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 .
Office Action: Notice
Any objection or rejection of record in the previous Office Action, mailed 9/8/2025,
which is not addressed in this action has been withdrawn in light of Applicants' amendments
and/or arguments. This action is FINAL
Claim Status
Claims 1, 3-4, 7, 10, and 13 have been amended (12/4/2025). Claim 14 has been cancelled (12/4/2025). No new matter was added. Thus, claims 1-13 and 15-20 are under examination (12/4/2025).
Priority
Claims 1-13 and 15-20 receive a priority date of 1/23/2020, the effective filing date of PCT/CN2020/073968.
Objections Withdrawn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The objections to the specification due to the use of a trademark or tradenames are withdrawn in view of Applicant’s amendments.
The objections to the specification due to the use of hyperlinks are withdrawn in view of Applicant’s amendments.
Sequence Listing (Specification):
The Specification is in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 after inserting adequate sequence identifiers (i.e., SEQ ID NO: X).
Claims:
The objections to write out abbreviations and edit minor informalities in claims 1, 3, 7 and 13 are withdrawn due to Applicant’s amendments.
Rejections Withdrawn
Claim Rejections - 35 USC § 112(b)
The rejections of claims 4 and 10-15 under 35 U.S.C. 112(b) or pre-AIA 35 U.S.C. 112, 2nd paragraph, are withdrawn in view of Applicant’s amendments of claims 4, 10 and 15, to address and further clarify indefiniteness, as well as the cancellation of claim 14.
Rejections Maintained
Claim Rejections - 35 USC § 102
Claims 1-13 and 15-20 are rejected under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Bell et al., (WO 2019/217099 A1, published 11/14/2019). Rejection has been modified to reflect Applicant’s amendments (12/4/2025).
Regarding claims 1-2, Bell teaches a droplet that includes: i) a mammalian cell
nucleus; and ii) a microbead presenting attached oligonucleotides, where the attached oligonucleotides include a nucleic acid sequence capable of hybridization and capture of genomic DNA and a microbead identification sequence or index sequence that is common to all oligonucleotides attached to the microbead, where the mammalian cell nucleus is accessible to the microbead-attached oligonucleotides to an extent sufficient to allow for genomic DNA capture and amplification of genomic DNA to occur within the droplet (Abstract). Further, Bell teaches a number of different barcodes, target capture sequences, or other sequence elements set forth herein as being unique (or sufficiently unique) to particular nucleic acid probes (p. 25, Paragraph 2). Bell teaches that a population of the microbeads can be configured such that each
microbead is attached to only one type of barcode e.g., a barcode that specifically identifies a
microbead and/or microbead-associated cell from which a sequence derived) and many different
microbeads each with a different barcode are present in the population and in this embodiment,
randomly distributing the microbeads to a population of droplets will result in randomly locating the nucleic acid probe-presenting microbeads (and their respective barcode sequences) in the population of droplets (p. 24, Paragraph 4). Further, Bell teaches that the captured nucleic acid probe can be amplified on the microbead such that the resulting cluster becomes a feature and although attachment is exemplified above as capture between a primer and a complementary portion of a probe, it will be understood that capture moieties other than primers can
be present at pre-formed features or as a lawn, where exemplary capture moieties include, but are
not limited to, chemical moieties capable of reacting with a nucleic acid probe to create a covalent bond or receptors capable of binding non-covalently to a ligand on a nucleic acid probe (p. 26, Paragraph 1).
Bell also teaches that the term “attached” refers to the state of two things being joined, fastened, adhered, connected or bound to each other and for example, such as a nucleic acid, can be attached to a material, such as a gel or solid support, by a covalent or non-covalent bond where a covalent bond is characterized by the sharing of pairs of electrons between atoms (p. 6, Paragraph 3).
Regarding claim 3, Bell teaches that the previously described droplet system encompasses a redundancy of barcodes within the droplet population, which will tend to produce a small population of droplets that exhibit redundancies, but it is contemplated that such redundant droplets can simply be eliminated from an ultimate population of single-cell-derived sequences produced by methods of the instant disclosure (for sperm cells especially, it will be clear which barcode sequences are actually associated with multiple sperm cells, rather than single sperm cells, due to an abundance of heterozygous SNPs within such sequences derived from multiple sperm cells) (p. 25, Paragraph 1).
Regarding claim 4, Bell teaches that the previously described droplet system includes a nucleic acid probe used in a composition or method set forth herein can include a target capture moiety where the target capture moiety is a target capture sequence and the target capture sequence is generally complementary to a target sequence such that target capture
occurs by formation of a probe-target hybrid complex (p. 34, Paragraphs 3-4).
Regarding claim 5, Bell teaches that the previously described droplet system includes one or more oligonucleotides can be added to the 3' or 5' end of a nucleic acid, for example, via chemical or enzymatic (i.e., ligase catalysis) methods (p. 35, Paragraphs 3-4).
Regarding claims 6-7, Bell teaches that the previously described droplet system includes certain aspects of the instant disclosure employ a nucleotide- or oligonucleotide-adorned bead (microbead), where the bead-attached oligonucleotide includes one or more of the following:
a linker (optionally a cleavable linker, optionally a photocleavable linker); an identical sequence for use as a sequencing priming site; a uniform or near-uniform nucleotide or oligonucleotide sequence; a Unique Molecular Identifier which differs for each priming site; a nucleic acid sequence capable of hybridizing to and capturing genomic DNA (optionally a random sequence that anneals to the targeted mammalian genome); and at least one oligonucleotide barcode which provides an substrate for identification of an individual bead's associated mammalian cell from which capture of genomic DNA has occurred (p. 32, Paragraph 3).
Further, Bell teaches that exemplary cleavage sites include, but are not limited to, moieties that are susceptible to a chemical, enzymatic or physical process that results in bond breakage, where the location can be a nucleotide sequence that is recognized by an endonuclease recognition sequence (p. 36, Paragraph 5). Additionally, Bell teaches that this anchor is used as a PCR primer, but because of the length of the template and its proximity to other nearby anchor oligonucleotides, extension by PCR results in the "arching over" of the molecule to hybridize
with an adjacent anchor oligonucleotide to form a bridge structure on the surface of the flow cell where these loops of DNA are denatured and cleaved and forward strands are then sequenced (p. 41, Paragraph 1).
Regarding claim 8, Bell teaches that the previously described droplet system includes single cell isolation and lysis, as well as double-stranded DNA molecules that are melted into single stranded forms (p. 29).
Regarding claims 9-10, Bell teaches that the previously described droplet system includes exemplary nucleic acid detection methods include, but are not limited to nucleic acid sequencing of a probe, hybridization of nucleic acids to a probe, ligation of nucleic acids that are hybridized to a probe, extension of nucleic acids that are hybridized to a probe, extension of a first nucleic acid that is hybridized to a probe followed by ligation of the extended nucleic acid to a second nucleic acid that is hybridized to the probe (p. 31, Paragraph 2).
Further, Bell teaches that the previously described droplet system includes a nucleic acid probe used in a composition or method set forth herein can include a target capture moiety where the target capture moiety is a target capture sequence and the target capture sequence is generally complementary to a target sequence such that target capture occurs by formation of a probe-target hybrid complex (p. 34, Paragraphs 3-4).
Bell also teaches that the term “attached” refers to the state of two things being joined, fastened, adhered, connected or bound to each other and for example, such as a nucleic acid, can be attached to a material, such as a gel or solid support, by a covalent or non-covalent bond where a covalent bond is characterized by the sharing of pairs of electrons between atoms (p. 6, Paragraph 3).
Regarding claim 11, Bell teaches that the previously described droplet system includes methodology incorporating disulfide bonds; specifically applying to a replication of a cellular process attempting to adapt such a sperm decondensation process to droplet-based sequencing of single sperm cells where the human egg then reduces the sperm genomic DNA's protamine disulfide bonds with glutathione and then accepts removed protamines, presumably via use of heparan sulfate - this disulfide reduction process and acceptance of removed protamines can also be achieved experimentally via administration of ~-mercaptoethanol (for disulfide reduction) and heparin (for both reduction and acceptance processes) (Figure 2; p. 50, Paragraph 1).
Regarding claims 12-13, Bell teaches that the previously described droplet system includes methodology to achieve single cell droplet-based capture of sperm genomic DNA and subsequent high-throughput sequencing, a reliable method of accessing decondensed nuclear
DNA from sperm, while retaining the single-cell character of each nucleus, where extraction and decondensation of sperm genomic DNA was attempted using agents such as dithiothreitol (DTT) and harsh salts, yet such agents failed to provide sperm nuclei (i.e., such treatments tended to burst the nucleus) having decondensed genomic DNA that could then be introduced to droplets to achieve single cell bead capture and amplification in droplets, followed by next-gen sequencing (p. 49, Paragraph 2).
Regarding claim 15, Bell teaches that the previously described droplet system includes the extension of probes can be carried out using methods exemplified herein or otherwise known
in the art for amplification of nucleic acids or sequencing of nucleic acids, where specific embodiments of one or more nucleotides can be added to the 3' end of a nucleic acid, for example, via polymerase catalysis (i.e., DNA polymerase) via chemical or enzymatic methods which can be used to add one or more nucleotide to the 3' or 5' end of a nucleic acid (p. 35, Paragraphs 3-4). Further, Bell teaches that a nucleic acid can be extended in a template directed manner, whereby the product of extension is complementary to a template nucleic acid that is hybridized to the nucleic acid that is extended (p. 35, Paragraphs 3-4). Additionally, Bell teaches that particularly useful functional analogs of nucleic acids are capable of hybridizing to a nucleic acid in a sequence specific fashion or capable of being used as a template for replication of a particular nucleotide sequence (p. 10, Paragraph 1). Bell also teaches that exemplary cleavage sites include, but are not limited to, moieties that are susceptible to a chemical, enzymatic or physical process that results in bond breakage where the location can be a nucleotide sequence that is recognized by an endonuclease (p. 36, Paragraph 5).
Regarding claim 16, Bell teaches that the previously described droplet system includes a step of hybridizing nucleic acid probes, that are on a microbead, to target genomic DNA of a single cell and a target-probe hybrid complex can form where the target nucleic acid encounters a complementary target capture sequence on a nucleic acid probe and the sequences of the target nucleic acids and associated barcodes applied during amplification steps will provide information about the cell of origin of a next-gen sequencing read obtained (p. 35, Paragraph 2). Bell also teaches that the previously described droplet system can be applied to oil-encapsulated droplets (p. 3, Paragraphs 4-5). Additionally, Bell teaches that the analyses via specified probes can be plotted as read depth in 1Mb bins as the number of observed reads in that bin divided by the number of reads expected in that bin based on library size and sequence context; a haploid read depth of 1 was expected at all chromosomes in sperm (Figure 17; p. 14, Paragraph 8).
Regarding claim 17, Bell teaches that the previously described droplet system includes all or part of a target nucleic acid that is hybridized to a nucleic acid probe can be copied by extension, where, for example, an extended probe can include at least, 1, 2, 5, 10, 25, 50, 100, 200, 500, 1000 or more nucleotides that are copied from a target nucleic acid (p. 36, Paragraphs 1-2). Bell further teaches that in each haploid cell, a median of 13,000 SNPs that were heterozygous in the sperm donor were genotyped and phased genomes were obtained for each donor and recombination and aneuploidy events in each sperm cell were thereby identified where
the instant disclosure provides for routinely making sequencing libraries for 1000-2000 individual sperm cells, sequencing them to low coverage (generally capturing 1-2% of the genome, though up to at least 10% is possible) (p. 23, Paragraphs 1-2). Further, Bell teaches that it is possible to achieve read lengths greater than or equal to 400 bases, and 106 sequence reads can be achieved, resulting in up to 500 million base pairs (Mb) of sequence (p. 40, Paragraph 3).
Regarding claim 18, Bell teaches that the previously described droplet system includes a droplet garnered from: i) a mammalian cell nucleus; and ii) a microbead presenting attached oligonucleotides, where the attached oligonucleotides include a nucleic acid sequence capable of hybridization and capture of genomic DNA and a microbead identification sequence or index sequence that is common to all oligonucleotides attached to the microbead, where the mammalian cell nucleus is accessible to the microbead-attached oligonucleotides to an extent sufficient to allow for genomic DNA capture and amplification of genomic DNA to occur within the droplet (Abstract). Further, Bell teaches a number of different barcodes, target capture sequences, or other sequence elements set forth herein as being unique (or sufficiently unique) to particular nucleic acid probes (p. 25, Paragraph 2).
Regarding claims 19-20, Bell teaches that the previously described droplet system includes certain aspects of the instant disclosure employ a nucleotide- or oligonucleotide-adorned bead (microbead), where the bead-attached oligonucleotide includes one or more of the following:
a linker (optionally a cleavable linker, optionally a photocleavable linker); an identical sequence for use as a sequencing priming site; a uniform or near-uniform nucleotide or oligonucleotide sequence; a Unique Molecular Identifier which differs for each priming site; a nucleic acid sequence capable of hybridizing to and capturing genomic DNA (optionally a random sequence that anneals to the targeted mammalian genome); and at least one oligonucleotide barcode which provides an substrate for identification of an individual bead's associated mammalian cell from which capture of genomic DNA has occurred (p. 32, Paragraph 3).
Further, Bell teaches that generally, all probes in a plurality will have the same length barcode (albeit with different sequences), but it is also possible to use different length barcodes for different probes or index sequences where a barcode sequence can be at least 2, 4, 6, 8, 10, 12, 15, 20 or more nucleotides in length and rather, alternatively or additionally, the length of the barcode sequence can be at most 20, 15, 12, 10, 8, 6, 4 or fewer nucleotides. (p. 31, Paragraph 2).
Bell teaches each and every limitation of claims 1-13 and 15-20, and therefore Bell anticipates claims 1-13 and 15-20.
Applicant’s Response: The Applicant argues that Bell fails to teach a droplet comprising (i) a droplet identification molecule carrying a droplet index sequence, and (ii) more than one first vector per droplet, each first vector carrying a capture sequence and a cell index sequence. The Applicant further asserts that Bell is limited to droplets containing a single mammalian nucleus and a single microbead, and therefore does not teach or suggest the claimed configuration enabling multiple capture vectors within one droplet. The Applicant additionally argues that the claimed configuration yields unexpected improvements in cell recovery and efficiency, rendering the amended claims patentable over Bell.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered but are not found persuasive, as discussed below.
Firstly, the Applicant argues that Bell does not teach a droplet identification molecule carrying a droplet index sequence. This argument is not persuasive because Bell reaches microbead-attached oligonucleotides comprising index or barcode sequences that identify droplets, microbeads, and associated cells during downstream sequencing (Abstract; p. 24-26, 32). Under the broadest reasonable interpretation, an index or barcode sequence that identifies a droplet or its contents constitutes a “droplet index sequence”, regardless of whether the sequence is described as bead-attached or otherwise. Functional identity, not nomenclature, governs anticipation. See MPEP 2111.
The Applicant further asserts that Bell teaches only a single microbead per droplet and therefore does not each “more than one first vector” per droplet. This argument is not persuasive because Bell teaches populations of microbeads bearing oligonucleotides that are randomly distributed into droplets (p. 24-25), and does not exclude presence of multiple microbeads within a single droplet. A reference need not expressly describe every possible embodiment where the claimed structure is inherently present or permitted by the disclosure. See MPEP 2112. Thus, Bell anticipates the claimed configuration under broadest reasonable interpretation.
However, the Applicant does acknowledge that Bell’s mammalian nucleus, microbead, capture sequences, and index sequences are equivalent to the claimed biological material, first vectors, capture sequences, and index sequences. As such, once equivalence is acknowledged, Bell teaches each structural and functional limitation of the claims. Differences in arrangement or descriptive terminology do not avoid anticipation where the same elements are present and perform the same functions. See MPEP 2131. As a result, Bell also teaches the additional limitations of the dependent claims, including capture probes, UMIs, cleavage sites, cleavage linkers, nucleic acid extension, sequencing library construction, barcode lengths, and sequencing methods (p. 10, 23, 31-36, 40). Accordingly, claims 2-13 and 15-20 are anticipated by Bell as previously set forth.
Notably, the Applicant’s assertions regarding improved recovery rates, reduced cell input, and increased efficiency are not persuasive. Unexpected results are not relevant to anticipation, as 35 USC § 102 requires only that the claimed invention be disclosed in a single prior art reference. See MPEP 2131. Moreover, the instant claims do not recite the alleged performance metrics or limitations necessary to establish a nexus between the instant claims and the asserted advantages. Therefore, these arguments do not overcome the rejection.
As a result, Bell discloses, either expressly or inherently, each and every limitation of claims 1-13 and 15-20, under their broadest reasonable interpretation. Accordingly, the 102 rejection is maintained.
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).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the date of this final action.
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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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached on 571-272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/ELIZABETH ROSE LAFAVE/ Examiner, Art Unit 1684
/HEATHER CALAMITA/ Supervisory Patent Examiner, Art Unit 1684
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