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 .
Priority
This application is a DIV of 16/422,837 filed on 05/24/2019 and awarded US. Patent No. 11414701. Application 16/422,837 has PRO 62/780,889 filed on 12/17/2018 and PRO 62/676,069 filed on 05/24/2018. The claims are being examined using earliest priority date of 05/24/2018 for all claims.
Election/Restrictions
Applicant’s election without traverse of Group I, including claims 124-131 in the reply filed on 6/15/2026 is acknowledged.
Applicant is reminded that upon the cancelation of claims to a non-elected invention, the inventorship must be corrected in compliance with 37 CFR 1.48(a) if one or more of the currently named inventors is no longer an inventor of at least one claim remaining in the application. A request to correct inventorship under 37 CFR 1.48(a) must be accompanied by an application data sheet in accordance with 37 CFR 1.76 that identifies each inventor by his or her legal name and by the processing fee required under 37 CFR 1.17(i).
Claim Status
Claims 124-131 and 163-166 are pending an under examination. Claims 1-123 were canceled prior to examination. Claims 132-162 have been cancelled in response to the restriction requirement sent 04/14/2026, in which Applicant elected Group I, including claims 124-131. Claims 163-166 are new. Claims 124 and 128 have been amended. Claim 124 is the only independent claim.
Claim Interpretation
For purposes of examination, certain claim terms that are broad or otherwise require construction have been interpreted in accordance with their broadest reasonable interpretation (BRI) consistent with the specification, as is required during prosecution. See MPEP 2111; In re Am. Acad. of Sci. Tech. Ctr., 367 F.3d 1359, 1369, 70 USPQ2d 1827, 1834 (Fed. Cir. 2004). The following claim constructions have been applied in this office action.
“Combinatorial split-and-pool [strategy]” – Under BRI, and consistent with the specification’s description of this strategy (see [0252], [0329]), this term is reasonably construed as a barcoding method comprising multiple, sequential rounds in which: (i) a population of cells, or nucleic acids therefrom, is s dived (“split”) across a plurality of physically separated pools (e.g., wells of a plate); (ii) each pool receives addition of a distinct tag or index sequences common to all members within that pool during that round; and (iii) the contents of all pools are then recombined (“pooled”) prior to the next round of splitting and tag addition. As explicitly described in paragraph [0329] (“after 3 cycles of split and pool ligation, the barcode on any given particle possesses the same one of 1003 possible barcodes”), the distinguishing characteristic of this species is that barcode uniqueness arises combinatorially from the particular ordered combination of tags accumulated across multiple, discrete, sequential tagging events rather than from a single tagging event of from physical isolation of each cell in a sealed compartment. Under this construction, a split-and-pool strategy does not require at any point during the barcoding process itself, that individual cells be physically isolated from one another in individual sealed volumes; physical separation exists only transiently, at the level of the pool (which contains many cells/particles) during each round.
“Droplet-based [strategy]” – Under the BRI, and consistent with the specification’s separately-titled “Droplet-Based Methods” description (see [0285]-[0288]), this term is reasonably construed as a barcoding method comprising: (i) physically isolating single cells into individual discrete volumes (e.g. emulsion droplets), each discrete volume being sealed off from all others; and (ii) incorporating a barcode into the resulting amplicon via a primer pair present within that discrete volume, wherein “at least one primer comprises a barcode sequence that uniquely identifies the individual discrete volume (see [0285]). Under this construction, the distinguishing characteristic of this species is that barcode uniqueness arises from the physical isolation of a single cell together with a single, pre-formed, uniquely barcoded primer within one sealed compartment, a single tagging/incorporation event occurring once the cell is isolated, rather than from multiple sequential tag additions events performed while the sample remains in bulk or pooled form.
Distinction: Because “sequential barcodes” is reasonably construed, in light of the specification to require multiple discrete, iterative tagging events producing a combinatorial barcode space, a feature the specification attributes specifically to the split-and-pool strategy and its multi-round split/tag/pool cycle, and because the specification’s droplet-based strategy is described as achieving barcode uniqueness through a single incorporation event within an isolated compartment rather than through iterative tag addition, the two constructions are not interchangeable with respect to the “sequential barcodes” limitation. A strategy satisfying the BRI of “droplet-based” as disclosed does not without more, satisfy the BRI of adding sequential barcodes as disclosed. This distinction is the basis or the written description rejection that follows.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claim 124 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 124(c) requires that the recited barcoding strategy, whether “combinatorial split-and-pool or droplet based”, “add sequential barcodes to the linked proximity dependent probes.” The instant specification does not support a droplet-based species. The specification’s “Split and Pool Methods” embodiment (see [0038], [0259]) is the only embodiment using the phrase “sequential barcodes,” reciting “using combinatorial split-and-pool strategies, such as ligation to add sequential barcodes to the linked proximity dependent probes.” By contrast, the specification’s separately titled “Droplet-Based Methods” embodiment (see [0045], [0300]-[0305]) recites isolating single cells into individual discrete volumes, each containing “a primer pair and amplification reagents, wherein the primer pair bind to the primer binding sites of the proximity dependent probes, and wherein at least one primer comprises a barcode sequence that uniquely identifies the individual discrete volume”, a single, one-steep barcode incorporation event per discrete volume, with no recitation of sequential or combinatorial barcode addition.
To the extent applicant may point to the “hydrogel droplet” embodiments elsewhere in the specification (see [0055]-[0057], [0362]) as support for a droplet-based species that adds sequential barcodes, those embodiments achieve combinatorial, sequential barcoding by performing split-pool ligation of barcodes onto primers while those primers are linked to a hydrogel matrix, i.e., they are an implementation of the combinatorial split-and-pool strategy using a hydrogel solid support, not a distinct “droplet based” barcoding mechanism separate from split-and-pool as the claim language of 124 (c) implies. As presently claimed, 124(c) recites split-and-pool and droplet-based as alternative species each independently capable of adding sequential barcodes but the specification’s genuinely droplet-based species discloses only single-step barcode incorporation.
To support the “droplet-based” species of 124(c), applicant should identify with particularity where the specification describes a droplet-based (as opposed to split-pool) strategy that adds sequential barcodes, since a single-step, one-barcode-per-droplet embodiment does not appear to fully support this limitation as claimed. The issue is particularly relevant given that “or droplet-based” was added to claim 124(c) by amendment after the prior restriction requirement and full support of the amendment as claimed should be confirmed.
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 124 rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 124 recites the limitation “the bound proximity probes” in step (b). There is insufficient antecedent basis for this limitation in the claim.
Claim 124 recites the limitation “the set of probes derived from single cells” in step (c). There is insufficient antecedent basis for this limitation in the claim.
Claim 124 recites the limitation "the primer pair" in step (d). There is insufficient antecedent basis for this limitation in the claim.
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 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 124-131 and 163-166 are rejected under 35 U.S.C. 103 as being unpatentable over Wedler et al. (US 2014/0336058 A1, published Nov. 13, 2014) in view of Seelig (US 2020/0263234 A1, published Aug. 20, 2020, with priority to Sep. 22, 2017).
Wedler is in the field of molecular biology and teaches methods for expression profiling including methods for determining the sequence and quantity of RNA in a sample. Wedler discloses a probe architecture and amplification/sequencing readout scheme, a two-part proximity dependent probe, each half comprising a target binding region hybridizing directly to RNA and an integral primer binding site, covalently linked by ligation, amplified using a primer pair, and quantified by sequencing. Seelig is in the similar field of identifying and labeling molecules within cells and cell nucleus and teaches methods for barcoding individual molecules, with single-cell resolution. Seelig teaches delivering probes/barcodes to intact cell populations and combinatorial split-pool barcoding to resolve cell of origin.
In regards to claim 124, the claim recites “a method for generating single-cell molecular analysis comprising: a) delivering one or more proximity dependent probes to a cell population, wherein each proximity dependent probe comprises: a target binding region configured to bind one or more target RNAs and a primer binding site region.” Wedler discloses a two-part nucleic acid hybridization probe delivered to a composition comprising target RNA, wherein each probe comprises a nucleic acid molecule with a target binding region and a tail which does not hybridize to an RNA in the composition but acts as a universal primer binding site (see Wedler [0041]).
Wedler’s disclosed embodiment delivers it probes to a cell-free composition of extracted RNA rather than to an intact cell population. Seelig, however, discloses delivering combinatorial barcoding reagents (including hybridization/ligation-based primers) directly to fixed, permeabilized cells retained as an intact population rather than to RNA extracted therefrom, specifically to preserve single-cell resolution during barcoding (see Seelig Fig. 2, [0003]). It would have obvious to a person of ordinary skill in the art to apply Wedler’s two-part hybridization probe chemistry directly to a fixed, permeabilized cell population as taught by Seelig, rather than to RNA pre-extracted from cells, in order to retain the cell-of-origin information necessary for the single-cell barcoding, with a reasonable expectation of success because Welder’s probe/target hybridization chemistry does not depend on the RNA being free in solution and applying known hybridization-probe chemistries to fixed, permeabilized cells in place of extracted nucleic acids was a well-established technique in the art by the priority date.
In regards to step (b) “linking the bound proximity dependent probes,” Wedler discloses “covalently linking the hybridized first nucleic acid molecule to the hybridized second nucleic acid, wherein the linking is done by means of reverse transcription and subsequent ligation.” (see Wedler [0031]). Wedler further discloses an embodiment in which the two probes hybridize immediately adjacent to one another on the target RNA without an intervening gap and are directly joined by ligation without a reverse-transcription step (see Wedler [0146]). This directly meets the “linking the bound proximity dependent probes” as claimed.
In regards to step (c) “using combinatorial split-and-pool or droplet-based strategies to add sequential barcodes to the linked proximity dependent probes to attach a unique barcode to the set of probes derived from single cells”, Seelig discloses a combinatorial split-pool barcoding method in which sequential rounds of dividing cells into aliquots, adding aliquot-specific nucleic acid tags, and recombining the aliquots to attach a unique combination of barcodes to nucleic acid molecules derived from each individual cell, enabling single-cell resolved analysis without physical isolation of individual cells (see Seelig [0035] disclosing “(b) dividing the plurality of cells into a number (n) of aliquots; (c) providing a plurality of nucleic acid tags to each of the n aliquots, wherein each labeling sequence of the plurality of nucleic acid tags provided into a given aliquot is the same, and wherein a different labeling sequence is provided into each of then aliquots …(e)combining then aliquots; and (f) repeating steps (b), (c), (d), and (e) with the combined aliquot.”)
It would have been obvious to a person of ordinary skill in the art before the effective filing date to combine Seelig’s combinatorial split-pool barcoding scheme with the linked, primer site bearing proximity probe products taught by Wedler, in order to obtain single-cell resolved digital quantification using Wedler’s efficient, low-step probe/ligation/sequencing chemistry, which Wedler identifies as improving on conventional RNA-seq library preparation by eliminating mRNA enrichment, rRNA depletion, and adapter ligation (see Wedler [0020]), at the throughput and cell-resolution scale that Seelig’s method was develop to provide. Combining a known, simplified probe-based RNA quantification chemistry with a known combinatorial single-cell barcoding scheme to obtain the predictable results of single-cell resolved digital transcript counting is the application of known techniques according to their established function.
In regards to step (d), “amplifying the probes using the primer pair, wherein the barcode is incorporated into each resulting amplicon,” Wedler explicitly disclosing amplification using a primer pair keyed to the two-tailed probe architecture (see Wedler [0025]). Seelig discloses that the sequential split-pool barcode tags are ligated into a continuous barcode sequence, such that each resulting sequencing product carries the barcode (see Seelig [0045]). The combination of Wedler’s primer-pair amplification of the linked probe with Seelig’s barcode-ligation scheme teaches this limitation.
In regards to step (e), “quantifying target RNAs in each individual cell based at least in part on sequencing the resulting amplicons,” Wedler explicitly discloses sequencing-based quantification of the amplified probe product (see Wedler [0025]). Seelig further discloses that this sequencing-based quantification is performed on an individual cell basis using the incorporated barcode combinations such that data can be gathered regarding RNA expression at the level of a single cell (see Seelig [0038]). The combination directly teaches this limitation.
As stated above, Wedler discloses nearly the entirety of the claimed probe architecture and amplification/sequencing readout scheme, a two-part proximity dependent probe, each half comprising a target binding region hybridizing directly to RNA and an integral primer binding site, covalently linked by ligation, amplified using a primer pair, and quantified by sequencing. Seelig supplies the single-cell resolution layer, delivery of probes to intact cell populations, and combinatorial split-and-pool barcoding to resolve cell of origin, that Wedler’s bulk-composition method lacks. Combining known prior art elements according to their established, complementary functions (efficient probe/ligation/sequencing chemistry + known single-cell combinatorial barcoding scheme) to yield the predictable result of single-cell resolved digital transcript quantification does not require more than ordinary skill in the art.
The motivation to combine Welder and Seelig is not merely that both are known elements in the same general field. Each reference’s own background section identifies a specific problem that the other references’ teaching directly and predictably solves. Wedler identifies that traditional RNA-seq sample preparation requires expensive and labor-intensive workflows and NGS platforms have limited sequencing capacity (see Wedler [0003]-[0019]) and teaches methods to account for a few of these hurdles, including direct RNA-hybridization probes (eliminating the need for mRNA enrichment, rRNA depletion, and cDNA synthesis) and using targeted gene expression profiling ensures only RNA of interest are counted (see Wedler [0020]). Meanwhile Seelig identified that bulk NGS methods in which “RNA transcripts are generally purified from lysed cells...all of the cDNA sequences are mixed together before sequencing, such that RNA expression is measured for a whole sample and individual sequences cannot be linked back to an individual cell” (see Seelig [0003]). Seelig’s solution of combinatorial split-pool barcoding of cellular contents while cells remain intact, solves the cell-of-origin resolution problem (see Seelig [0056]).
A person of ordinary skill reading Wedler would recognize that Wedler’s method, while efficient, provides no way to resolve which cell a given transcript count came from, exactly the limitation Seelig’s background section identifies as the central shortcoming of bulk profiling. Conversely, a person of ordinary skill reading Seelig would recognize that Seelig’s split-pool barcoding, while solving cell-of-origin resolution, still requires reverse-transcribing RNA into cDNA and barcoding a large fraction of the transcriptome (or relies on non-targeted RT priming), consuming sequencing capacity across many genes of no interest to a given panel-based study, exactly the read-capacity problem Wedler’s background section identifies and Wedler’s targeted, ligation-based probe panel was specifically designed to solve. Theis complementary fit is not a matter of hindsight reconstruction: the read capacity problem is more acute in a single-cell context, because the total sequencing budget must be divided across many individual cells rather than one bulk sample, meaning a person of ordinary skill implementing Seelig’s single-cell barcoding scheme would have had heightened motivation to pair it with a targeted, low-read burden assay chemistry like Wedler’s in order to make efficient use of the limited per-cell sequencing depth inherent to any single cell method.
Beyond the complementary problem-solution fit, the two chemistries are structurally compatible without modification of either references core mechanism. Wedler’s probe architecture terminates in defined non-target-hybridizing “tail” sequences that Welder identifies as universal priming sites configured to bind PCR primers for amplification, i.e., a defined single-stranded overhand sequence designed for downstream engagement (see Wedler [0041]). Seelig’s split-pool tagging mechanism operates generically on any such defined overhang, using “a first portion complementary to at least one of the 5’ hybridization sequences and the 5’ overhang sequences” of whatever upstream-generated molecule is present to it (see Seelig [0036]) to hybridize and ligate its aliquot-specific barcode tags. A person of ordinary skill would therefore have had a reasonable expectation of success in applying Seelig’s barcode-tagging chemistry directly to the free tail/overhang already present on Wedler’s linked primer site bearing probe products without need to redesign either references core probe or tagging chemistry. Therefore, each reference’s own state problem is the other’s solution, and the two chemistries are structurally compatible, giving a person of ordinary skill in the art both reason to combine and a reasonable expectation of success in doing so.
In regards to claim 125 and 126, Seelig discloses labeling of cellular proteins with DNA-tagged antibodies or aptamers, integrated into the identical combinatorial barcoding workflow used for RNA (see Seelig [0065] (disclosing “the molecules are protein, peptide, and/or antigen, and the adapter sequences may be bound to unique identifiers sequence… that is coupled to an antibody… In certain embodiments… the adapter sequence may be integrated in an aptamer”). Seelig further discloses that “A sequencing reaction can reveal a unique identifier sequence associated with a given protein as well as the label or barcode associated with a unique cell or cells.” (see Seelig [0067]), i.e., DNA-tagged protein binding molecules, amplified and sequenced to quantify target protein abundance on a per-cell basis, substantially as recited in claim 125, and using either an antibody or aptamer as recited in claim 126.
In regards to claim 127, Wedler disclosed linking bound proximity probes by enzymatic ligation, as discussed above with respect to 124(b), including an embodiment using ligation alone without an intervening reverse-transcription/gap-filling step (see Wedler [0031]).
In regards to claim 128, Wedler’s probe architecture, two physically separate oligonucleotides molecules each independently hybridizing to the target RNA, which subsequently joined by ligation to form a single linked product, directly reads on “split-ligation probes” of claim 128.
In regards to claim 129, Wedler discloses methods in which a large number of probes are used in a multiplex fashion wherein “A plurality of these probes is used and means 10 or more of such probes. Preferably between 15 and 100, more preferably between 100 and 500, even more preferably between 100 and 1000” (see Wedler [0040]). This disclosed range directly overlaps and exceeds the lower thresholds recited in claim 129. To the extent the claims upper thresholds (“at least 10,000”) exceeds Wedler’s expressly disclosed range, the additional scaling represents the product of routine optimization of a result-effective variable and would have been obvious to one of ordinary skill in the art.
In regards to claims 130-131, Wedler teaches that placement of probes on exon-exon junctions serves a specific stated purpose beyond simple target capture “By placing of probes on exon-exon junctions and adjustment of suitable hybridization conditions the selectivity to distinct mRNAs can be increased. Furthermore, it allows expression profiling of different splice variants of a mRNA.” (see Wedler [0130]). A person of ordinary skill reading Wedler would understand from this passage that different splice isoforms of the same gene/target RNA are distinguished by designing separate probe sets keyed to different exon-exon junctions unique to each isoform, necessarily meaning that more than one proximity dependent probe (or probe pair) is applied to the same target transcript locus in order to resolve which splice variant is present, rather than a single probe set applied once per gen. A person of ordinary skill implementing Welder’s targeted panel would have had reason to deploy multiple (2-100), differently positioned probe sets against a single transcript of interest specifically to resolve splice-variant identity and abundance. The specific numerical range is a routine optimization of the number of probes used per target, motivated by Wedler’s splice-variant profiling rationale, would scale the number of probe sets applied to a given transcript with the number of splice isoforms of interest, which would suggest to a person of ordinary skill that increasing the probe number per target could improve detection sensitivity and per-cell data completeness, consistent with In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
In regards to claim 163, Wedler explicitly discloses incorporation of a molecular barcode into each probe (see Wedler [0046]). Wedler further explains that this barcode functions a UMI (see Wedler [0048] disclosing “These molecular barcodes are generated by introduction of random nucleotides between universal tail and target specific sequences of the probe. It allows differentiation between fragments derived from a target RNA molecule and copies generated during PCR amplification”).
In regards to claims 164-165, these claims recite that the proximity dependent probes detect gene expression markers on one or more cell signaling pathways, including cell development pathway, a cancer signaling pathway, or an immune response signaling pathway. This is a non-limiting field of use/intended use limitation directed to the choice of target genes assayed by the method of claim 124, rather h to any structural or procedural modification of the method itself. Selection of a target gene panel directed to known, well-characterized cell development, cancer, or immune signaling pathways would have been an obvious application of the methods of claim 124 (as rendered obvious above) to targets of known clinical and research interest well within the routine skill of a person of ordinary skill in the art seeking to apply a general-purpose multiplexed transcript detection method to biologically relevant gene panels. Furthermore, Wedler, discloses measuring RPL13A expression (see Wedler Fig. 8), “RPL13A acts in the GAIT complex after interferon-gamma activation” and “participates in selective translational repression during inflammatory signaling”1 directly reading on an immune response signaling pathway.
In regards to claim 166, Seelig’s split-pool barcoding scheme is expressly ligation-based (see Seelig [0044]).
Conclusion
No claim is allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Weibrecht et al. (“In situ detection of individual mRNA molecules and protein complexes or post-translational modifications using padlock probes combined with the in-situ proximity ligation assay”, Nature Protocols, Vol. 8, No. 2, pgs. 355-372, 2013). Discloses a single-cell workflow combining padlock-probe RNA detection with antibody-based in situ proximity ligation assay (PLA) protein/PTM detection, including DNA-tagged antibody probe architecture, circularization-oligo ligation, and RCA based amplification.
Nilsson et al. (“Enhanced detection and distinction of RNA by enzymatic probe ligation”, Nature Biotechnology, Vol. 18, pgs. 791-793, 2000). Discloses pairs of DNA oligonucleotide probes hybridizing directly to, and enzymatically ligated upon, an RNA target strand without an intervening cDNA synthesis step.
Larsson et al. (“In situ detection and genotyping of individual mRNA molecules”, Nature Methods, Vol. 7, No. 5, pgs. 395-400, 2010). Discloses padlock probe based in situ transcript detection and genotyping via target primed rolling circle amplification, including use of two different probes targeting different regions of the same transcript.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Matthew H Raymonda whose telephone number is (703)756-5807. The examiner can normally be reached Monday - Friday 10:00 am - 4:00 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached at 571-272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684
/AARON A PRIEST/Primary Examiner, Art Unit 1681
1 Genecard Human Gene Database, https://www.genecards.org/card/RPL13A
Prosecution Insights