, a gene-specific sequence, a target-specific sequence, a multimer sequence, a random multimer sequence, and a complement thereof.
26. The composition of claim 1, wherein said single bead comprises silica gel, controlled pore glass, Wang resin, Merrifield resin, a Dynabead, a Sephadex bead, a Sepharose bead, a cellulose bead, polystyrene bead, or any combination thereof.
27. The composition of claim 1, wherein said single bead comprises a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, a paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, cellulose, nylon, silicone, and any combination thereof.
28. The composition of claim 1, wherein said single bead comprises a hydrogel bead, a magnetic bead, or a combination thereof.
29. A partition comprising:
a. a composition of claim 1; and
b. a single cell, or a lysate thereof.
30. The partition of claim 29, wherein the partition comprises a well or a droplet.
Instant claims 27-40, 44 and 46-47 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over conflicting claims 1-30 of U.S. Patent No. US 10151003.
The conflicting claims anticipate the instant claims because the conflicting claims teach wells with (a) cell and (b) barcoded bead with cellular label, sample label, molecular label and target hybridizing sequence. Specifically, the conflicting claims teach:
1. A method comprising: introducing a single bead with a single cell into a partition, wherein said single bead comprises a plurality of oligonucleotides, wherein each of said plurality of oligonucleotides comprises an identical cellular label sequence, a molecular label sequence, and a target-binding region, wherein the cellular label sequence comprises at least 6 nucleotides, wherein the molecular label sequence comprises at least 6 nucleotides, and wherein at least 100 of said plurality of oligonucleotides comprise different molecular label sequences.
2. The method of claim 1, wherein the partition comprises a well or a droplet.
3. The method of claim 1, comprising lysing said single cell after associating said single bead with said single cell.
4. The method of claim 1, comprising lysing said single cell before associating said single bead with said single cell.
5. The method of claim 1, comprising attaching nucleic acid target molecules associated with said single cell to said plurality of oligonucleotides.
6. The method of claim 5, comprising performing a nucleic acid extension reaction on nucleic acid target molecules attached to said plurality of oligonucleotides.
7. The method of claim 6, wherein the nucleic acid extension reaction comprises a reverse transcription reaction.
8. The method of claim 6, wherein the nucleic acid extension reaction is performed using a reverse transcriptase, a DNA polymerase, or a combination thereof.
9. The method of claim 5, wherein said nucleic acid target molecules comprise a messenger ribonucleic acid (mRNA) molecule.
10. The method of claim 5, wherein said nucleic acid target molecules comprise a deoxyribonucleic acid (DNA) molecule.
11. The method of claim 5, wherein said nucleic acid target molecules comprise a sample tag oligonucleotide.
12. The method of claim 11, wherein the sample tag oligonucleotide is 50-500 nucleotides in length.
13. The method of claim 1, wherein the molecular label sequence is 6-30 nucleotides in length.
14. The method of claim 1, wherein the cellular label sequence is 6-30 nucleotides in length.
15. The method of claim 1, wherein at least 10,000 of said plurality of oligonucleotides comprise different molecular label sequences.
16. The method of claim 1, wherein at least 700,000 of said plurality of oligonucleotides comprise different molecular label sequences.
17. The method of claim 1, wherein about 1,000,000 of said plurality of oligonucleotides comprise different molecular label sequences.
18. The method of claim 11, comprising labeling the sample tag oligonucleotide using an oligonucleotide of said plurality of oligonucleotides.
19. The method of claim 1, comprising labeling target nucleic acid molecules associated with said single cell using said plurality of oligonucleotides.
20. The method of claim 19, comprising stochastically labeling target nucleic acid molecules associated with said single cell using said plurality of oligonucleotides.
21. The method of claim 5, wherein a nucleic acid target molecule of said nucleic acid target molecules is associated with said single cell via a peptide.
22. The method of claim 21, wherein the peptide comprises an antibody.
23. The method of claim 21, comprising contacting the peptide with said single cell, whereby the peptide binds to said single cell.
24. The method of claim 1, wherein said target-binding region comprises a sequence selected from the group consisting of an oligo-dT sequence, a gene-specific sequence, a target-specific sequence, a multimer sequence, a random multimer sequence, and a complement thereof.
25. The method of claim 1, wherein said single bead comprises silica gel, controlled pore glass, Wang resin, Merrifield resin, a Dynabead, a Sephadex bead, a Sepharose bead, a cellulose bead, a polystyrene bead, or any combination thereof.
26. The method of claim 1, wherein said single bead comprises a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, a paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof.
27. The method of claim 1, wherein said single bead comprises a hydrogel bead, a magnetic bead, or a combination thereof.
28. The method of claim 1, comprising estimating the number of said target nucleic acid molecules.
29. The method of claim 28, wherein said estimating comprises counting the number of unique oligonucleotides associated with a distinct target nucleic acid molecule of said target nucleic acid molecules associated with said single cell.
30. The method of claim 11, comprising determining an origin of said single cell based on the sequence of the sample tag oligonucleotide, or a portion thereof.
Instant claims 27-40, 44 and 46-47 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over conflicting claims 1-30 of U.S. Patent No. US 10208356.
The conflicting claims anticipate the instant claims because the conflicting claims teach wells with (a) cell and (b) barcoded bead with cellular label, sample label, molecular label and target hybridizing sequence. Specifically, the conflicting claims teach:
1. A composition comprising:
a single bead, wherein the bead comprises a plurality of oligonucleotides,
wherein each of the plurality of oligonucleotides comprises a cellular label sequence, a molecular label sequence, and a target-binding region, wherein the cellular label sequence of each of the plurality of oligonucleotides is the same, wherein the cellular label sequence comprises 4-300 nucleotides, wherein the molecular label sequence comprises 4-300 nucleotides, and at least 100 of the plurality of oligonucleotides comprise different molecular label sequences.
2. The composition of claim 1, further comprising a single cell, or a lysate of a single cell.
3. The composition of claim 2, wherein said plurality of oligonucleotides is capable of labeling individual occurrences of target molecules associated with said single cell.
4. The composition of claim 3, wherein said plurality of oligonucleotides is capable of labeling the individual occurrences of said target molecules associated with said single cell via hybridization of the individual occurrences of said target molecules to the target-binding regions of said plurality of oligonucleotides.
5. The composition of claim 3, wherein said target molecules comprise nucleic acid molecules.
6. The composition of claim 5, wherein said plurality of oligonucleotides is capable of labeling the individual occurrences of said target molecules associated with said single cell via a nucleic acid extension reaction.
7. The composition of claim 6, wherein the nucleic acid extension reaction comprises a reverse transcription reaction.
8. The composition of claim 6, wherein the nucleic acid extension reaction is performed using a reverse transcriptase, a DNA polymerase, or a combination thereof.
9. The composition of claim 3, wherein a target molecule of said target molecules comprises a messenger ribonucleic acid (mRNA) molecule.
10. The composition of claim 3, wherein a target molecule of said target molecules comprises a deoxyribonucleic acid (DNA) molecule.
11. The composition of claim 3, wherein a target molecule of said target molecules comprises a sample tag oligonucleotide.
12. The composition of claim 11, wherein the sample tag oligonucleotide is 25-300 nucleotides in length.
13. The composition of claim 1, wherein the molecular label sequence is 4-30 nucleotides in length.
14. The composition of claim 1, wherein the cellular label sequence is 4-30 nucleotides in length.
15. The composition of claim 1, wherein at least 10,000 of said plurality of oligonucleotides comprise different molecular label sequences.
16. The composition of claim 1, wherein about 1,000,000 of said plurality of oligonucleotides comprise different molecular label sequences.
17. The composition of claim 3, wherein a target molecule of said target molecules is associated with said single cell via a peptide.
18. The composition of claim 17, wherein the peptide comprises an antibody.
19. The composition of claim 18, wherein the peptide is capable of binding to said single cell.
20. The composition of claim 1, comprising a peptide.
21. The composition of claim 20, wherein the peptide comprises an antibody.
22. The composition of claim 20, wherein the peptide is associated with a sample tag oligonucleotide.
23. The composition of claim 22, wherein the plurality of oligonucleotides is capable of labeling the sample tag oligonucleotide.
24. The composition of claim 1, wherein said plurality of oligonucleotides comprises at least 700,000 oligonucleotide.
25. The composition of claim 1, wherein said plurality of oligonucleotides comprises about 1,000,000 oligonucleotide.
26. The composition of claim 1, wherein said target-binding region comprises a sequence selected from the group consisting of an oligo-dT sequence, a gene-specific sequence, a target-specific sequence, a multimer sequence, a random multimer sequence, and a complement thereof.
27. The composition of claim 1, wherein said single bead comprises silica gel, Wang resin, Merrifield resin, polydimethylsiloxane (PDMS), polystyrene, glass, controlled pore glass, polypropylene, agarose, gelatin, hydrogel, a paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sephadex, Sepharose, cellulose, nylon, silicone, or a combination thereof.
28. The composition of claim 1, wherein said single bead is a hydrogel bead, a magnetic bead, or a combination thereof.
29. A partition comprising:
a. a composition of claim 1; and
b. a single cell, or a lysate of a single cell.
30. The partition of claim 29, wherein the partition is a well or a droplet.
Instant claims 27-40, 44 and 46-47 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over conflicting claims 1-30 of U.S. Patent No. US 10253375.
The conflicting claims anticipate the instant claims because the conflicting claims teach wells with (a) cell and (b) barcoded bead with cellular label, sample label, molecular label and target hybridizing sequence. Specifically, the conflicting claims teach:
1. A method comprising: introducing a single bead and a single cell into a partition, wherein said single bead comprises a plurality of oligonucleotides, wherein each of said plurality of oligonucleotides comprises an identical cellular label sequence, a molecular label sequence, and a target-binding region, wherein the cellular label sequence comprises 4-300 nucleotides, wherein the molecular label sequence comprises 4-300 nucleotides, and wherein at least 100 of said plurality of oligonucleotides comprise different molecular label sequences.
2. The method of claim 1, wherein the partition is a well or a droplet.
3. The method of claim 1, comprising lysing said single cell after introducing said single bead and said single cell into said partition.
4. The method of claim 1, comprising lysing said single cell before introducing said single bead and said single cell into said partition.
5. The method of claim 1, comprising attaching target molecules associated with said single cell to said plurality of oligonucleotides.
6. The method of claim 5, wherein said attaching comprises hybridizing the target molecules to the target-binding regions of said plurality of oligonucleotides.
7. The method of claim 5, wherein said target molecules comprise nucleic acid molecules.
8. The method of claim 7, wherein said attaching comprises performing a nucleic acid extension reaction on the target molecules attached to said plurality of oligonucleotides.
9. The method of claim 8, wherein the nucleic acid extension reaction comprises a reverse transcription reaction.
10. The method of claim 8, wherein the nucleic acid extension reaction is performed using a reverse transcriptase, a DNA polymerase, or a combination thereof.
11. The method of claim 5, wherein said target molecules comprise a messenger ribonucleic acid (mRNA) molecule.
12. The method of claim 5, wherein said target molecules comprise a deoxyribonucleic acid (DNA) molecule.
13. The method of claim 5, wherein said target molecules comprise a sample tag oligonucleotide.
14. The method of claim 13, wherein the sample tag oligonucleotide is 25-300 nucleotides in length.
15. The method of claim 1, wherein the molecular label sequence is 4-30 nucleotides in length.
16. The method of claim 1, wherein the cellular label sequence is 4-30 nucleotides in length.
17. The method of claim 1, wherein at least 10,000 of said plurality of oligonucleotides comprise different molecular label sequences.
18. The method of claim 1, wherein about 1,000,000 of said plurality of oligonucleotides comprise different molecular label sequences.
19. The method of claim 13, comprising labeling the sample tag oligonucleotide using an oligonucleotide of said plurality of oligonucleotides.
20. The method of claim 1, comprising labeling target molecules associated with said single cell using said plurality of oligonucleotides.
21. The method of claim 20, wherein labeling target molecules comprises stochastically labeling the target molecules associated with said single cell using said plurality of oligonucleotides.
22. The method of claim 5, wherein a target molecule of said target molecules is associated with said single cell via a peptide.
23. The method of claim 22, wherein the peptide comprises an antibody.
24. The method of claim 22, comprising contacting the peptide with said single cell, whereby the peptide binds to said single cell.
25. The method of claim 1, wherein said target-binding region comprises a sequence selected from the group consisting of an oligo-dT sequence, a gene-specific sequence, a target-specific sequence, a multimer sequence, a random multimer sequence, and a complement thereof.
26. The method of claim 1, wherein said single bead comprises silica gel, Wang resin, Merrifield resin, polydimethylsiloxane (PDMS), polystyrene, glass, controlled pore glass, polypropylene, agarose, gelatin, hydrogel, a paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sephadex, Sepharose, cellulose, nylon, silicone, or a combination thereof.
27. The method of claim 1, wherein said single bead is a hydrogel bead, a magnetic bead, or a combination thereof.
28. The method of claim 23, comprising estimating the number of said target molecules.
29. The method of claim 28, wherein said estimating comprises counting the number of unique molecular label sequences associated with a distinct target molecule of said target molecules associated with said single cell.
30. The method of claim 13, comprising determining an origin of said single cell based on the sequence of the sample tag oligonucleotide, or a portion thereof.
Instant claims 27-40, 44 and 46-47 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over conflicting claims 1-17 of U.S. Patent No. US 10927419.
The conflicting claims anticipate the instant claims because the conflicting claims teach wells with (a) cell and (b) barcoded bead with cellular label, sample label, molecular label and target hybridizing sequence. Specifically, the conflicting claims teach:
1. A method for processing messenger ribonucleic acid (mRNA) molecules from a single cell, comprising:
(a) partitioning a plurality of cells and a plurality of beads in a plurality of partitions, wherein a partition of said plurality of partitions comprises a single cell from said plurality of cells and a single bead from said plurality of beads,
wherein said single bead comprises a plurality of nucleic acid barcode molecules each comprising a first barcode sequence a second barcode sequence, a RNA priming sequence,
wherein the first barcode sequence of each of the plurality of nucleic acid barcode molecules is the same; and wherein at least 1000 nucleic acid barcode molecules of the plurality of said nucleic acid barcode molecules comprise different second barcode sequences, wherein at least 1000 of the plurality of beads comprises a plurality of nucleic acid barcode molecules comprising first barcode sequences that are different across the at least 1000 beads;
(b) in said partition comprising said single cell and said single bead, releasing messenger ribonucleic acid (mRNA) molecules from said single cell, thereby said mRNA molecules attach to the nucleic acid barcode molecules via the RNA priming sequence;
(c) subjecting said mRNA molecules to reverse transcription to yield complementary deoxyribonucleic acid (cDNA) molecules each comprising said first barcode sequence and a second barcode sequence; and
(d) subjecting said cDNA molecules to one or more reactions to generate a set of nucleic acid molecules for nucleic acid sequencing.
2. The method of claim 1, wherein (c) is performed in said partition comprising said single cell and said single bead, and wherein subsequent to (c), said cDNA molecules, or derivatives thereof, are removed from said given partition.
3. The method of claim 1, wherein, prior to (c), said mRNA molecules are removed from said partition comprising said single cell and said single bead.
4. The method of claim 1, wherein each of said plurality of nucleic acid barcode molecules comprises a universal primer sequence.
5. The method of claim 1, wherein said single bead is a single magnetic bead, and wherein said plurality of nucleic acid barcode molecules are attached to said single magnetic bead.
6. The method of claim 1, wherein said single bead comprises hydrogel.
7. The method of claim 6, wherein said plurality of nucleic acid barcode molecules are covalently or non-covalently attached to the single bead.
8. The method of claim 1, wherein said RNA priming sequence is an oligo(dT) sequence.
9. The method of claim 8, wherein in step (b) said mRNA molecules attach to the oligo(dT) sequence of said nucleic acid barcode molecules by hybridization.
10. The method of claim 1, wherein the first barcode sequences of at least 10,000 of said plurality of beads are different across said at least 10,000 beads.
11. The method of claim 1, wherein said different first barcode sequences are capable of distinguishing nucleic acid molecules in different partitions comprising said 1,000 beads.
12. The method of claim 9, further comprising, prior to (c), (i) pooling said mRNA molecules attached to said nucleic acid barcode molecules and (ii) performing said one or more reactions in bulk.
13. The method of claim 1, wherein said one or more reactions comprise nucleic acid amplification that generates amplified products from said plurality of cDNA molecules.
14. The method of claim 1, comprising performing said nucleic acid sequencing on said set of nucleic acid molecules, or derivatives thereof, to generate a plurality of sequences comprising sequences corresponding to said mRNA molecules and said first barcode sequence.
15. The method of claim 1, wherein said partition comprises a droplet.
16. The method of claim 1, wherein in step (b) said mRNA molecules attach to the RNA priming sequence of said nucleic acid barcode molecules by hybridization.
17. The method of claim 1, wherein said partition comprises a well.
Response to Arguments
The Office is not persuaded of error by Applicants’ arguments because only “objections or requirements as to form not necessary to further consideration of the claims” may be held in abeyance. MPEP § 714.02.
Prior Art
The following prior art also teaches optically transparent windows on microwell arrays for imaging single cells and/or single beads: US20140323330; US20120065082.
Conclusion
No claims are allowed.
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/AARON A PRIEST/Primary Examiner, Art Unit 1681
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