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 .
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 were previously cancelled (8/8/2022). Claims 1, 2, 46 and 59-61 have been amended (4/8/2026). Thus, claims 1-2, 9, 16, 21, 30, 36, 39, 45-50, 53, 59-62 and 85 are under examination (4/8/2026).
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.
Objections Withdrawn
Specification:
The objections to the specification due to the use of a trademark or tradenames are withdrawn in view of Applicant’s amendments.
Claims:
The objection to claim 2 is withdrawn in view of Applicant’s amendment to claim 2 for grammatical purposes.
Rejections Withdrawn
Claim Rejections - 35 USC § 112
The rejections of claims 1, 46-50, 59-61 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or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention are withdrawn in view of Applicant’s amendments to said claims to address issues of antecedent basis.
Claim Rejections – 35 USC § 102
The 102 (a) (1) and 102 (a) (2) rejections of claims 1, 46 and 61 are withdrawn in view of Applicant’s arguments and amendments (4/8/2026). Specifically, amended independent claims 1 now recites, inter alia, pooling a first subset of barcoded nucleic acids while retaining a second subset of barcoded nucleic acids in the partition, determining a barcode sequence from the retained subset, and matching the retained barcode sequence with the pooled barcoded nucleic acids, limitations that are not expressly or inherently disclosed by Cater in the claimed arrangement. Likewise, amended dependent claims 46 and 61 (please note these are dependent on claim 2) recite determining the partition and linking an expression profile using matching between retained barcoded nucleic acids in the partition and pooled barcoded nucleic acids, limitations that are likewise not expressly or inherently disclosed by Cater in the claimed arrangement.
Rejections Maintained
Claim Rejections – 35 USC § 102
Claims 2, 9, 16, 21, 30, 36, 39, 45, 47-50, 53, 59-60, 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 2 and 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 claim 45, 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 59-60, 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 2, 9, 16, 21, 30, 36, 39, 45, 47-50, 53, 59-60, 62 and 85, and therefore Cater anticipates claims 2, 9, 16, 21, 30, 36, 39, 45, 47-50, 53, 59-60, 62 and 85.
Applicant’s Response: The Applicant argues that Cater fails to disclose or suggest the amended limitations requiring retention of a subset of barcoded nucleic acids within the partition, matching barcode sequences of retained and pooled barcoded nucleic acids, and associating the resulting expression profile with the identified cell-cell interaction. Applicant further contends that Cater’s teachings of releasing partition contents for pooling and sequencing is inconsistent with the claimed retained-versus-pooled workflow and therefore does not anticipate the amended claims.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered and are found partially persuasive, as discussed below.
Firstly, Applicant argues that Cater fails to disclose retaining a subset of barcoded nucleic acids within the partition while pooling a separate subset, matching retained and pooled barcode sequences, determining the partition location using the retained barcoded nucleic acids, and determining an expression profile based on the matched retained and pooled barcode sequences. Applicant’s arguments are persuasive to amended independent claim 1 and dependent claims 46 and 61 (note that these are dependent on independent claim 2) because those claims now require additional limitations requiring retention of a subset of barcoded nucleic acids within the partition, pooling a separate subset for sequencing, and matching the retained and pooled barcode sequences, limitations that are not expressly or inherently disclosed by Cater. Accordingly, the rejection of claims 1, 46 and 61 under 35 USC 102 is withdrawn, as highlighted above.
However, Applicant’s remaining arguments are not persuasive with respect to claim 2 and the remainder of dependent claims therefrom. As an initial matter, Applicant’s arguments are not fully commensurate in scope with independent claim 2. See MPEP 2145 (II). Although Applicant relies extensively on limitations directed to retained barcoded nucleic acids, pooled barcoded nucleic acids, matching retained and pooled barcode sequences, and determining expression profiles from the matched sequences, claim 2 instead broadly recites partitioning first and second cells into partitions, introducing barcode molecules comprising identical first barcode sequences and second barcode sequences, barcoding sample nucleic acids, sequencing the barcoded nucleic acids, and determining the location of the partition using the first barcode sequences determined from the sequenced barcoded nucleic acids. Claim 2 does not require retaining a subset of barcoded nucleic acids in the partition, pooling a separate subset for sequencing, matching retained and pooled barcode sequences, or determining an expression profile from matched retained and pooled barcode sequences. Arguments directed to subject matter that is not positively recited in the claim cannot distinguish the claimed invention.
Applicant further argues that Cater does not determine the location of a partition. This argument is not persuasive. During examination, claims are given their broadest reasonable interpretation consistent with the specification. See MPEP 2111 and 2111.01. Under the BRI, determining the location of the partition does not require determining a physical x-y coordinate or visually identifying a droplet. Rather, the limitation broadly encompasses determining from which partition the barcoded nucleic acids originated. Cater teaches partition ID-tag oligonucleotides that uniquely identify individual partitions, and teaches that nucleic acids associated with the same partition ID tag sequence are determined to have originated from the same partition (Paragraphs 77, 103-104). Accordingly, identifying the partition ID necessarily identifies the partition from which the nucleic acids originated. Thus, to the extent the determination of the partition is not expressly stated, it is inherent in Cater’s disclosed partition identification system because it is the necessary result of the disclosed method because determination necessarily flows from the disclosed partition -identification process. See MPEP 2112. Thus, Cater teaches determining the partition associated with the barcoded nucleic acids as broadly claimed.
Applicant additionally argues that Cater fails to teach the claimed barcode molecules. This argument is likewise not persuasive. Cater teaches barcode molecules comprising a partition-specific barcode together with unique molecular identifiers (UMIs), wherein the partition barcode is shared among barcode molecules associated with the same partition while the UMIs distinguish individual nucleic acid molecules (Paragraphs 2 and 94). Under BRI, this teaches the claimed first barcode sequence that is identical among barcode molecules and the claimed second barcode sequence that differs between barcode molecules.
Applicant further argues that Cater fails to disclose the claimed workflow. The Examiner agrees that the amended workflow recited in claims 1, 46, and 61, introduced additional workflow limitations requiring retention of one subset of barcoded nucleic acids in the partition, pooling a separate subset of barcoded nucleic acids in the partition, pooling a separate subset for sequencing, and subsequently matching retained and pooled barcode sequences. Those newly added limitations are not expressly or inherently disclosed by Cater and therefore the rejection of claims 1, 46, and 61 has been withdrawn as previously noted. For that reason, the rejection of those amended claims has been withdrawn. However, claim 2 does require that any subset of barcoded nucleic acids remain physically retained within the partition after another subset is pooled, nor does claim 2 require sequencing both retained and pooled subsets, matching barcode sequences between those subsets, or using the matched sequences to determine the location of the partition or an expression profile. Rather, claim 2 broadly recites sequencing the plurality of barcoded nucleic acids and determining the location of the partition using first barcode sequences determined from the sequenced barcoded nucleic acids.
Under BRI consistent with the specification, claim 2 encompasses any method in which barcoded nucleic acids are sequences and the partition associated with those nucleic acids is determined using the first barcode sequence. The claim does not recite a particular order of retaining and pooling nucleic acids, nor does it require a comparison between retained and pooled barcode sequences before determining the partition. Cater teaches sequencing barcoded nucleic acids after pooling partition contents and teaches the use of partition ID tag sequences to identify the partition from which the sequenced nucleic acids originated (Paragraphs 2, 77, 94, 103-104, 114). Notably, under BRI, this satisfies the recited determination of the partition using the first barcode sequences. To the extent Applicant argues that Cater does not expressly describe the claimed determination, such determination is at least inherent because identifying a unique partition ID necessarily identifies the partition associated with the sequenced nucleic acids. See MPEP 2112. Accordingly, Applicant’s arguments regarding the amended workflow are not commensurate in scope with claim 2 and therefore do not overcome the rejection.
To place the claims in a position to overcome the existing rejection, Applicant may amend claim 2 to incorporate one or more of the limitations of claim 1 (i.e., requiring retaining a subset of the barcoded nucleic acids within the partition while pooling a separate subset for sequencing, determining the retained barcode sequences, matching the retained and pooled barcode sequences, and determining the partition and expression profile using the matched retained and pooled barcode sequences. Alternatively, Applicant may amend claim 2 in dependent form if Applicant intends both claims to cover the same inventive workflow, as suggested in the Applicant’s argument. Notably, Applicant’s remarks appear to rely on workflow limitations now recited in amended independent claim 1, but not presently recited in independent claim 2. Consequently, the arguments are not commensurate in scope with the rejected claims.
However, such amendments would narrow claim 2 to the subject matter upon which the withdrawal of the rejection of claims 1, 46, and 61 is based, as well as render Applicant’s arguments commensurate in scope with the pending claim.
New Rejections
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
The factual inquiries 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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 46 and 61 are rejected under 35 U.S.C. 103 as being unpatentable over Cater et al., (WO 2019152395 A1, published 8/8/2019), as applied to claims 2, 9, 16, 21, 30, 36, 39, 45, 47-50, 53, 59-60, 62 and 85 above, and Guimaraes et al. (“Ionizable lipid nanoparticles encapsulating barcoded mRNA for accelerated in vivo delivery screening”, Journal of Controlled Release, published 12/28/2019), in further view of Wu et al. (“INSIGHT: A population-scale COVID-19 testing strategy combining point-of-care diagnosis with centralized high-throughput sequencing”, Science Advances, published 2/12/2021) and Ebong et al. (“Molecular Species Delimitation and Morphology of Aquatic and Sub-Aquatic Bugs (Heteroptera) in Cameroon”, PLOS One, published 5/5/2016).
As previously highlighted, and specifically regarding claim 1, 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 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 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).
Cater does not teach or suggest the amended workflow requiring retention of barcoded nucleic acids within the partition while a separate subset is pooled for sequencing and subsequently matched to the retained subset to determine the partition and/or expression profile.
Guimaraes teaches that engineered lipid nanoparticle formulations can encapsulate functional barcoded mRNA molecules, wherein the barcoded mRNA includes barcodes and UMIs that permit delivery and identity to be quantified by deep sequencing (Abstract; Introduction, Paragraphs 1-4; Figure 1B-1D). Thus, Guimaraes teaches pooled analysis of separately barcoded nucleic acid populations while preserving the ability to identify individual members of the pool by barcode sequencing.
Wu teaches a two-stage barcoded sequencing workflow in which a first-stage assay provides an initial readout while simultaneously incorporating sample-specific barcodes, and the same reaction products form many samples are subsequently pooled for a second-stage multiplexed sequencing assay (Abstract). Further, Wu teaches that second-stage sequencing may be performed using pooling strategies including “all pool” containing all samples and a “negative pool” selected based on first-stage results (Results, Paragraphs 2-14; Figures 3-4). Thus, Wu teaches staged analysis in which sample-specific barcoded products are first generated and then pooled for centralized sequencing while preserving sample identity through barcode analysis.
Ebong teaches separating specimens into independent batches, analyzing one batch by morphology and another batch by COI DNA sequencing/DNA barcoding, and then integrating into morphological and molecular barcode information to reconcile specimens and associate juvenile/nymph forms with corresponding adult species (Abstract; Molecular-Based Species Delimination). Thus, Ebong teaches the general workflow of dividing a population into separate subsets, subjecting the subsets to different analyses, and correlating the results from those analyses using DNA barcode information.
It would have been obvious to one of ordinary skill in the art at the time of the invention to modify Cater in view of these references by retaining a portion of the barcoded nucleic acids while pooling another portion for sequencing and correlating the resulting barcode information. A person of ordinary skill in the art would have recognized that barcoded nucleic acid populations could be separately handled, pooled for sequencing, and subsequently correlated using their associated barcode sequences, as demonstrated by the cited secondary references. Likewise, the cited secondary references collectively demonstrate that independently barcoded nucleic acid populations may be pooled for sequencing while preserving sample identity through barcode information, and that barcode information can subsequently be used to associate sequencing results with the originating sample or population. Applying these known barcode-based pooling and sample correlation techniques to Cater’s partition-based barcoding system would have represented the predictable use of prior art elements according to their established functions to improve sequencing throughput while preserving sample identity.
One of ordinary skill in the art would have had a reasonable expectation of success because each reference relies upon the same underlying principle of uniquely identifying nucleic acid populations through barcode sequences before or after pooled sequencing. Combining Cater’s partition-based barcoding with the known pooled strategies and barcode-based correlation techniques disclosed by the secondary references would merely have involved the application of familiar sequencing and sample-management techniques using conventional barcode technology. The proposed combination therefore would have yielded predictable results while preserving the ability to identify the partition associated with each sequenced nucleic acid population through its corresponding barcode sequence.
Conclusions
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
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/ELIZABETH ROSE LAFAVE/ Examiner, Art Unit 1684 /HEATHER CALAMITA/ Supervisory Patent Examiner, Art Unit 1684
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