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
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 4/15/2026 has been entered.
Claim Status
Claims 1 has been amended (4/15/2026). Claim 14 was previously cancelled (12/4/2025) and claims 4, 9 are newly cancelled (4/15/2026). No new matter was added. Thus, claims 1-3, 5-8, 10-13 and 15-20 are under examination (4/15/2026).
Priority
Claims 1-3, 5-8, 10-13 and 15-20 receive a priority date of 1/23/2020, the effective filing date of PCT/CN2020/073968.
Rejections Withdrawn
Claim Rejections - 35 USC § 102
The rejection to claims 1-13 and 15-20 under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Bell et al., (WO 2019/217099 A1, published 11/14/2019) is withdrawn due to Applicant’s amendments of independent claim 1. Specifically, amended independent claim 1 recites additional limitations directed to a droplet identification molecule comprising a captured sequence covalently bonded to a droplet index sequence and a second vector carrying a plurality of droplet identification molecules, which are not expressly disclosed by Bell. Accordingly, Bell no longer anticipates the claims as presently amended.
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 1-3, 5-8, 10-13 and 15-20 are rejected under 35 U.S.C. 103 as being unpatentable over Bell et al., (WO 2019/217099 A1, published 11/14/2019) and in view of Zilionis et al. (“Single-cell barcoding and sequencing using droplet microfluidics”, Nature Protocols, published 12/8/2016).
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).
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 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 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 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 claim 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 does not teach or suggest a droplet identification molecule comprising a captured sequence covalently bonded to a droplet index sequence, nor does Bell teach a second vector carrying a plurality of droplet identification molecules.
Zilionis teaches a method called inDrops, which has the capability to index >15,000 cells in an hour where a suspension of cells is first encapsulated into nanoliter droplets with hydrogel beads (HBs) bearing barcoding DNA primers and cells are then lysed and mRNA is barcoded (indexed) by a reverse transcription (RT) reaction (Abstract). Specifically, Zilionis teaches details for (i) establishing an inDrops platform (1 d); (ii) performing hydrogel bead synthesis (4 d); (iii) encapsulating and barcoding cells (1 d); and (iv) RNA-seq library preparation (2 d) (Abstract). Further, Zilionis teaches that the basic principle of the technology is easy to understand: a mixture of cells is encapsulated into microfluidic droplets together with barcoding oligonucleotide primers (attached to HBs), and a mix of RT and lysis reagents (Fig. 1) and the mRNA released from lysed cells remains trapped inside the same droplet and is tagged (barcoded) with oligonucleotide primers during the RT reaction (Introduction: Paragraphs 3-5). Further Zilionis teaches that after barcoding, the material from all cells is pooled by breaking the droplets, and the cDNA library is processed for next-generation sequencing and in this process it is essential that each droplet carry primers encoding only one unique barcode, which should be different from the barcodes in other droplets and to achieve this, they developed a combinatorial approach to produce a library of barcoding hydrogel beads (BHBs) that are coencapsulated with the cells where each bead carries covalently coupled, photoreleasable primers encoding one predefined barcode and further where the barcode in this context encodes two parts: a 'cellular barcode', which indicates the cell from which mRNA is captured, and a 'unique molecular identifier' (UMI), which provides a quantitative measure of absolute transcript levels in a given cell (Introduction: Paragraphs 3-5; Figure 1).
It would have been obvious to one of ordinary skill in the art at the time of the invention to modify the droplet-based sequencing system of Bell to incorporate the barcoding hydrogel bead architecture taught by Zilionis (inDrops). Specifically, Bell teaches droplet-based nucleic acid capture and sequencing using barcoded oligonucleotides, while Zilionis teaches hydrogel beads carrying a plurality of covalently attached barcoding primers that include both a barcode region and a capture region for capturing nucleic acids within individual droplets. Incorporating the bead-based barcoding architecture of Zilionis into Bell’s system would have predictably enabled identification and association of nucleic acids originating from a common droplet while maintaining Bell’s high-throughput droplet sequencing workflow.
A person of ordinary skill in the art would have been motivated to combine these teachings because both references are directed to droplet microfluidic sequencing systems that use barcoded oligonucleotides to track the origin of captured nucleic acids and improve sequencing throughput and data attribution. Such a combination would merely substitute one known droplet-indexing and capture architecture for another known architecture performing the same function within the same technological field.
Further, there would have been a reasonable expectation of success because Bell and Zilionis employ compatible droplet-based microfluidic platforms, utilize nucleic-acid capture oligonucleotides and barcode sequences, and rely on established molecular biology techniques for nucleic acid capture, reverse transcription, amplification, and sequencing. The modification would have involved the predictable use of known barcoding hydrogel beads in Bell’s droplet environment to obtain the expected benefit of associating captured nucleic acids with a unique droplet-associated barcode.
Applicant’s Response: The Applicant argues that Bell fails to teach a droplet identification molecule carrying a droplet index sequence that is separate from the first vectors carrying capture and cell index sequences, and further fails to disclose the newly added limitations requiring a captured sequence covalently bonded to a droplet index sequence and a second vector carrying a plurality of droplet identification molecules. Applicant further contends that Bell’s disclosure is directed to a one-cell/one-microbead architecture and does not teach or suggest the claimed arrangement involving more than one first vector within a droplet.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered but are found to be partially persuasive, as discussed below.
As previously stated, the Applicant argues that Bell does not teach a droplet identification molecule comprising a captured sequence covalently bonded to a droplet index sequence, a second vector carrying a plurality of droplet identification molecules, or the claimed arrangement involving more than one first vector. These arguments are persuasive with respect to the anticipation rejection, which has been withdrawn. However, the arguments are not persuasive as to the present obviousness rejection because Zilionis teaches barcoding hydrogel beads carrying a plurality of covalently attached primers, wherein the primers comprise a barcode region and a capture region for capturing nucleic acids within droplets. Zilionis further teaches that each droplet carries primers encoding a unique barcode, such that nucleic acids captured within a droplet are associated with that barcode.
Accordingly, Zilionis teaches the additional droplet-identification architecture lacking in Bell, including a second vector in the form of a hydrogel bead carrying a plurality of barcode-containing primers and capture sequences linked on the same oligonucleotide. One of ordinary skill in the art would have been motivated to incorporate the hydrogel bead barcoding architecture of Zilionis into Bell’s droplet-based sequencing system in order to provide droplet-associated identification and tracking of captured nucleic acids while maintaining the known benefits of high-throughput droplet sequencing. The combination merely applies a known barcoding and capture architecture to a known droplet sequencing platform for its established purpose and would have yielded predictable results. Furthermore, there would have been a reasonable expectation of success because Bell and Zilionis are directed to compatible droplet microfluidic sequencing systems that utilize barcode-containing oligonucleotides, nucleic acid capture, amplification, and sequencing using well-understood and routine molecular biology techniques. See MPEP 2143.
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
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