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 371 of PCT/US2022/043139 filed 09/09/2022. PCT/US2022/043139 has PRO 63/244,957 filed 09/16/2021. All claims are examined using the earliest possible priority date of 09/16/2021.
Claim Status
Claims 1-17 are pending and under examination. Claim 1 is the only independent claim.
Specification
The specification is objected to because the use of improperly demarcated trademarks has been noted in this application. Although the use of trademarks is permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner that might adversely affect their validity as trademarks. See MPEP §608. 01(v). 9. Examples of such an improperly demarcated trademarks “MiSeq”, “HiSeq”, and “NovaSeq” which appears in the present specification in paragraphs [0004]. Examiner notes that the provided examples are not meant to be a complete list of improperly demarcated trademarks found in the present specification. Applicant should review the entire specification and correct all instances of improperly demarcated trademarks. Appropriate corrections required. Each letter of a trademark should be capitalized or otherwise the trademark should be demarcated with the appropriate symbol indicating its proprietary nature (e.g., ™, ©, ®) and accompanied by generic terminology. Applicants may identify trademarks using the USPTO's trademark database. Trademark Electronic Search System (TESS), on the Internet at https://tmsearch.uspto.go
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 3,5-10, 12-15, and 17 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.
Claim 1, step (a), recites “each first polynucleotide independently and optionally comprising one or more non-silent mutations with respect to a reference sequence of the protein-coding region and/or region of interest, at least one of the first polynucleotides encoding the protein-coding region or region of interest comprising at least one silent mutation with respect to the reference sequence, the at least one silent mutation or a combination of silent mutations in a given protein-coding region and/or region of interest providing a first barcode.”
The term optionally is placed at the outset of this recitation, before any mutation, silent or non-silent, has been introduced into the claim. Because “optionally” precedes and is not grammatically confined to the “non-silent mutations” that happen to follow it in the same clause, the claim is amenable to at least two plausible constructions: (1) “optionally” modifies only the recited non-silent mutation, such that every first polynucleotide is nevertheless mandatorily subject to the separately recited requirement that: “at least one of the first polynucleotides” comprises “at least one silent mutation” providing the first barcode; or (2) “optionally” together with “independently” modifies the entire mutation framework that follows, non-silent and silent alike, such that whether any given first polynucleotide carries a mutation of either kind is itself optional and independently variable from molecule to molecule. See Ex parte Miyazaki, 89 USPQ2d 1207 (BPAI 2008) ("[R]ather than requiring that the claims are insolubly ambiguous, we hold that if a claim is amenable to two or more plausible claim constructions, the USPTO is justified in requiring the applicant to more precisely define the metes and bounds of the claimed invention by holding the claim unpatentable under 35 U.S.C. §112, second paragraph, as indefinite.").
This construction issue also affects the proper reading of claims 9, 10, 12, and 13, each of which recites back to “non-silent mutation(s)” as thought their presences in the first polynucleotide library were assured.
Clarification and correction are required. Applicant may wish to amend claim 1 to expressly states whether: (a) each first polynucleotide’s non-silent mutation content is optional while the silent-mutation is mandatory for at least one polynucleotide of the library, consistent with what appears to be the intended scope; or (b) some other relationship between the “independently and optionally” language and the recited silent/non-silent mutations is intended, stated in terms that do not require resort to the placement of a single modifier to resolve the scope of two materially different limitations.
Claim 1 recites in step (b), “a unique second randomized barcode nucleotide sequence” and refers to this element in steps (c) and (d) as the “second randomized barcode.” Claims 3,5-6, 8, 13-15, and 17 recite the limitation "second barcode(s)". There is insufficient antecedent basis for this limitation in the claim as “second barcode(s)” is not the term introduced in claim 1 and it is unclear whether the “second barcode” is intended to refer back to the “second randomized barcode nucleotide sequence”/”second randomized barcode” of claim 1 or to some other, distinct element. Clarification and correction are required. An appropriate correction would be to amend “second barcode(s)” to “second randomized barcode(s) throughout.
Claim 7 recites “the second (randomized) barcode”, this parenthetical insertion makes it unclear whether the parenthetical term is a limitation or just an aside. It is unclear whether “(randomized)” is meant to tie back to the “second randomized barcode” from claim 1, or whether it is introducing a new claim element.
Claim 13 recites “the two molecules” in line 2, there is insufficient antecedent basis for this limitation in the claim. The claim recites “two or more polynucleotides”, but it is not clear whether “the two molecules” refers to these polynucleotides, any two of the “two or more”, or to all of them collectively, or represents an inconsistency with the “two or more” language itself. Clarification and correction are required.
The term “about” in claims 5 and 6 is a relative term which renders the claim indefinite. The term “about” 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. Claim 5 recites that the first and second barcodes are separated by “more than about 300 nucleotides,” and claim 6 recites that they are separated by “less than about 600 nucleotides.” The specification defines “the terms "approximately," "about," "proximate," "minor variation," and similar terms generally refer to ranges that include the identified value within a margin of 20%, 10% or preferably 5% in certain embodiments, and any values therebetween.” This definition does not specify which of the three enumerated margins (or which intermediate value) applies to any given recitation of “about” in the claims. Because the scope of claims 5 and 6 depends entirely on which margin is selected, a person of ordinary skill in the art cannot determine the scope of claims 5 and 6 with reasonable certainty. See Nautilus, Inc. v. Biosig Instruments, Inc., 134 S. Ct. 2120, 110 USPQ2d 1688 (2014); MPEP 2173.05(b).
Claim Interpretation
BRI for Claims 14-17
For purposes of examination, certain claim aspects that require construction have been interpreted in accordance with their broadest reasonable interpretation 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.
Claim 1 recites synthesize, pairing, sequencing, and mapping steps performed on first and second polynucleotide library, but does not recite introducing that library, or any member thereof into a cell. Claims 14-17 presuppose a cellular context without ever positively reciting the step that creates it. Under BRI, and reading claims 14-17 in the manner most consistent with claim 1 and the specification (rather than as introducing an entirely new, unclaimed cell-based method), the examiner construes:
Claim 14 is interpreted as though the second polynucleotide library of claim 1 were transformed/transfected into a population of cells, such that a protein-coding region can be identified, within a given cell, by sequencing its associated barcodes.
Claim 15 is interpreted as requiring that two such protein-coding regions/second barcode pairs are present, and separately identifiable by their second barcodes, within a single cell.
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 1-17 are rejected under 35 U.S.C. 103 as being unpatentable over Dever et al. (WO 2020/210225 A1, published Oct. 15, 2020, claiming priority to Apr. 12, 2019), in view of Peikon et al. (US 10,017,758 B1, published Jul. 10, 2018).
Regarding claim 1, Dever discloses a method of synthesizing a plurality of homology recombination donor template polynucleotide sequences (a first polynucleotide library), each comprising a coding sequence corresponding to a protein-coding region or region of interest (e.g., the HBB coding sequence), the coding sequence independently and optionally comprising a non-silent mutation with respect to a wild-type/reference coding sequence, for example, correction of the sickle-cell causing E6V mutation, itself a single-nucleotide, non-silent substitution relative to the patient’s reference sequence (see [0033], [0168]), and at eat one of the polynucleotides comprising at least one silent mutation with respect to the reference sequence, wherein the silent mutation, or a combination of silent mutations, provides a first barcode. Dever expressly states that “one can introduce a barcode sequence into the targeted modification such that independent, otherwise identical, targeted modifications can be monitored by the presence of different barcode sequences,” and that “the barcode sequence is introduced as part of an introduced coding sequence as silent mutations,” made possible “in view of an identical protein” (see [0062]). Devers further discloses combinatorial silent-0mutation barcoding across up to nine amino acid codon position of the HBB coding sequence, yielding a theoretical maximum of 36,864 distinct in-frame synonymous donor sequences (see [0033], [0168]), corresponding to claim 1’s “combination of silent mutations… providing a first barcode”.
Dever further discloses that “alternatively to the silent mutation barcoding discussed above or in combination,” a separate, physically distinct barcode sequence can also be included in the same donor construct, outside the coding sequence. This second barcode is disclosed as a random or semi-randomized nucleotide sequence (see [0073]) and the working AAVS1 example at [0156], in which a 12-nucleotide degenerate region is introduced 3’ of the reporter cassette, yielding a theoretical library of 531,441 unique donor templates (see [0181]). This corresponds to claim 1, step (b) randomly pairing each first polynucleotide with a unique second randomized barcode nucleotide sequence, to produce a second polynucleotide library.
Dever further discloses sequencing the resulting barcoded polynucleotides, or at least the barcode containing regions thereof, using next-generation sequencing of paired end reads spanning the barcoded region of the genome (see [0137], [0140]-[0142], [0164]-[0165]), corresponding to claim 1, step (c).
Dever further discloses mapping the barcodes to the corresponding coding region, and expressly discloses that once this association is established, the barcode sequence alone is thereafter used to identify and track the corresponding polynucleotide: “Genome nucleotide sequencing can be used to determine the barcode sequence in each cell in a lineage or following cell division. The quantity of different barcode sequences will indicate the relative accumulation of cells having different donor template polynucleotides,” (see [0134]). This is the operative principle underlying the entire “TRACE-Seq” methodology disclosed in the reference, barcode-only sequencing is used at scale to track and quantify individual clones without resequencing the full coding region for every read (see [0168]-[0186], describing quantification of thousands of individual barcoded clones by amplicon sequencing of the barcode region alone). This discloses claim 1, step (d), including the “can be identified by sequencing only the second randomized barcode” limitation.
To the extent Applicant argues that Dever does not, in a single disclosed embodiment combine the silent-mutation first barcode and the physically separate second randomized barcode within the identical working example, [0072]’s express teaching that the two barcoding strategies may be used “in combination” provides the requisite motivation, a person of ordinary skill, aware of Dever’s own disclosure that its two barcoding schemes (silent mutation based and physically separate) are combinable, and aware of its own express statement that combining them was contemplated, would have combined them with a reasonable expectation of success, as the reference discloses no impediment to doing so and each element performs its already-recognized function (identity encoding via the coding sequence itself, and randomized tag encoding via a separate sequence) predictably when combined.
In regards to claim 2, Dever discloses that “any type of genomic nucleotide sequencing can be used,” and expressly lists long-read sequencing platforms among the discloses options, including SMRT sequencing (see [0135]-[0136], citing Eid et al. Science 323:133-138 (2009)). It would have been obvious to a person of ordinary skill to select a long-read platform such as SMRT sequencing from Dever’s own disclosed list of suitable sequencing technologies, particularly where doing so predictably enables single-read association of a barcode with a more distant coding region, a known advantage of long-read sequencing that Dever itself makes available as an alternative to the short-read (Illumina) approach used in its examples.
In regards to claim 3, Dever’s actual working examples sequence the second barcode region using Illumina MiSeq and HiSeq platforms, which are short-read next generation sequencing platforms (see [0136], [0139]-[0142], [0164]-[0165]).
In regards to claim 4, Dever discloses determining the relative abundance of polynucleotides in a population by sequencing their barcodes and using the resulting barcode read percentage/counts to quantify clonal representation (see [0134], [0172]-[0174], [0178]).
In regards to claims 5 and 6, Dever discloses homology arms ranging from about 10 bp up to about 4kb in length (see [0065]), and further discloses that the second barcode may be positioned after the poly-A sequence or between the coding sequence and the poly-A sequence, i.e., variably spaced from the first (silent mutation) barcode located within the N-terminal coding sequence (see [0006], [0015], [0021], and [0072]). The working HBB example places the silent mutation barcode within a homology region of approximately 645 bases (see [0155]), and the AAVS1 example places its outside coding barcode following an entire BFP reporter cassette (see [0156], [0181]). Under the broadest reasonable interpretation of “about 300 nucleotide” and “about 600 nucleotides,” informed by the specification’s own definition of “about” as encompassing a margin of up to 20%, a barcode separation on the order of 645 nucleotides as disclosed by Dever’s donor architecture falls within “more than about 300 nucleotides” (claim 5) under any of the specification’s disclosed margins, and within “less than 600 nucleotides” (claim 6) under the specifications 20% and 10% margins. To whatever extent Applicant contends a narrower margin controls, Dever’s express disclosure of homology arms ranging from about 10 base pairs to about 4 kilobases (see [0065]), renders the specific numerical spacing recited in claims 5 and 6 as a routine, result effective variable within a range already disclosed and rendered obvious by Dever, particularly in the absence of any disclosed criticality for either boundary in the specification.
In regards to claim 7, Dever’s disclosed workflow amplifies and sequences in a single amplicon read, the region spanning both the CRISPR cut site/anchor bases (within the coding sequence, adjacent to the silent-mutation barcode) and the barcode region using short-read paired-end sequencing (see [0137], [0140]-[0142], [0164]-[0165]), such that both the coding-region proximal silent mutation barcode and in embodiments combining both barcoding schemes per [0072], the second randomized barcode would be captured within the same short-read sequencing run.
In regards to claim 8, as with claim 2, Dever’s disclosure that any suitable sequencing technology may be used, including long-read platforms such as SMRT sequencing (see [0135]-[0136]), renders obvious an embodiment in which both the first and second barcode of a combined barcoding donor construct are read out via a single long-read sequencing step, consistent with the general proposition in the art that long-read sequencing is used precisely to span greater distances between linked sequence elements than short-read technology allows.
In regards to claim 9, Dever’s HBB donor library corrects the E6V mutation, a single amino acid substitution relative to the wild-type/reference HBB protein sequence (see [0033], [0168]).
In regards to claim 10, the E6V sickle-cell mutation corrected by Dever’s donor library is well known in the art, and disclosed in the reference’s own figures, to result from a single-nucleotide substitution (GAG[Wingdings font/0xE0]GTG; see [0033]).
In regards to claim 11, Dever’s silent-mutation barcoding scheme spans up to nine amino acid codon positions of the HBB coding sequence, each independently variable among degenerate codons, yielding combinations of far more than three silent mutations defining each first barcode (see [0033], [0168]: “36,864 in frame, synonymous mutations”).
In regards to claim 12, in Dever’s HBB donor library, each donor polynucleotide carries up to nine silent-mutation bearing codon positions (defining the first barcode) together with only the single non-silent E6V correcting mutation, such that the number of silent mutations exceeds the number of non-silent mutations as recited (see [0033], [0168]).
In regards to claim 13, Dever discloses that a plurality of donor polynucleotides each carrying the identical non-silent (E6V-correcting) mutation but a different, second randomized barcode, are introduced into different cells, and that these otherwise identical corrected polynucleotides/cells are thereafter distinguished from one another exclusively by sequencing their respective barcodes (see [0003]-[0004], [0061]-[0062], [0132]-[0134]). Dever’s stated purpose is precisely to track “independent, otherwise identical, targeted modification”, i.e., multiple molecules/cells sharing an identical corrective (non-silent) mutation, “by the presence of different barcode sequences” (see [0062]), and its examples confirm that hundreds to thousands of distinct barcoded clones, all carrying the identical HBB correction, are identified and quantified by barcode sequencing alone (see [0168], [0174]).
In regards to claim 14, Dever teaches introducing polynucleotides of a barcoded donor template library into a population of cells, and identifying a protein-coding region within a given cell by sequencing its associated second randomized barcodes (see [0003]-[0004], [0061]-[0062]). Dever’s disclosed cells each contain a barcoded donor template comprising a protein-coding regions, and that protein coding region is identified in the cell by sequencing the associated barcode.
In regards to claims 15-17, Dever teaches identifying a single protein-coding region within a cell by sequencing its associated second barcode (see [0134], as discussed above for claim 14), but each cell disclosed by Dever receives only a single donor template polynucleotide corresponding to a single protein-coding region and single associated barcode. Dever does not teach two distinct protein-coding regions, each with its own second barcode, both present and separately identified within one and the same cell.
Peikon teaches this missing element. Peikon discloses a diploid yeast cell formed by mating two haploid yeast cells, each independently carrying a distinct plasmid comprising a unique molecular barcode operatively linked to a distinct protein-coding sequence, such that the resulting diploid cell contains both barcoded protein coding sequences, and discloses identifying the associated barcoded from that single diploid cell, either by direct co-detection of the two distinct barcode sequences (see Col. 14, lines 10-16), or in a further disclosed embodiment, by first joining the two barcode via Cre-Lox recombination into a single recombined sequence prior to sequencing (see Col. 13 lines 25-31). Either disclosed approach in Peikon teaches two protein-coding regions being identified, within a single cell, by sequencing two second barcodes contained within that cell.
A person of ordinary skill in the art would have been motivated to combine Peikon’s teaching of housing two independently barcoded protein coding constructs within a single cell with Dever’s barcoded library/barcode-sequencing identification architecture because both references solve the same underlying problem, using a short DNA barcode as a sequencing based proxy for the identity of an associated larger protein coding construct, so that identification can be accomplished by barcode sequencing rather than by resequencing the full construct. Extending Dever’s single-barcode per cell identification scheme to a two-barcode per cell scheme using Peikon’s known mating-based cell combination technique would have predictably yielded the ability to identify two protein coding regions from one cell, since each barcode in Peikon’s system functions and is read out independently of the other, exactly as each barcode functions independently in Dever’s system. A person of ordinary skill would have had a reasonable expectation of success in this combination because Peikon’s mating and barcode-recovery techniques were already established, working methods for combining two independently-barcoded genetic elements in a single yeast cell and successfully reading out both by sequencing require no more than routine application of that known technique to Dever’s barcoded constructs.
In regards to claims 16-17, as mentioned above in regards to claim 15, Peikon teaches a system of mating haploid yeast each containing one barcoded protein-coding region together to generate a diploid yeast cell containing both barcoded protein-coding regions (see col. 1 lines 55-65, col. 6 lines 49-52)
Double Patenting
A rejection based on double patenting of the “same invention” type finds its support in the language of 35 U.S.C. 101 which states that “whoever invents or discovers any new and useful process... may obtain a patent therefor...” (Emphasis added). Thus, the term “same invention,” in this context, means an invention drawn to identical subject matter. See Miller v. Eagle Mfg. Co., 151 U.S. 186 (1894); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Ockert, 245 F.2d 467, 114 USPQ 330 (CCPA 1957).
A statutory type (35 U.S.C. 101) double patenting rejection can be overcome by canceling or amending the claims that are directed to the same invention so they are no longer coextensive in scope. The filing of a terminal disclaimer cannot overcome a double patenting rejection based upon 35 U.S.C. 101.
Claims 1-17 provisionally rejected under 35 U.S.C. 101 as claiming the same invention as that of claim 1-17 of copending Application No. 18/423,075 (reference application). This is a provisional statutory double patenting rejection since the claims directed to the same invention have not in fact been patented.
Claim #
Application No. 18/291,791 (Claim 1/24/2024)
Application No. 18/423,075 (Claims 1/25/2024)
1
A method comprising:
a. synthesizing a first polynucleotide library comprising multiple first polynucleotides each comprising at least one protein-coding region, and/or encoding at least one region of interest within a protein-coding region, each first polynucleotide independently and optionally comprising one or more non-silent mutations with respect to a reference sequence of the protein-coding region and/or region of interest, at least one of the first polynucleotides encoding the protein-coding region or region of interest comprising at least one silent mutation with respect to the reference sequence, the at least one silent mutation or a combination of silent mutations in a given protein-coding region and/or region of interest providing a first barcode;
b. randomly pairing each first polynucleotide to a unique second randomized barcode nucleotide sequence to produce a second polynucleotide library comprised of second polynucleotides;
c. sequencing the second polynucleotides or at least the first barcode and second randomized barcode thereof; and,
d. mapping each second randomized barcode to a protein-coding region and/or region of interest of a first polynucleotide; wherein a polynucleotide of the second polynucleotide library can be identified by sequencing only the second randomized barcode.
A method comprising:
a. synthesizing a first polynucleotide library comprising multiple first polynucleotides each comprising at least one protein-coding region, and/or encoding at least one region of interest within a protein-coding region, each first polynucleotide independently and optionally comprising one or more non-silent mutations with respect to a reference sequence of the protein-coding region and/or region of interest, at least one of the first polynucleotides encoding the protein-coding region or region of interest comprising at least one silent mutation with respect to the reference sequence, the at least one silent mutation or a combination of silent mutations in a given protein-coding region and/or region of interest providing a first barcode;
b. randomly pairing each first polynucleotide to a unique second randomized barcode nucleotide sequence to produce a second polynucleotide library comprised of second polynucleotides;
c. sequencing the second polynucleotides or at least the first barcode and second randomized barcode thereof; and,
d. mapping each second randomized barcode to a protein-coding region and/or region of interest of a first polynucleotide; wherein a polynucleotide of the second polynucleotide library can be identified by sequencing only the second randomized barcode.
2
The method of claim 1 wherein the second polynucleotides are sequenced by long-read next generation sequencing.
The method of claim 1 wherein the second polynucleotides are sequenced by long-read next generation sequencing.
3
The method of claim 1 wherein the second barcode is sequenced using short-read next generation sequencing.
The method of claim 1 wherein the second barcode is sequenced using short-read next generation sequencing.
4
The method of claim 1 further comprising determining the identities and relative abundances of polynucleotides encoding one or more protein-coding sequences by sequencing the second randomized barcodes thereof.
The method of claim 1 further comprising determining the identities and relative abundances of polynucleotides encoding one or more protein-coding sequences by sequencing the second randomized barcodes thereof.
5
The method of claim 1 wherein the polynucleotides of the second polynucleotide library contain first barcodes and second barcodes that are separated by more than about 300 nucleotides.
The method of claim 1 wherein the polynucleotides of the second polynucleotide library contain first barcodes and second barcodes that are separated by more than about 300 nucleotides.
6
The method of claim 1 wherein the polynucleotides of the second polynucleotide library contain first barcodes and second barcodes separated by less than about 600 nucleotides.
The method of claim 1 wherein the polynucleotides of the second polynucleotide library contain first barcodes and second barcodes separated by less than about 600 nucleotides.
7
The method of claim 6 wherein both the first barcode and the second (randomized) barcode contained within the polynucleotides of the second polynucleotide library are sequenced by short-read next-generation sequencing.
The method of claim 6 wherein both the first barcode and the second (randomized) barcode contained within the polynucleotides of the second polynucleotide library are sequenced by short-read next-generation sequencing.
8
The method of claim 1 wherein both the first and second barcode contained within each polynucleotide of the second polynucleotide library are sequenced by long-read next-generation sequencing.
The method of claim 1 wherein both the first and second barcode contained within each polynucleotide of the second polynucleotide library are sequenced by long-read next-generation sequencing.
9
The method of claim 1 wherein the first polynucleotide library contains one or more polynucleotides that contain a protein-coding region coding for a protein with a single amino acid mutation with respect to a reference protein sequence.
The method of claim 1 wherein the first polynucleotide library contains one or more polynucleotides that contain a protein-coding region coding for a protein with a single amino acid mutation with respect to a reference protein sequence.
10
The method of claim 9 wherein one or more polynucleotides from the first polynucleotide library contains a single non silent mutation resulting from a single nucleic acid substitution.
The method of claim 9 wherein one or more polynucleotides from the first polynucleotide library contains a single non silent mutation resulting from a single nucleic acid substitution.
11
The method of claim 1 wherein the first barcode comprises three or more silent mutations.
The method of claim 1 wherein the first barcode comprises three or more silent mutations.
12
The method of claim 1 wherein one or more polynucleotides from the first polynucleotide library contains more silent mutations with respect to a reference protein sequence as compared to the number of non-silent nucleic acid mutations with respect to the reference protein sequence.
The method of claim 1 wherein one or more polynucleotides from the first polynucleotide library contains more silent mutations with respect to a reference protein sequence as compared to the number of non-silent nucleic acid mutations with respect to the reference protein sequence.
13
The method of claim 1 wherein two or more polynucleotides from the second polynucleotide library contain identical non-silent mutations with respect to a reference protein sequence but different second barcodes such that the two molecules encoding an identical amino acid sequence are identified by sequencing the second barcodes.
The method of claim 1 wherein two or more polynucleotides from the second polynucleotide library contain identical non-silent mutations with respect to a reference protein sequence but different second barcodes such that the two molecules encoding an identical amino acid sequence are identified by sequencing the second barcodes.
14
The method of claim 1 wherein one or more protein coding regions in one or more cells are identified by sequencing one or more second barcodes.
The method of claim 1 wherein one or more protein coding regions in one or more cells are identified by sequencing one or more second barcodes.
15
The method of claim 14 wherein two protein coding regions in one or more cells are identified by sequencing two second barcodes contained within the same cell.
The method of claim 14 wherein two protein coding regions in one or more cells are identified by sequencing two second barcodes contained within the same cell.
16
The method of claim 15 wherein the cell is a yeast diploid cell.
The method of claim 15 wherein the cell is a yeast diploid cell.
17
The method of claim 16 wherein the yeast diploid cell was produced through the mating of two yeast haploid cells, each comprising one second barcode.
The method of claim 16 wherein the yeast diploid cell was produced through the mating of two yeast haploid cells, each comprising one second barcode.
Pursuant to 37 CFR 1.78(f), when two or more applications filed by the same applicant or assignee contain patentably indistinct claims, elimination of such claims from all but one application may be required in the absence of good and sufficient reason for their retention during pendency in more than one application. Applicant is required to either cancel the patentably indistinct claims from all but one application or maintain a clear line of demarcation between the applications. See MPEP § 822.
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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