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 claims benefit to provisional application 63/451,769 filed 03/13/2023. All claims are afforded the earlier filing date for examination.
Claim status
Claims 1-5, 15, 31, 33, 39-41, 43-46, 48-50, 57 and 63 are pending and under examination. Claims 6-14. 16-30, 32, 34-38, 42, 47, 51-56, and 58-62 were cancelled prior to examination. Claim 1 is the only independent claim.
Claim Rejections - 35 USC § 112
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.
Claims 4 and 63 are 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.
The term “diverse set” in claim 4 is a relative term which renders the claim indefinite. The term “diverse set” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The specification uses the identical unbounded phrase at [0006] and [0102] without further definition, and nowhere ties “diverse” to any quantifiable metric, e.g., minimum edit distance between barcode sequences, a minimum number of distinct sequences in the set, or a minimum ratio of set diversity to the size of the population being barcoded. Absent such a definition a person of ordinary skill in the art has no way to determine, for a given set of barcode sequences, whether that set crosses the threshold from “not diverse” to “diverse” as required by the claim. Two sequences different at a single position and ten thousand sequences differing extensively could each be argued to satisfy or fail the limitation with no boundary drawn by the claim language itself.
In regards to claim 63, the claim recites the alternative expression “A composition/kit” naming two different statutory categories of invention, a composition and a kit, as alternatives within a single claim, without specifying which is being claimed. MPEP § 2173.05 permits alternative language in claim (“or”-type or Markush-type expressions) only when the alternatives are of a similar nature and do not render the scope of the claim unclear. Here, the two named alternatives are not of a similar nature. The specification itself distinguishes a “composition,” described as comprising “a plurality of beads and/or a plurality of barcode oligonucleotides” (see [0120]), from a “kit,” described as additionally comprising “instructions of using the composition for determining the binding strength between each of the at lest one protein and the target” (see [0121]). The claims scope therefore varies depending on which alternative is read into it, and neither the claim nor the specification indicates which alternative governs. This is precisely the kind of ambiguity that renders alternative claim language indefinite under § 112(b).
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 63 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim 63 recites “A composition/kit comprising a bead and a plurality of barcode oligonucleotides of claim 39.” Claim 39, from which claim 63 purports to depend, is a method claim “the method of claim wherein barcoding the nucleic acids from the vectors that are bound to the target comprises barcoding the nucleic acids from the vectors that are bound to the target with a plurality of barcode oligonucleotides.”
Under 35 USC 112(d), a dependent claim must “contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed.” A claim directed to a composition or kit, a product, does not further limit a method claim directed toa series of process steps, because the teow claims are directed to different statutory categories of invention under 35 USC 101 (a product versus a process), and reciting a bead and barcode oligonucleotides as physical components of a composition or kit does not narrow, restrict, or add a limitation to any of the process steps recited in claim 39 (i.e., providing vectors, contacting with a target, separating, barcoding, analyzing , and determining binding). See MPEP 608.01, which explains that a dependent claim must incorporate by reference all the limitations of the claim to which it refers, and that this reference and limitation relationship is generally inapplicable between claims of different statutory classes, since a claim toa physical product cannot “specify a further limitation” of a claim to a series of acts performed by a person or apparatus.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-5 and 57 are rejected under 35 U.S.C. 102(a)(1)/102(a)(2) as being anticipated by Xue et al. (US 2020/0262914 A1, published Aug. 20, 2020).
Xue discloses a method of determining binding between one or more proteins and a target, comprising the following steps, mapped to claim 1 in turn.
Xue discloses “providing a plurality of vectors each comprises a nucleic acid encoding a protein”. Xue discloses a library of yeast cells, each displaying a different BH3 peptide encoded by a plasmid contained within that cell. The library was constructed by cloning BH3-motif-derived peptide sequences into a yeast surface display plasmid (“Puma PCT plasmid”), transforming yest cells such that each transformed cell carries and expresses one peptide-encoded sequences (see Xue [0098], [0121]). The instant specification’s own definition of “vector” specifically states “the vector is a eukaryotic cell, a procaryotic cell, or a non-cell vector” (see [0012] of the instant specification), and separately identifies “the display library can be a cell surface display library (e.g., yeast display library)” (see [0073] of the instant specification) as a contemplated embodiment of the claimed invention. Xue’s yeast cells are therefore vector as broadly defined by the Applicant.
Xue further discloses “wherein each of the plurality of vectors displays the encoded protein on its surface and the encoded proteins from at least two of the plurality of vectors are different.” Xue’s input library had a theoretical diversity of 27,696,384 members (see Xue [0098]), of which approximately 10,000 clones were assayed (see Xue [0123]), and 5,769 unique peptides were quantified in a single experiment (see Xue [0100]). Each displayed peptide is expressed on the yeast cell surface, as confirmed by detection using anti-HA and anti-c-myc antibody labeling of the surface displayed constructs (see Xue [0120]). The encoded proteins across this library overwhelmingly different from one another, far exceeding the “at least two” of the claim.
Furthermore, Xue discloses “contacting the plurality of vectors with a target in a condition allowing the proteins displayed on the surfaces of the vectors to specifically bind the target.” Xue discloses the induced yeast library is “resuspended in BSS with least 10 - fold molar excess target protein and incubated in the filter plate for 2 h at room temperature with gentle shaking” (see Xue [0120], [0213]), using Bcl-xL, Mcl-1, or Bfl-1 as the target proteins in separate experiments (see Xue [0098], [0123]).
Additionally, Xue discloses “separating the vectors that are bound to the target from the vectors that are not bound to the target.” Xue discloses that “cells were sorted into 12 gates to separate bindings of different affinities”, using fluorescence activated cells sorting (FACS; see Xue [0123]). Xue further classifies the sorted population by reference to a bound/unbound spectrum, vectors in gate 1 are classified as “unresolvable tight”, gates 2-10 are classified as “resolvable”, and gates 11-12 classified as “unresolvable weak” (see Xue [0141]). This 12-gate architecture necessarily performs, as a specific and more granular species, the separation of target-bound vectors from non-bound vectors recited in the claim.
Xue further discloses “barcoding the nucleic acids from the vectors that are bound to the target to generate barcoded nucleic acids.” Xue’s computational processing section discloses that “deep sequencing datasets were filtered to retain only reads with at least 99% base call accuracy that included the specific multiplex barcodes used to identify each experiment” (see Xue [0136]), confirming that barcode sequences are incorporated into the plasmid-derived nucleic acid recovered from sorted (i.e., bound) yeast cells prior to sequencing, consistent with standard preparation of a barcoded sequencing library from recovered display-vector DNA (see Xue [0122]-[0124]), describing recovery, growth, and sequencing of cells sorted into each affinity gate).
Xue further discloses “analyzing the barcoded nucleic acids.” Xue discloses deep sequencing of the barcoded DNA recovered from each gate, followed by extensive computational analysis, filtering for read quality, clustering by sequence similarity, and reconstruction of each clone’s distribution across the 12 sorting gates (see Xue [0136]-[0139]).
Furthermore, Xue teaches “determining the binding between the proteins encoded by the plurality of vectors and the target.” Xue converts each clone’s gate-distribution profile into a “mean affinity coordinate” (see Xue [0139]) and further converts this into an apparent cell-surface binding free energy I kcal/mol using calibration standards, thereby determining the binding between each encoded peptide and the target protein (see Xue [0099], [0143]).
In regards to claim 2, Xue discloses that “amped SORTCERY can quantify apparent cell surface binding energies for thousands of diverse ligands in high-throughput. We used this technique to measure peptide binding to three related proteins : Bfl-1 , Mcl-1 , and Bcl-xL.” (see Xue [0112]). Xue reports binding energies (in kcal/mol) individually for 5,769 peptides across three separate target proteins (see Xue [0100]), i.e., binding affinity value determined for each of the encoded proteins in the library with respect to a given target. This is squarely “determining the binding affinity between each of the encoded proteins and the target” as recited in claim 2.
In regards to claim 3, Xue disclosed FACS sorting into 12 gates operates by physically diverting cells into separate collection vessels according to gate criteria. Cells whose binding signal places them in the “unresolvable weak” gate i.e., non-binders, are thereby excluded from the collected, resolvable binder pools that are grown, barcoded, sequenced, and carried forward for affinity determination. (see Xue [0122]-[0124], [0141]). This discloses removing the vectors displaying a protein not bound to the target from the pool of interest.
In regards to claim 4, Xue’s sequencing reads include both the peptide-coding insert and a multiplex barcode sequence used to distinguish reads from different experiments (see Xue [0136], [0139]. The term “diverse set of barcode sequences” as used in the claim is broad and unbound, as the specification does not quantify what makes a barcode “diverse”. Under this broad, unbounded reading, any set of more than one mutually distinguishable barcode sequence, such as the set of multiplex barcodes Xue uses to distinguish it’s twelve gates across multiple targets and replicate experiments, falls within “a diverse set of barcode sequences” as claimed.
In regards to claim 5, Xue discloses both alternatives. Sequencing information: Xue performs deep sequencing of the recovered, barcoded DNA (see Xue [0136]). Abundance: Xue’s computational pipeline calculates a per-clone, per-gate frequency directly from barcoded nucleic acid read counts “we first calculated the clone's frequency in that gate as the number of reads for sequence x in gate i… divided by the sum of all reads for all sequences in gate I” (see Xue [0138]).
In regards to claim 57, Xue discloses “Based on sparse titrations, the apparent dissociation constants (KD_app) for the designs binding to their targets on the cell surface were estimated to be < 100 nM; there were 28 examples with KD_app < 10 nM, and 5 with KD_app < 1 nM. Off-target affinities were weak for all designed peptides, with estimated KD_app values > 1000 nM” (see Xue [0107]). Xue additionally reports a range of measured dissociation constants directly in paragraph [0099], and separately reports solution phase Ki and Kd values determined by an orthogonal competition fluorescence polarization assay for a subset of peptides (see Xue [0107], Table X2).
Xues disclose of computing an “apparent dissociation constant (KD_app)” from display-library sequencing data is a verbatim match to the claim term “apparent dissociation constant” and claim 57 is anticipated by Xue.
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.
Claim(s) 1-5, 15, 31, 33, 39-41, 43-46, and 48-50 are rejected under 35 U.S.C. 103 as being unpatentable over Li et al. (US 2011/0207628 A1, published Aug. 25, 2011) in view of Wang et al. (US 2018/0002738 A1, published Jan. 4, 2018).
In regards to claim 1, Li discloses methods of determining binding between one or more proteins and a target comprising: “providing a plurality of vectors each comprises a nucleic acid encoding a protein, wherein each of the plurality of vectors displays the encoded protein on its surface and the encoded proteins from at least two of the plurality of vectors are different.” Li discloses an ORF phage display cDNA library in which foreign cDNA is genetically fused toa phage capsid protein, such that library proteins are expressed as coat fusion proteins and displayed on the phage surface (see Li [0043]). Li’s libraries generated from mouse eye and embryo tissue, comprise thousands to millions of phage clones each displaying a different cDNA-encoded protein (see Li [0053], Tables 2-4 showing dozens of distinct identified proteins from a single library).
Li further discloses “contacting the plurality of vectors with a target in a condition allowing the proteins displayed on the surfaces of the vectors to specifically bind the target.” Li discloses incubating the phage display library with immobilized bait (target) under conditions permitting binding (see Li [0043] disclosing “purified bait is immobilized, for example, on 96-well ELISA plates (or microbeads), blocked, and incubated with phage display library”). Li further discloses “separating the vectors that are bound to the target from the vectors that are not bound to the target.” Li discloses that “after washing, bound phages are eluted” (see Li [0043]).
Additionally, Li discloses “determining the binding between the proteins encoded by the plurality of vectors and the target.” Li discloses using the eluted phage for quantification, and using conventional means such as PCR screening of individual plaques with primers annealing to the phage vector and to the cDNA insert (see Li [0036], [0043], [0058]-[0061], Table 2).
Li does not discloses barcoding the recovered phage nucleic acids with a molecular tag and is silent to sequencing-based, single-molecule quantification of the population of bound phage.
Wang cures this deficiency. Wang discloses a method for amplifying target nucleic acids in a nucleic acid sample comprising: extending a plurality of barcode primers (“BC primers”) using the target nucleic acids as templates, wherein each barcode primer comprises a universal primer sequence, a molecular tag sequence, and a target-specific sequence (see Wang [0011]-[0012], [0053]); separating unused barcode primers from the extension product (see Wang [0011], [0076]-[0078]); and amplifying the extension products with a second set of primers to generate barcoded amplification products suitable for sequencing (see Wang [0011], [0079]-[0091]).
Wang explicitly teaches that molecular barcoding solves the problem of distinguishing an original template molecule from PCR resampling of that same molecule, and that “the target quantification can also be better achieved by counting the number of unique molecular barcodes in the reads rather than counting the number of total reads, as total read counts are more likely skewed for targets by non-uniform amplification” (see Wang [0008]).
It would be obvious to one of ordinary skill in the art at the time of filing to combine Li’s phage display, protein identification method with Wang’s barcoding technique to arrive at the claimed invention. Li identifies the very problem that Wang’s barcoding technique solves. Li describes plaque assay, in its own disclosed method of quantifying bound phage, as “[a] major rate-limiting step” involving “the tedious procedure of plaque assay includes serial dilution of phages, infecting the host cells, plating, culturing and plaque counting” (see Li [0058]), and states that this limitation “substantially limit[s] T7 phage application with high throughput screening by pharmaceutical and biotech industries” (see Li [0045]). The Applicant’s own specification confirms that combining display technologies such as phage display with next-generation sequencing to screen for desirable sequences was already known in the art before the instant invention, stating that “[v]arious display technologies such as phage, yeast, or ribosome display, coupled with next-generation sequencing (NGS) can be used to screen for desirable sequences” (see [0049]). A person having ordinary skill in the art seeking to overcome the throughput limitation the Li itself identifies, would have been motivated to apply Wang’s known molecular barcoding plus NGS technique to the population of phage recovered from Li’s binding assay, in order to quantify the relative abundance of each distinct protein-displaying clone in the bound population, with a reasonable expectation if success, since Wang’s technique is expressly designed to be broadly applicable to “any nucleic acid of interest” (see Wang [0049]) and Li’s recovered phage nucleic acids are unremarkable substrates for such amplification. Therefore teaching the “barcoding the nucleic acids from the vectors that are bound to the target to generate barcoded nucleic acids; analyzing the barcoded nucleic acids” of the instant claim, and reading on the limitations of claim 1.
In regards to claim 2, Li disclose quantifying binding as a relative measure (e.g., “binding index”, “phagocytosis index”, fold-enrichment over control (see Li [0059], [0227]). The combination with Wang, which enable quantification via unique barcode counts before and after selection, further supports a finding that determining a comparative binding measure between each encoded protein and the target would have been obvious.
In regards to claim 3, Li explicitly discloses that separating bound from unbound vectors is accomplished by washing away non-specifically bound and unbound phage, i.e., “removing the vectors displaying a protein not bound to the target” (see Li [0043], [0052]).
In regards to claim 4, Wang discloses that the molecular tag (barcode) sequence is selected such that “the diversity of the MT sequence of each BC primer needs to be at least 10 fold higher than the original number of DNA copies in the input nucleic acid sample,” so that each copy of the starting nucleic acid receives a unique tag (see Wang [0065]). This teaches selection of a barcode sequence from a set of sequences whose diversity is deliberately sized relative to the population being tagged, i.e., a “diverse set of barcode sequences” as recited. It would have been obvious to apply this same design principle to the barcode sequences appended to Li’s recovered phage nucleic acids, for the same reason Wang gives: to ensure that each original phage-derived template molecules receives a distinguishable tag.
In regards to claim 5, the claim recites two alternative (or combinable) limitations on the analyzing step of claim 1. (a) obtaining sequence information of the barcoded nucleic acids, and/or (b) determining abundance of the barcoded nucleic acids generated from the vectors that are bound to the target. Wang teaches both.
Sequence information: Wang’s method culminates in preparing a sequencing library from the barcoded/amplified nucleic acids and sequencing it. “The method disclosed herein may further comprise step (f) to sequence the amplification products from step (e)” (see Wang [0104]), using high-throughput platforms such as Illumina (see Wang [0108]). This directly discloses obtaining sequence information of the barcoded nucleic acids.”
Determining abundance: Wang discloses that “the method disclosed herein may further comprise step (g) that determine the copy number of one or more target nucleic acid(s) in a nucleic acid sample… Preferably, the copy number may be determine by counting unique molecular barcodes linked to the target nucleic acid(s)” (see Wang [0110). Wang further demonstrates this quantification in Example 3, measuring the abundance of RNA transcripts by counting unique molecular barcodes and showing improved accuracy over raw read counts, particularly for low-abundance targets (see Wang [0157]-[0160]).
Applying Wang’s sequencing and barcode-counting steps to the barcoded nucleic acids recovered from Li’s target-bound phage (per the combination already established for claim 1) renders obvious both alternatives recited in claim 5, for the same motivation to combine discussed above, Li’s own stated need for a higher-throughput, more quantitative alternative to plaque assay is satisfied precisely by Wang’s sequence-based abundance determination.
In regards to claim 15, Li discloses solid supports for immobilizing the binding pair target including “plastic, magnetic beads, glass beads, filter membranes, filter paper, and polymeric beads” (see Li [0041]), and states that “purified bait is immobilized, for example on 96-well ELISA plates (or microbeads)” (see Li [0043]).
In regards to claim 31, Li’s entire disclosure concerns phage display, with T7 phage as a preferred embodiment (see Li [0045]).
In regards to claim 33, Li discloses that cDNA insert is ligated into the phage vector and packaged into phage such that the coding sequence resides within, and is recoverable from, the phage genome itself (see Li [0036], [0052]-[0053]; PCR amplification of cDNA insert directly from phage genomic DNA at [0136], [0141]).
In regards to claim 39, Wang discloses a plurality of barcode primers, specifying “at least 20 different barcode primers” are used to apply barcodes to nucleic acids (see Wang [0011], [0069]).
In regards to claim 40, Wang’s barcode primers comprise from 5’ to 3’, a universal primer sequence, a molecular tag sequences (the barcode sequence), and a target-specific sequence (reading on the recited “barcoding primer”), which “specifically binds to a target nucleic acid, which allows the extension of the barcode primer using the target nucleic acid as the template” (see Wang [0053], [0061]). The Target nucleic acids region to which the primer binds reads on the recited “barcoding primer binding region.”
In regards to claim 41, Wang discloses the molecular tag sequence lengths as “from 3 to 20 nucleotides, such as from 5 to 15 nucleotides” (see Wang [0057]), a range squarely overlapping the claimed values. Overlapping ranges gives rise to a presumption of obviousness absent evidence of criticality (see MPEP 2144.05); no such evidence appears in the specification, which itself frames these lengths as a routine listing of operable values (see [0014]).
In regards to claim 43, Wang expressly teaches that “to ensure that each copy of starting nucleic acid receives a unique MT sequence, the diversity of the MT sequences of each BC primer needs to be at least 10 fold… higher than the original number of DNA copies” (see Wang [0065]), i.e., a design rule expressly directed to achieving the claimed uniqueness.
In regards to claim 44, Wang’s barcode primer includes the universal primer sequence, “used for further PCR amplification” (see Wang [0056]), and Wang further discloses appending an adapter sequence platforms (e.g. Illumina compatible adapters) via universal adapter primers (see Wang [0094]-[0098]).
In regards to claim 45, Wang’s target specific sequence is “at least substantially and preferably completely complementary to a region of the target nucleic acid of interest,” permitting hybridization based annealing (see Wang [0061]-[0063]).
In regards to claim 46, Li’s own vector design already discloses PCR primers annealing to defined positions withing the phage vector backbone flanking the cDNA insert at known distances (e.g., T7SelectUp/T7SelectDown primers annealing to the phage vector outside the insert, (see Li [0048], [0136]), establishing that position an annealing primer at a defined distance upstream of a coding insert was routine in phage vector design. Wang further discloses target-specific primer sequences of length “from 10 to 40 nucleotides, preferably from 15 to 25 nucleotides” (see Wang [0063]), overlapping the claimed 5-100 bp range. Selecting a specific upstream distance and primer binding region length within these known, overlapping ranges is a matter of routine primer design and optimization within the ordinary skill of the art (see MPEP 2144.05), particularly as the specification does not identify any criticality or unexpected result associated with the specific numerical ranges claimed.
In regards to claim 48, Wang’s step (a) discloses “extending each of a plurality of barcode primers (BC primers) to obtain extension products using the target nucleic acid as templates” (see Wang [0011], [0046]).
In regards to claim 49, Wang discloses that “BC primer extension is limited to just one cycle to strictly avoid ‘barcode resampling’” (see Wang [0075]). An explicitly teaching, accompanied by an express rationale (avoiding the same template molecule being independently resampled and mis-tagged, which would corrupt molecule counting), directly applicable to the goal of Li’s (the instant combination’s) single-molecule quantification of bound phage.
In regards to claim 50, Li discloses monitoring phage enrichment by comparing a population before and after selection (see Li [0035] comparing diluted input phage to phage recovered after selection, Fig. 24 comparing total phagocytosed phage across rounds). In combination with Wang’s barcode-based quantification (which enables sequencing based abundance measurements of any nucleic acid population, including a pre-selection aliquot (see Wang [0110]-[0113]). It would have been obvious to apply the same barcoding and sequencing quantification to a portion of Li’s phage population set aside before contacting with the target, in order to obtain a pre-binding abundance for comparison against the post-binding abundance, consistent with Li’s own practice of comparing enrichment across selection stages.
In regards to claim 63, it would have been obvious over the combination of Li, teaching a bead as solid support for the binding assay (see Li [0041],[0043]) and Wang, teaching the barcode primer/oligonucleotide structure discussed above to include the items necessary to perform the method as a composition or a kit for the same reasons of combination given to claim 39, for which claim 63 depends.
Conclusion
No claim is allowed.
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/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684 /AARON A PRIEST/Primary Examiner, Art Unit 1681
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