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 .
Claim Status
Claims 3-8, 10-15, 17-20, 22-29, 31-35, 37-38, 40-44, 51-52, 54-58, 63-84 and 86-95 have been cancelled (8/8/2022). Claims 21, 30, 36, 39, 45-46, 49-50, 53, 59, 61-62 and 85 have been amended (8/8/2022). Thus, claims 1-2, 9, 16, 21, 30, 36, 39, 45-50, 53, 59-62 and 85 are under examination (8/8/2022).
Priority
Claims 1-2, 9, 16, 21, 30, 36, 39, 45-50, 53, 59-62 and 85 receive a priority date of 5/24/2021, the effective filing date of US Provisional Patent 63/192,432.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure
statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information
submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be
incorporated into the specification but must be submitted in a separate paper." Therefore, unless
the references have been cited by the examiner on form PTO-892, they have not been
considered.
Information disclosure statements (IDS) were submitted on 6/26/2023 and 5/28/2024 and are being considered by the examiner.
Specification
The disclosure is objected to because of the following informalities (see MPEP § 608.01):
The use of the terms “Teflon” (p. 23), “TritonX-100” (p. 31), “Tween 20” (p. 31), “Sylgard 184” (p. 59), “Nextera” (p. 62), “Agilent Bioanalyzer” (p. 62), “HiSeq 2500” (p. 62), “Seurat” (p. 62), “Texas Red” (p. 64) which are trade names or marks used in commerce, have been noted in this application. The terms 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.
Claim Objections
Claim 2 is objected to because of the following informality:
Claim 2 at step (e) at line 1; “the partition the plurality” should be replaced with “the partition of the plurality” for grammatical accuracy.
Claim Rejections - 35 USC § 112
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.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 46-50, 59-61 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 applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 is rejected. Claim 1 recites the limitation "the pooled barcoded nucleic acids" in line 17. There is insufficient antecedent basis for this limitation in the claim.
Claim 46 is rejected. Claim 46 recites the limitation "the pooled barcoded nucleic acids" in line 5. There is insufficient antecedent basis for this limitation in the claim.
Claims 47-50 are included in this limitation due to their dependency on claim 46.
Claim 59 is rejected. Claim 59 recites the limitation "the second sequences" in line 5. There is insufficient antecedent basis for this limitation in the claim.
Claims 59-61 are included in this limitation due to their dependency on claim 59.
Claim 60 is further rejected. Claim 60 recites the limitation "the expression profile" in line 1. There is insufficient antecedent basis for this limitation in the claim.
Claim 61 is further rejected. Claim 61 recites the limitation "the sequencing data" in line 6. There is insufficient antecedent basis for this limitation in the claim.
Claim Rejections - 35 USC § 102
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 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)(1) 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.
(a)(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 1-2, 9, 16, 21, 30, 36, 39, 45-50, 53, 59-62 and 85 are rejected under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Cater et al., (WO 2019152395 A1, published 8/8/2019).
Regarding claims 1-2, 9, Cater teaches methods of assessing whether multiple differently-barcodes primers are in partitions (Abstract). Further, Cater teaches beads conjugated to oligonucleotides are used in microfluidic detection applications such as high-throughput sequencing having many different partitions (i.e., droplets) and in order to uniquely identify each partition, the beads can be labeled with unique barcode sequences; however, in order to ensure that partitions have only one bead and thus are uniquely labeled by the barcode (Paragraph 2, lines 1-5). Further, Cater teaches that the oligonucleotide primers conjugated to a particular bead comprise a barcode sequence that is the same or substantially the same among the plurality of oligonucleotides on a bead, but unique or substantially unique as compared to the plurality of oligonucleotides on other beads (Paragraph 2, lines 1-5).
Cater also teaches that the previously described method of assessing or profiling whether multiple differently-barcodes primers are in partitions includes forward primers that also comprise a capture sequence, typically at the 3’ end of the forward primer, that hybridizes under the conditions of the assay with a target sequence or a reverse complement thereof, where the target sequence can be 100% complementary or partially (i.e., at least 95%, 90%, 80%, etc.) complementary depending on the desired results and conditions and in some embodiments, the capture sequence is a sequence of about 6 to about 20 nucleotides (Paragraph 94, lines 1-5). Specifically, Cater teaches that the forward primers can comprise a capture sequence complementary to the 3’ sequence or a reverse complement thereof, of a target nucleic acid and in addition, the forward primer can include one or more other sequences where the forward primers each include a unique molecular identifier (UMI) sequence so each copy can be separately tracked and counted (Paragraph 94, lines 1-10).
Cater also teaches that the previously described method of assessing or profiling whether multiple differently-barcodes primers are in partitions includes following the molecular reactions in the partitions (i.e., amplification or other linkage of the forward primer to partition ID tag oligonucleotides in the partitions and optional amplification or other manipulation of target nucleic acids in the partitions), the contents of the partitions are released prior to the downstream application, i.e., to pool multiple partitions for a downstream application such as a sequencing reaction (Paragraph 114, lines 1-5). Additionally, Cater teaches that a second oligonucleotide primer that functions as a reverse primer in combination with the first oligonucleotide primer on a target nucleic acid can be included in the partitions, or alternatively following combining of partitions into a bulk reaction where the target reverse primer, for example, will include a sequence that hybridizes to a reverse complement sequence on the target under the conditions of the assay to allow, for example, for polymerase-based extension (Paragraph 97, lines 1-5). Specifically, Cater teaches that for example, if a target sequence comprising a 5’ sequence and a 3’ sequence is present in a partition, the target reverse primer will have a 3’ end identical to the 5’ sequence of the target which will allow hybridization of the 3’ end of the target reverse primer to the extension product of the forward primer that uses the target nucleic acid as a template where alternatively, the target reverse primer can initiate extension using the target nucleic acid as a template in which case that target reverse primer 3’ sequence will be the reverse complement of the 5’ sequence of the target nucleic acid (Paragraph 97, lines 5-10). Cater also teaches that the target reverse primer can also have, for example, near or at its 5’ end, a universal sequence (also referred to as a “PCR handle” or “adaptor” sequence) (Paragraph 97, lines 1-10).
Cater also teaches that the previously described method of assessing or profiling whether multiple differently-barcodes primers are in partitions includes samples that can be collected at one location, partitioned into droplets containing enzymes, buffers, and/or primers or other probes, optionally one or more polymerization reactions can be performed, the partitions can then be heated to perform microencapsulation, and the microcapsules can be stored or transported for further analysis (Paragraph 85, lines 5-10). Further, Cater teaches that the number of copies is enough to get good confirmation of bead occupancy in each partition, but few enough to conserve the majority of sequencing space for assay samples where the partition ID tag oligonucleotide can be added to partitions as free
oligonucleotide, or linked to a solid support (either a solid support different from or the same as the solid support linked to the forward primer) (Paragraph 103, lines 10-15). Specifically, Cater teaches that the partition ID tag oligonucleotide is delivered to the partition as a free oligonucleotide (not linked to the solid support), whereas in such embodiments, the partition ID tag oligonucleotide can be single-stranded (Figure 2) or partially (Figure 7) or fully double-stranded (Paragraph 104, lines 5-10). Further Cater teaches that exemplary partial double stranded options include providing two partition ID tag oligonucleotides that hybridize at the partition ID tag sequence, one of the partition ID tag oligonucleotides having a reverse complement of the partition ID tag sequence, or portion thereof, of the partition ID tag sequence of the other partition ID tag oligonucleotide (Figures 2, 7-9; Paragraph 104, lines 15-20). Additionally, Cater teaches that in these embodiments, the partially double-stranded molecules have 3’ overhangs that comprise the capture sequence or a reverse complement thereof, leaving the capture sequences available for hybridization or otherwise interact with the capture sequence of the forward primer and this is advantageous for example in a situation where there are not multiple rounds of capture and extension in an assay where the double-stranded version allows each strand (forward and reverse-complement of tag sequence) to combine with a different forward primer in the same capture/extension step (Paragraph 104, lines 5-25).
Regarding claim 16, Cater also teaches that the previously described method of assessing or profiling whether multiple differently-barcodes primers are in partitions includes a partition-specific barcode that should be unique for that partition as compared to barcodes present in other partitions, where for example, partitions containing target RNA from single cells can be subjected to reverse transcription conditions using primers that contain a different partition-specific barcode sequence in each partition, thus incorporating a copy of a unique "cellular barcode" into the reverse transcribed nucleic acids of each partition (Paragraph 58, lines 1-10).
Regarding claim 21, Cater also teaches that the previously described method of assessing or profiling whether multiple differently-barcodes primers are in partitions includes following the molecular reactions in the partitions (i.e., amplification or other linkage of the forward primer to partition ID tag oligonucleotides in the partitions and optional amplification or other manipulation of target nucleic acids in the partitions), where the contents of the partitions are released prior to the downstream application, i.e., to pool multiple partitions for a downstream application such as a sequencing reaction (Paragraph 114, lines 1-5).
Regarding claim 30, Cater also teaches that the previously described method of assessing or profiling whether multiple differently-barcodes primers are in partitions includes in either option, in the partition, ID tag oligonucleotides are allowed to hybridize to at forward primers to form a hybridized product where the hybridized product can then be extended or otherwise amplified either within the partitions, or in bulk and, in addition to linkage (and optionally amplification) of the forward primer to a target nucleic acid, if present, the reaction will also link available copies of the partition ID tag oligonucleotide to some copies of the forward primers in the partition, thereby forming polynucleotides comprising a forward primer including the forward primer barcode sequence in the partition with the partition ID tag (Paragraph 111, lines 10-15).
Regarding claim 36, Cater also teaches that the previously described method of assessing or profiling whether multiple differently-barcodes primers are in partitions includes following the molecular reactions in the partitions (i.e., amplification or other linkage of the forward primer to partition ID tag oligonucleotides in the partitions and optional amplification or other manipulation of target nucleic acids in the partitions), where the contents of the partitions are released prior to the downstream application, i.e., to pool multiple partitions for a downstream application such as a sequencing reaction. (Paragraph 114, lines 1-5)
Regarding claim 39, Cater also teaches that the previously described method of assessing or profiling whether multiple differently-barcodes primers are in partitions includes providing a 5' phosphate group for ligation to interrogation probes containing two probe-specific bases followed by 6 degenerate bases and one of four fluorescent labels where fluor color, and thus identity of each probe, corresponds to specified color-space coding schemes (Paragraph 123, lines 5-10).
Regarding claims 45-46, Cater also teaches that the previously described method of assessing or profiling whether multiple differently-barcodes primers are in partitions includes methods of detecting the presence or absence of multiple barcodes in a partition are provided, where forming partitions comprising forward primers comprising a barcode and a capture sequence complementary to the 3’ sequence, or a reverse complement thereof, of the target nucleic acid, wherein different partitions contain different forward primers comprising different barcode sequences, a partition ID tag oligonucleotide comprising a 5’ binding sequence and a 3’ variable partition ID tag sequence; linking the target nucleic acid with the forward primers and the target reverse primers, the target reverse primers having a 3’ sequence identical to, or a reverse complement of, the 5’ sequence of the target nucleic acid wherein the linking also results in some products in which a forward primer is linked to the partition ID tag oligonucleotide; and sequencing the products, wherein if different forward primers form products with the same variable partition ID tag sequence, the different forward primers are considered to be from the same partition, wherein the forward primer and partition ID tag oligonucleotide are linked to the same bead when delivered to the partitions; or the partition ID tag oligonucleotide has a blocked 3’ end such that a polymerase cannot extend the blocked 3’ end during amplification; or the partition ID tag oligonucleotide comprises a double-stranded variable partition ID tag sequence and one or two single-stranded 3’ ends comprising the reverse complement of the capture sequence (Paragraph 20, lines 1-5).
Further, Cater teaches that following the molecular reactions in the partitions (i.e., amplification or other linkage of the forward primer to partition ID tag oligonucleotides in the partitions and optional amplification or other manipulation of target nucleic acids in the partitions), where the contents of the partitions are released prior to the downstream application, i.e., to pool multiple partitions for a downstream application such as a sequencing reaction (Paragraph 114, lines 1-5).
Regarding claim 47, Cater also teaches that the previously described method of assessing or profiling whether multiple differently-barcodes primers are in partitions includes an example where samples can be collected at one location, partitioned into droplets containing enzymes, buffers, and/or primers or other probes, optionally one or more polymerization reactions can be performed, the partitions can then be heated to perform microencapsulation, and the microcapsules can be stored or transported for further analysis (Paragraph 85, lines 5-10).
Regarding claim 48, Cater also teaches that the previously described method of assessing or profiling whether multiple differently-barcodes primers are in partitions includes depicting two forward primers with different barcodes are in the same partition, where the forward primers are depicted as linked to beads, that aspect is not required and included in the partition are some copies of a single-stranded partition ID tag oligonucleotide (having capture and partition ID tag sequences) as well as target nucleic acids (having capture and assay (e.g., a sequence to be determined) sequences) (Figure 3; Paragraph 77, lines 1-5). Additionally, Cater teaches that some copies of the forward primer are linked via the capture sequences to the target nucleic acids and other copies of the forward primers are linked via the capture sequence to the partition ID tag oligonucleotides and the linkage of different barcodes (BC1 and BC2) with the same partition ID tag sequence (TAG1) indicates two different forward primers (associated with BC1 and BC2) were present in the same partition (Paragraph 77, lines 1-5; Figure 3).
Regarding claim 49, Cater also teaches that the previously described method of assessing or profiling whether multiple differently-barcodes primers are in partitions includes sequencing reactions that are performed using immobilized template, modified phi29 DNA polymerase, and high local concentrations of fluorescently labeled dNTPs and high local concentrations and continuous reaction conditions allow incorporation events to be captured in real time by fluor signal detection using laser excitation, an optical waveguide, and a CCD camera (Paragraph 129, lines 5-10).
Regarding claims 50 and 53, Cater also teaches that the previously described method of assessing or profiling whether multiple differently-barcodes primers are in partitions includes multiple rounds (usually 7) of probe annealing, ligation, and fluor detection are followed by denaturation, and then a second round of sequencing using a primer that is offset by one base relative to the initial primer and in this manner, the template sequence can be computationally re-constructed, and template bases are interrogated twice, resulting in increased accuracy (Paragraph 123, lines 10-15).
Regarding claims 59-61, Cater teaches methods of assessing whether multiple differently-barcodes primers are in partitions (Abstract). Further, Cater teaches Beads conjugated to oligonucleotides are used in microfluidic detection applications such as high-throughput sequencing having many different partitions (e.g., droplets). In order to uniquely identify each partition, the beads can be labeled with unique barcode sequences. However, in order to ensure that partitions have only one bead and thus are uniquely labeled by the barcode (Paragraph 2, lines 1-5). he oligonucleotide primers conjugated to a particular bead comprise a barcode sequence that is the same or substantially the same among the plurality of oligonucleotides on a bead, but unique or substantially unique as compared to the plurality of oligonucleotides on other beads.
Cater also teaches that the previously described method of assessing or profiling whether multiple differently-barcodes primers are in partitions includes forward primers that also comprise a capture sequence, typically at the 3’ end of the forward primer, that hybridizes under the conditions of the assay with a target sequence or a reverse complement thereof where the target sequence can be 100% complementary or partially complementary depending on the desired results and conditions and in some embodiments, the capture sequence is a sequence of about 6 to about 20 nucleotides (Paragraph 94, lines 1-5). Cater also teaches that for example, the forward primers can comprise a capture sequence complementary to the 3’ sequence or a reverse complement thereof, of a target nucleic acid where the forward primer can include one or more other sequences and, in some embodiments, the forward primers each include a unique molecular identifier (UMI) sequence so each copy can be separately tracked and counted (Paragraph 94, lines 1-10).
Additionally, Cater teaches that in some embodiments, methods of detecting the presence or absence of multiple barcodes in a partition are provided and the method comprises forming partitions comprising forward primers comprising a barcode and a capture sequence complementary to the 3’ sequence, or a reverse complement thereof, of the target nucleic acid, wherein different partitions contain different forward primers comprising different barcode sequences, a partition ID tag oligonucleotide comprising a 5’ binding sequence and a 3’ variable partition ID tag sequence; linking the target nucleic acid with the forward primers and the target reverse primers, the target reverse primers having a 3’ sequence identical to, or a reverse complement of, the 5’ sequence of the target nucleic acid wherein the linking also results in some products in which a forward primer is linked to the partition ID tag oligonucleotide; and sequencing the products, wherein if different forward primers form products with the same variable partition ID tag sequence, the different forward primers are considered to be from the same partition, wherein the forward primer and partition ID tag oligonucleotide are linked to the same bead when delivered to the partitions; or the partition ID tag oligonucleotide has a blocked 3’ end such that a polymerase cannot extend the blocked 3’ end during amplification; or the partition ID tag oligonucleotide comprises a double-stranded variable partition ID tag sequence and one or two single-stranded 3’ ends comprising the reverse complement of the capture sequence (Paragraph 20, lines 1-5).
Regarding claim 62, Cater also teaches that the previously described method of assessing or profiling whether multiple differently-barcodes primers are in partitions includes a nucleic acid from a partition is analyzed by a sequencing or genotyping method (high throughput sequencing) where nucleic acids comprising the forward primer sequence (including the forward primer barcode) and the partition ID tag oligonucleotide (including the tag sequence) are sequenced and when multiple forward primer barcodes are associated with the same tag sequence, one can then assume that those multiple forward primers were within the same partition (Paragraph 117, lines 1-5).
Regarding claim 85, Cater also teaches that the previously described method of assessing or profiling whether multiple differently-barcodes primers are in partitions includes the addition of one or more reagents during droplet formation or to the droplets after the droplets are formed and methods and compositions for delivering reagents to one or more partitions include microfluidic methods as known in the art; droplet or microcapsule combining, coalescing, fusing, bursting, or degrading; droplet injection methods (Paragraph 79, lines 1-5). Specifically, Cater teaches that the partitions can be picowells, nanowells, or microwells or the partitions can be droplets, i.e., emulsion droplets (Paragraph 80, lines 1-5).
Cater teaches each and every limitation of claims 1-2, 9, 16, 21, 30, 36, 39, 45-50, 53, 59-62 and 85, and therefore Cater anticipates claims 1-2, 9, 16, 21, 30, 36, 39, 45-50, 53, 59-62 and 85.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ELIZABETH ROSE LAFAVE whose telephone number is (703)756-4747. The examiner can normally be reached Compressed Bi-Week: M-F 7:30-4:30.
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/ELIZABETH ROSE LAFAVE/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684