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 .
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . Applicant’s amendment and arguments filed 3/6/2026 is acknowledged. Claims 10-12, 14-17, 19, 22, 24, 31, 34-45, 47, 49-50, 52-55, 58, 63-70 and 72 have been canceled. Claims 1-9, 13, 18, 20, 21, 23, 25-30, 32-34, 46, 48, 51, 56-57, 59-62, 71 and 73 are pending and under examination.
All of the amendment and arguments have been thoroughly reviewed and considered.
Any rejection not reiterated in this action has been withdrawn as being obviated by the amendment of the claims.
This action is made Final.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Previous Rejections
The objection to the specification is withdrawn in view of Applicant’s amendment to the specification. The claim rejection under 102(b) is maintained and discussed below.
Claim Rejections - 35 USC § 102
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim(s) 1-9, 13, 18, 20-21, 23, 25-30, 32-34, 46, 48, 51, 56-57, 59-62, 71 and 73 is/are rejected under 35 U.S.C. 102(a)(1) and/or alternatively 35 U.S.C. 102(a)(2) as being anticipated by Belhocine et al {Belhocine, used interchangeably herein} (US20190361010, November 28, 2019).
Regarding claim 1, Belhocine teaches a method comprising: a) providing a partition comprising: (i) a biological particle comprising a heterologous nucleic acid molecule, wherein the heterologous nucleic acid molecule comprises an exogenous insert and endogenous flanking sequences that flank the exogenous insert; (ii) a plurality of nucleic acid barcode molecules comprising a partition- specific barcode sequence and a capture sequence; (iii) a first insert primer, comprising from 5' to 3', a splinting sequence and a sequence complementary to a first sequence of the exogenous insert, and (iv) a splint oligonucleotide comprising a sequence complementary to the capture sequence of a first nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules and a sequence complementary to the splinting sequence of the first insert primer; b) subjecting the partition to conditions sufficient for: (i) coupling the first nucleic acid barcode molecule to the first insert primer to generate a barcoded first insert primer; and (ii) hybridizing the barcoded first insert primer to the first sequence of the exogenous insert of the heterologous nucleic acid molecule; and c) generating a first barcoded nucleic acid molecule comprising: (i) the partition- specific barcode sequence, or a reverse complement thereof, and (ii) a first endogenous flanking sequence of the endogenous genomic flanking sequences, or a reverse complement thereof [e.g., [0006] – [0019], [0096] – [0119], [[0208] – [0209], [0250]- [0251], [0336] – [0337], [0372]-[0376], [0603], [0802], [0938], [0945].
Regarding claim 2, Belhocine teaches wherein the heterologous nucleic acid molecule comprises a double-stranded deoxyribonucleic acid (dsDNA) molecule [e.g., [0007] and [0099]).
Regarding claim 3, Belhocine teaches wherein the exogenous insert comprises a recognition site for a restriction enzyme (RE), wherein the recognition site is at a 5' or 3' end of the exogenous insert [e.g., [0007] - [0119].
Regarding claim 4, Belhocine teaches wherein the recognition site for the RE differs from a cleavage site for the RE ([0007] – [0119]).
Regarding claim 5, Belhocine teaches wherein the conditions are further sufficient for denaturing the dsDNA molecule [0145].
Regarding claim 6, Belhocine teaches wherein coupling the first nucleic acid barcode molecule to the first insert primer comprises hybridizing: (i) the first nucleic acid barcode molecule to the splint oligonucleotide; and (ii) the first insert primer to the splint oligonucleotide ([0007] – [0119]).
Regarding claim 7, Belhocine teaches wherein: (i) the sequence complementary to the capture sequence of the first nucleic acid barcode molecule of the splint oligonucleotide hybridizes to the capture sequence of the first nucleic acid barcode molecule; and (ii) the sequence complementary to the splinting sequence of the splint oligonucleotide hybridizes to the splinting sequence of the first insert primer [e.g., [0007] – [0119]).
Regarding claim 8, Belhocine teaches wherein coupling the first nucleic acid barcode molecule to the first insert primer comprises ligating the first nucleic acid barcode molecule to the first insert primer [e.g., [0007] – [01119]).
Regarding claim 9, Belhocine teaches wherein i) the ligating is performed by an enzyme capable of nick repair, and/or ii) the enzyme comprises a ligase or polymerase, and/or iii) the enzyme comprises a DNA polymerase, a reverse transcriptase, or a DNA ligase [0251], [0255] and [0276].
Regarding claim 13, Belhocine teaches wherein the barcoded first insert primer hybridized to the first sequence of the exogenous insert of the heterologous nucleic acid molecule is extended into the first endogenous flanking sequence to produce the first barcoded nucleic acid molecule [0007] – [0119].
Regarding claim 18, Belhocine teaches further comprising subjecting the first barcoded nucleic acid molecule to second strand synthesis to yield a double stranded (ds) derivative of the first barcoded nucleic acid molecule [0007] – [0119].
Regarding claim 20, Belhocine teaches further comprising fragmenting the ds derivative of the first barcoded nucleic acid molecule to yield a barcoded fragment thereof ([0006] – [0019], see also [0096] – [0119], [[0208] – [0209]).
Regarding claim 21, Belhocine teaches wherein the fragmenting is performed by an RE ([0006] – [0019], see also [0096] – [0119], [[0208] – [0209]).
Regarding claim 23, Belhocine teaches further comprising attaching a first adaptor to the first barcoded nucleic acid molecule, or a derivative, or amplicon, or a barcoded fragment thereof to generate an adapted barcoded product, wherein the first adaptor comprises a reverse primer binding site ([0006] – [0019], see also [0096] – [0119], [[0208] – [0209], [0518]- [0519]).
Regarding claim 25, Belhocine teaches further comprising amplifying the adapted barcoded product with a first primer that hybridizes to the reverse primer-binding site [0006] – [0019], see also [0096] – [0119], [[0208] – [0209]).
Regarding claim 26, Belhocine teaches wherein the plurality of nucleic acid barcode molecules further comprise a forward primer binding site or reverse complement thereof, and wherein the amplifying further comprises amplifying with a second primer that hybridizes to the forward primer binding site [0006] – [0019], see also [0096] – [0119], [[0208] – [0209]).
Regarding claim 27, Belhocine teaches wherein the sequence complementary to the first sequence of the exogenous insert is within about 20 to 50 nucleotides of the junction of the exogenous insert and the first endogenous flanking sequence [0281], [0748].
Regarding claim 28, Belhocine teaches wherein the exogenous insert comprises a recognition site for an RE, wherein the recognition site is: (i) in the sequence complementary to the first sequence of the exogenous insert of the first insert primer, or (ii) between the sequence complementary to the first sequence of the exogenous insert of the first primer and the junction of the exogenous insert and the first endogenous flanking sequence [0006] – [0019], see also [0096] – [0119], [[0208] – [0209] and [0282]).
Regarding claim 29, Belhocine teaches wherein the recognition site for the RE differs from the cleavage site for the RE, and wherein the RE cleavage site is in the first endogenous flanking sequence [0006] – [0019], see also [0096] – [0119], [[0208] – [0209]).
Regarding claim 30, Belhocine teaches wherein the exogenous insert encodes a polypeptide or a functional RNA e.g., [0006] – [0019], [0096] – [0119], [[0208] – [0209], [0336] – [0337], [0372]-[0376].
Regarding claim 32, Belhocine teaches wherein the polypeptide comprises a chimeric antigen receptor (e.g., [0357], [0414]).
Regarding claim 33, Belhocine teaches further comprising determining expression of an analyte of the biological particle, (e.g., [0006] – [0019], [0096] – [0119], [[0208] – [0209], [0336] – [0337], [0344] [0664], [0886], [0889]).
Regarding claim 34, Belhocine teaches wherein the analyte is an mRNA analyte, and expression of the mRNA analyte is determined via generation of a second barcoded nucleic acid molecule, wherein the second barcoded nucleic acid molecule comprises: (i) the partition- specific barcode sequence, or a reverse complement thereof; and (ii) at least a portion of a nucleic acid sequence of the mRNA analyte, or a reverse complement thereof ([0006] – [0019], [0096] – [0119], [[0208] – [0209] and [0372]-[0376]).
Regarding claim 46, Belhocine teaches wherein the analyte is a peptide or protein analyte, wherein the expression of the peptide or protein analyte is determined by: further providing in the partition, a labeling agent operatively coupled to a first reporter oligonucleotide, wherein the labeling agent binds the peptide or protein analyte [0006] – [0019], [0096] – [0119], [[0208] – [0209] and [0371]-[0376].
Regarding claim 48, Belhocine teaches wherein (i) the first reporter oligonucleotide comprises a first reporter barcode sequence and a capture handle sequence, and/or (ii) the plurality of nucleic acid barcode molecules further comprises a further nucleic acid barcode molecule comprising the partition-specific barcode sequence and a further capture sequence, wherein the further capture sequence is configured to couple to the capture handle sequence [0378] – [0395].
Regarding claim 51, Belhocine teaches further comprising generating a further barcoded nucleic acid molecule wherein the further barcoded nucleic acid molecule comprises: (i) the partition-specific barcode sequence, or a reverse complement thereof, and (ii) the first reporter barcode sequence, or a reverse complement thereof, wherein the further barcoded nucleic acid molecule is used to determine expression of the peptide or protein analyte ([0006] – [0019], [0096] – [0119], [[0208] – [0209], [0372]-[0376], [0398] – [0400]).
Regarding claim 56, Belhocine teaches further comprising determining a sequence of the first endogenous flanking sequence of the first barcoded nucleic acid molecule.
Regarding 57, Belhocine teaches further comprising mapping the exogenous insert as inserted into a location in endogenous sequences of the biological particle based on the determined sequence of the first endogenous flanking sequence, wherein the biological particle is characterized as comprising the exogenous insert at the location in the endogenous sequences ([0006] – [0019], [0096] – [0119], [[0208] – [0209], [0337], [0372]-[0376], [0398] – [0400]).
Regarding claim 59, Belhocine teaches further comprising identifying the location as capable of dysregulating expression of one or more endogenous genes of the biological particle, wherein the identifying comprises determining the biological particle is unsuitable for administration as a therapeutic agent to a subject in need thereof [0337], [0372]-[0376], [0398] – [0400]) .
Regarding claim 60, Belhocine teaches wherein the dysregulating comprises: (i) activation of an adjacent endogenous gene, wherein the adjacent endogenous gene comprises an oncogene; (ii) interruption of an endogenous gene; or (iii) silencing an endogenous gene ([0006] – [0019], [0096] – [0119], [[0208] – [0209], [0337], [0372]-[0376], [0398] – [0400]).
Regarding claim 61, Belhocine teaches wherein the biological particle is a cell or a nucleus of a cell ([0006] – [0019], [0096] – [0119], [[0208] – [0209], [0337], [0372]-[0376], [0398] – [0400]).
Regarding claim 62, Belhocine teaches wherein the cell:(i) is a T cell, bone marrow progenitor cell, an induced progenitor stem cell, a plasma cell or a retinal cell, and/or (ii) is for administration to a subject, and/or (iii) is autologous or allogeneic to the subject, and/or (iv) has been modified to comprise the exogenous insert by a gene editing protein having sequence-specific integration characteristics ([0006] – [0019], [0096] – [0119], [[0208] – [0209], [0337], [0372]-[0376], [0398] – [0400]).
Regarding claim 71, Belhocine teaches wherein the cell is characterized as comprising on-target insertion of the exogenous insert if the first endogenous flanking sequence reflects the sequence-specific integration characteristics of the gene editing protein, and the cell is characterized as comprising off-target insertion of the exogenous insert if the first endogenous flanking sequence does not reflect the sequence-specific integration characteristics of the gene editing protein ([0006] – [0019], [0096] – [0119], [[0208] – [0209], [0337], [0372]-[0376], [0398] – [0400]).
Regarding claim 73, Belhocine teaches wherein the partition is a well, microwell or droplet (e.g., [0004]). Therefore, Belhocine meets the limitations of the claimed invention.
Response to Arguments
Applicant’s Traversal
Applicant traverses the rejections on the following grounds:
(a) Applicant submits that Belhocine does not disclose a partition comprising a biological particle comprising a heterologous nucleic acid molecule, wherein the heterologous nucleic acid molecule comprises an exogenous insert and endogenous flanking sequences that flank the exogenous insert.
(b) In that same partition, Belhocine does not disclose the presence of the first insert primer of claim 1,
(c) Belhocine disclose a splint oligonucleotide comprising a sequence complementary to the capture sequence of a first nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules and a sequence complementary to the splinting sequence of the first insert primer.
(d) Furthermore, Belhocine also fails to disclose the coupling and hybridization of part (b) of claim 1 within the context of the elements of part (a), that is, as Belhocine does not disclose the components of part (a), the coupling and hybridization could not proceed as recited in part (b).
Examiner’s Response
8. All of the amendment and arguments have been thoroughly reviewed and considered but are not found persuasive for the reasons that follows: The examiner acknowledges Applicant’s arguments but respectfully disagree.
(i) With regards to Applicant’s arguments at (a) –(d) above, the specification defines the following limitations recited in the claims as follows:
[00711] The term "partition," as used herein, generally, refers to a space or volume that may be suitable to contain one or more species or conduct one or more reactions. A partition can be a physical container, compartment, or vessel, such as a droplet, a flow cell, a reaction chamber, a reaction compartment, a tube, a well, or a microwell. The partition may isolate space or volume from another space or volume. The droplet may be a first phase (e.g., aqueous phase) in a second phase (e.g., oil) immiscible with the first phase. The droplet may be a first phase in a second phase that does not phase separate from the first phase, such as, for example, a capsule or liposome in an aqueous phase. A partition may comprise one or more other (inner) partitions. In some cases, a partition may be a virtual compartment that can be defined and identified by an index (e.g., indexed libraries) across multiple and/or remote physical compartments. For example, a physical compartment may comprise a plurality of virtual compartments.
[0005] biological particle, e.g., cell or nucleus, modified to include an exogenous nucleic acid, e.g., gene of interest, inserted in its genomic, e.g., endogenous, nucleic acids [0005].
[0028] In some embodiments, the cell has been modified to comprise the exogenous insert by a gene editing protein having sequence-specific integration characteristics. In some embodiments, the gene editing protein is a zinc finger protease, a transcription activator-like effector nuclease (TALEN) or an RNA-guided endonuclease. In some embodiments, the RNA-guided endonuclease is a Cas protein. In some embodiments, the Cas protein is a Cas 9 protein.
[0081] The biological particle, e.g., cell, cell derivative or cell organelle, in the provided partition may include a heterologous nucleic acid molecule. The heterologous nucleic acid molecule may include an exogenous insert, e.g., exogenous nucleic acids, and endogenous, e.g., genomic, flanking sequences that flank the exogenous insert.
In some embodiments, the heterologous nucleic acid molecule, including the exogenous insert and endogenous flanking sequences flanking the insert, may be a double-stranded deoxyribonucleic acid (dsDNA) molecule. In other embodiments, it may be either a single-stranded DNA (ssDNA) or single- stranded ribonucleic acid (ssRNA) molecule.
[0082] The exogenous insert of the heterologous nucleic acid molecule may include a nucleic acid sequence that encodes a polypeptide or a functional RNA.
[0085] In some embodiments, the exogenous insert of the heterologous nucleic acid molecule may include nucleic acid sequences in addition those encoding the polypeptide or functional RNA. These sequences may include a restriction enzyme (RE) recognition site.
[0240] In 620a, the bead comprises nucleic acid barcode molecules that are attached thereto, and sample nucleic acid molecules (e.g., RNA, DNA) may attach, e.g., via hybridization of ligation, to the nucleic acid barcode molecules. Such attachment may occur on the bead. In process 630, the beads 604 from multiple wells 602 may be collected and pooled. Further processing may be performed in process 640. For example, one or more nucleic acid reactions may be performed, such as reverse transcription, nucleic acid extension, amplification, ligation, transposition, etc.
(a-i) In response to Applicant’s arguments that Belhocine does not teach the limitations at (a)-d) above, Belhocine teaches the following citations below, in conjunction with the citations noted in the prior office action, that given the broadest reasonable interpretation falls within the scope of the instant invention in light of the teachings of the specification which broadly defines terminologies recited and depicted by the claim. See below:
[0234] The nucleic acid molecule 802 may comprise a unique molecular identifying sequence 816 (e.g., unique molecular identifier (UMI)). In some cases, the unique molecular identifying sequence 816 may comprise from about 5 to about 8 nucleotides. Alternatively, the unique molecular identifying sequence 816 may compress less than about 5 or more than about 8 nucleotides. The unique molecular identifying sequence 816 may be a unique sequence that varies across individual nucleic acid molecules (e.g., 802, 818, 820, etc.) coupled to a single bead (e.g., bead 804). In some cases, the unique molecular identifying sequence 816 may be a random sequence (e.g., such as a random N-mer sequence). For example, the UMI may provide a unique identifier of the starting mRNA molecule that was captured, in order to allow quantitation of the number of original expressed RNA. As will be appreciated, although FIG. 8 shows three nucleic acid molecules 802, 818, 820 coupled to the surface of the bead 804, an individual bead may be coupled to any number of individual nucleic acid molecules, for example, from one to tens to hundreds of thousands or even millions of individual nucleic acid molecules. The respective barcodes for the individual nucleic acid molecules can comprise both common sequence segments or relatively common sequence segments (e.g., 808, 810, 812, etc.) and variable or unique sequence segments (e.g., 816) between different individual nucleic acid molecules coupled to the same bead. (see Figure which gives a structure that could be akin the structural components of step a).
[0235] In operation, a biological particle (e.g., cell, DNA, RNA, etc.) can be co-partitioned along with a barcode bearing bead 804. The barcoded nucleic acid molecules 802, 818, 820 can be released from the bead 804 in the partition. By way of example, in the context of analyzing sample RNA, the poly-T segment (e.g., 812) of one of the released nucleic acid molecules (e.g., 802) can hybridize to the poly-A tail of a mRNA molecule. Reverse transcription may result in a cDNA transcript of the mRNA, but which transcript includes each of the sequence segments 808, 810, 816 of the nucleic acid molecules 802. Because the nucleic acid molecule 802 comprises an anchoring sequence 814, it will more likely hybridize to and prime reverse transcription at the sequence end of the poly-A tail of the mRNA. Within any given partition, all of the cDNA transcripts of the individual mRNA molecules may include a common barcode sequence segment 810. However, the transcripts made from the different mRNA molecules within a given partition may vary at the unique molecular identifying sequence 812 segment (e.g., UMI segment). Beneficially, even following any subsequent amplification of the contents of a given partition, the number of different UMIs can be indicative of the quantity of mRNA originating from a given partition, and thus from the biological particle (e.g., cell). As noted above, the transcripts can be amplified, cleaned up and sequenced to identify the sequence of the cDNA transcript of the mRNA, as well as to sequence the barcode segment and the UMI segment. While a poly-T primer sequence is described, other targeted or random priming sequences may also be used in priming the reverse transcription reaction. Likewise, although described as releasing the barcoded oligonucleotides into the partition, in some cases, the nucleic acid molecules bound to the bead (e.g., gel bead) may be used to hybridize and capture the mRNA on the solid phase of the bead, for example, in order to facilitate the separation of the RNA from other cell contents. (These teachings suggest operations involving the coupling and hybridization steps).
[0371] Unique identifiers, e.g., barcodes, may be previously, subsequently, or concurrently delivered to the partitions that hold the compartmentalized or partitioned cells, in order to allow for the later attribution of the characteristics of the individual cells to the particular compartment. Further, unique identifiers, e.g., barcodes, may be coupled to the analytes and previously, subsequently, or concurrently delivered to the partitions that hold the compartmentalized or partitioned cells, in order to allow for the later attribution of the characteristics of the individual cells to the particular compartment. Barcodes may be delivered, for example on an oligonucleotide, to a partition via any suitable mechanism (e.g., attached to a gel bead as described herein). In accordance with the methods and systems described herein, analytes of individual cells can be provided with unique identifiers such that, upon characterization of those analytes they may be attributed as having been derived from the same cell or cells. The ability to attribute characteristics to individual cells or groups of cells is provided by the assignment of unique identifiers specifically to an individual cell or groups of cells. Unique identifiers, e.g., in the form of nucleic acid barcodes can be assigned or associated with individual cells or populations of cells, in order to tag or label the cell's components (and as a result, its characteristics) with the unique identifiers. These unique identifiers can then be used to attribute the cell's components and characteristics to an individual cell or group of cells. In some aspects, this is carried out by co-partitioning the individual cells or groups of cells with the unique identifiers. In some aspects, the unique identifiers are provided in the form of oligonucleotides (also referred to herein as herein oligonucleotides or reporter oligonucleotides) that comprise nucleic acid barcode sequences that may be attached to or otherwise associated with the nucleic acid contents of individual cells, or to other components of the cells, and particularly to fragments of those nucleic acids. The oligonucleotides may be partitioned such that as between oligonucleotides in a given partition, the nucleic acid barcode sequences contained therein are the same, but as between different partitions, the oligonucleotides can, and do have differing barcode sequences, or at least represent a large number of different barcode sequences across all of the partitions in a given analysis. In some aspects, only one nucleic acid barcode sequence can be associated with a given partition, although in some cases, two or more different barcode sequences may be present.
[0276] Additional reagents may also be co-partitioned with the biological particles, such as endonucleases to fragment a biological particle's DNA, DNA polymerase enzymes and dNTPs used to amplify the biological particle's nucleic acid fragments and to attach the barcode molecular tags to the amplified fragments. Other enzymes may be co-partitioned including without limitation, polymerase, transposase, ligase, proteinase K, DNAse, etc. Additional reagents may also include reverse transcriptase enzymes, including enzymes with terminal transferase activity, primers and oligonucleotides, and switch oligonucleotides (also referred to herein as “switch oligos” or “template switching oligonucleotides”) which can be used for template switching. In some cases, template switching can be used to increase the length of a cDNA. In some cases, template switching can be used to append a predefined nucleic acid sequence to the cDNA. In an example of template switching, cDNA can be generated from reverse transcription of a template, e.g., cellular mRNA, where a reverse transcriptase with terminal transferase activity can add additional nucleotides, e.g., polyC, to the cDNA in a template independent manner. Switch oligos can include sequences complementary to the additional nucleotides, e.g., polyG. The additional nucleotides (e.g., polyC) on the cDNA can hybridize to the additional nucleotides (e.g., polyG) on the switch oligo, whereby the switch oligo can be used by the reverse transcriptase as template to further extend the cDNA. Template switching oligonucleotides may comprise a hybridization region and a template region. The hybridization region can comprise any sequence capable of hybridizing to the target. (These passages suggested that additional components may be associated with the partition and having the ability to function in the manner claimed).
[0373] In other embodiments, a given partition comprises (a) a first plurality of oligonucleotides comprising a first barcode sequence; and (b) a second plurality of oligonucleotides comprising a second barcode sequence; wherein said first plurality of oligonucleotides are capable of coupling to a first analyte (e.g., a first adapter sequence present in, e.g., a CRISPR sgRNA molecule) and wherein said second plurality of oligonucleotides are capable of capturing at least two additional analytes (e.g., an mRNA molecule and an adapter sequence of a labelling agent oligonucleotide, e.g., a barcoded antibody). In some embodiments, said first plurality of oligonucleotides comprise a first capture sequence (e.g., a sequence complementary to an adapter sequence present in, e.g., a CRISPR sgRNA molecule) and said second plurality of oligonucleotides comprise a second sequence (e.g., a rGrGrG sequence complementary to a CCC sequence of a labelling agent oligonucleotide and a CCC sequence present on the 5′ end of a cDNA molecule).
[0747] In an aspect, the present disclosure provides a method for processing a nucleic acid molecule, comprising (a) loading a transposase molecule with a pair of nucleic acid adapters, where each of the pair of nucleic acid adapters comprises a first single-stranded portion comprising a first nucleic acid sequence and a second single-stranded portion comprising a second nucleic acid sequence, where the first nucleic acid sequence is different from the second nucleic acid sequence; (b) bringing the transposase molecules in contact with the nucleic acid molecule under conditions sufficient to generate a nucleic acid fragment, where the nucleic acid fragment (i) comprises at each of the first end and the second end, both the first nucleic acid sequence and the second nucleic acid sequence and (ii) is at least partially double-stranded; and (c) subjecting the nucleic acid fragment under conditions sufficient to generate a processed nucleic acid fragment, where the processed nucleic acid fragment comprises (i) the first nucleic acid sequence at the first end and the second nucleic acid sequence at the second end, or (ii) the second nucleic acid sequence at the first end and the first nucleic acid sequence at the second end.
[0748] In some cases, the first single-stranded portion and the second double-stranded portion are adjacent to a same end of the double-stranded portion. In some embodiment, the first single-stranded portion and the second single-stranded portion may be attached via a linker comprising a restriction enzyme recognition site. (These passages all teach limitations which falls within the embodiments of the teachings of the instant invention in light of the teachings of the specification).
[0990] In parallel to the chromatin workflow of panel 12400, an RNA molecule deriving from the same cell, cell bead, or cell nucleus may be processed. As shown in panel 12450, RNA molecule 12458 comprising RNA sequence 12460 and polyA sequence 12462 may be contacted 12464 with primer molecule 12452 comprising polyT sequence 12454 and additional primer sequence 12456. RNA molecule 12458 may then be reverse transcribed 12466 off of polyT sequence 12454 using a reverse transcriptase with terminal transferase activity, which reverse transcriptase may append sequence 12468 to the resultant cDNA molecule comprising cDNA sequence 12470. Sequence 12468 may be a polyC sequence. A template switch oligonucleotide 12472 comprising sequencing primer or portion thereof or complement thereof 12474, unique molecule identifier sequence or complement thereof 12476, and capture sequence (e.g., polyG sequence) 12478 may then hybridize 12480 to the RNA-cDNA molecule and template switching may take place. The partition may include a gel bead 12416b coupled to a nucleic acid barcode molecule 12418b. Nucleic acid barcode molecule 12418b may comprise a flow cell adapter sequence 12420b (e.g., a P5 sequence), a barcode sequence 12422b, and a sequencing primer or portion thereof or complement thereof 12474′. Gel bead 12416b may be the same as gel bead 12416a such that partition comprises a single gel bead. In such a case, nucleic acid barcode molecule 12418b and nucleic acid barcode molecule 12418a may have the same sequences. Sequence 12474′ may hybridize to sequence 12474 of the RNA-cDNA molecule, or its complement, and undergo primer extension 12482 to yield a strand comprising sequences 12420b, 12422b, 12474′, 12476 or a complement thereof, 12468 or a complement thereof, and insert sequence 12470 or a complement thereof. The contents of the partition may then be recovered in bulk solution (e.g., a droplet may be broken) to provide the strand in bulk solution. This strand may undergo amplification (e.g., PCR) 12484 to provide a double-stranded amplification product 12486 that includes sequences of the nucleic acid barcode molecule 12418b, the original RNA molecule or cDNA corresponding thereto, and, optionally, an additional sequence 12488 that may comprise a sequencing primer or portion thereof (e.g., an R2 sequence) 12490, a sample index sequence 12492, and a flow cell adapter sequence (e.g., a P7 sequence) 12494. (These passages including e.g., [1056] teach embodiments that falls within the teachings of the instant invention given the broadest reasonable interpretation of the claims in light of the teachings of the specification.
Like the instant specification, the teachings of Belhocine provides multiple embodiments of the instant invention that falls with the definition of terminologies recited in the claim and therefore meets the limitations of the claims. Applicant’s arguments are not found persuasive and accordingly, the rejections are maintained.
Conclusion
9. No claims are allowed. THIS ACTION IS MADE FINAL. 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 nonprovisional extension fee (37 CFR 1.17(a)) 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 mailing date of this final action.
Conclusion
10. Any inquiry concerning this communication or earlier communications from the examiner should be directed to CYNTHIA B WILDER whose telephone number is (571)272-0791. The examiner can normally be reached Flexible.
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/CYNTHIA B WILDER/ Primary Examiner, Art Unit 1681