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 .
Applicant previously canceled claims 5-24, 26-40, 43, 46-48, 50-54, 59, 63, 65, 67, 70, 74, 78-79, 81-99, 101-106, 108 and 110-128. Claims 1-4, 25, 41-42, 44-45, 49, 55-58, 60-62, 64, 66, 68-69, 71-73, 75-77, 80, 100, 107 and 109 are currently pending and under examination.
Information Disclosure Statement
The Information Disclosure Statements filed May 02, 2023; and October 30, 2024 have been considered.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/patents-application- process/filing-online/legal-framework-efs-web), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency - The incorporation by reference paragraph required by 37 CFR 1.834(c)(1),
1.835(a)(2), or 1.835(b)(2) is missing, defective or incomplete.
Required response – Applicant must:
Amend the Sequence Listing Incorporation by Reference paragraph at page 1 of the
specification. It is noted the Sequence Listing Incorporation by Reference paragraph lists the size of the ASCII text file as 1,477,536 bytes, whereas the ASCII text file itself lists the size as 28,800 bytes.
Specification
The use of the term USER® (see Page 48, [0159] and Page 93, [0290]-[0291]), which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claims 1, 3, 60, 66, 80, 100, 107 and 109 are objected to because of the following informalities:
In claim 1, line 11, “sequence capable of hybridizing to” should read “sequence that hybridizes to”.
In claim 1, line 13, “the second polynucleotide is capable of forming a hairpin”, should read “the second polynucleotide forms a hairpin”.
In claim 3, lines 1-2, “the first polynucleotide is capable of forming one or”, should read “the first polynucleotide forms one or”.
In claim 60, line 7, “sequence capable of hybridizing to” should read “sequence that hybridizes to”.
In claim 60, line 9, “polynucleotide is capable of forming a hairpin” should read “polynucleotide forms a hairpin”.
In claim 66, line 12, “sequence capable of hybridizing to” should read “sequence that hybridizes to”.
In claim 66, line 14, “polynucleotide is capable of forming a hairpin” should read “polynucleotide forms a hairpin”.
In claim 80, lines 8 and 12, “sequence capable of hybridizing to” should read “sequence that hybridizes to”.
In claim 100, lines 10, 14, 19 and 23, “sequence capable of hybridizing to” should read “sequence that hybridizes to”.
In claim 107, lines 8 and 12, “sequence capable of hybridizing to” should read “sequence that hybridizes to”.
In claim 109, line 12, “sequence capable of hybridizing to” should read “sequence that hybridizes to”.
In claim 109, line16, “is capable of forming a hairpin” should read “forms a hairpin”.
Claim 109: Each claim begins with a capital letter and end with a period. Periods may not be used elsewhere in the claims except for abbreviations. Where a claim sets forth a plurality of elements or steps, each element or step of the claim should be separated be a line indentation (see the requirements of 37 CFR 1.75 (i) for “full, clear, concise, and exact terms”. See MPEP 608.01(m).
Appropriate correction is required.
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 109 is 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 109 is considered vague and indefinite for the following reasons:
In claim 109, lines 3-4, 6, 8, 14, 16 and 20-22, “…” are unclear and confusing. It is unclear what the ellipses are meant to indicate? It is a range? Are they additional sequences, targets, beads and capture moieties? It is additionally unclear as to how many sets of polynucleotides, subsequences, beads, tags and target sequences the claim is disclosing?
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 1-4, 25, 41-42, 45, 49, 55-58, 61-62, 64, 66, 68-69, 71-72 are rejected under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Schatz et al. (WIPO International Patent Application Publication WO 00/75368 A2, published December 14, 2000), cited on the IDS filed May 02, 2023.
Regarding claim 1, Schatz teaches a method of assembling a target polynucleotide (Abstract, Page 4, Seventh Paragraph). Schatz teaches partitioning a plurality of polynucleotides into a contained reaction volume (Page 3, Second Paragraph and Page 6, Ninth Paragraph). Schatz teaches the plurality of polynucleotides comprise a first polynucleotide and a second polynucleotide and the second polynucleotide is attached to a support (Splinker Oligo and Anchor oligo attached to a solid support, Fig. 1). Schatz teaches the first polynucleotide comprises a first subsequence of a target polynucleotide, wherein the first polynucleotide comprises a single-stranded 3' end sequence (Splinker oligo ligated to an anchor oligo to form a larger nucleic acid (Target Polynucleotide), Page 3, Second Paragraph, Page 6, Second and Seventh Paragraphs, Page 7, Last Two Paragraphs and Fig. 1). Schatz teaches the second polynucleotide comprises, in the 3' to 5' direction, a single-stranded 3' end sequence, a second subsequence of the target polynucleotide (Splinker oligo ligated to an anchor oligo to form a larger nucleic acid (Target Polynucleotide)) a Type IIS restriction enzyme recognition sequence, and a complementary sequence capable of hybridizing to all or a portion of the second subsequence (Page 2, Fifth Paragraph, Page 3, Second Paragraph, Page 4, Last Paragraph, Page 6, Second, Seventh and Ninth Paragraphs, Page 7, Last Two Paragraphs, Page 13, Second Paragraph and Fig.1). Schatz teaches the second polynucleotide is capable of forming a hairpin molecule comprising a 3' overhang, a stem formed by intramolecular nucleotide base pairing between all or a portion of the second subsequence and the complementary sequence, and a loop, wherein the hairpin molecule is in a configuration that is not cleaved by a Type IIS restriction enzyme (Page 3, Second Paragraph and Fig. 1). Schatz teaches the first and second polynucleotides are connected within the contained reaction volume, thereby assembling the first and second subsequences (Splinker oligo ligated to an anchor oligo to form a larger nucleic acid (Target Polynucleotide), Page 3, Second Paragraph, Page 6, Second, Seventh and Ninth Paragraphs, Page 7, Last Two Paragraphs and Fig.1).
Regarding claim 2, Schatz teaches the first polynucleotide comprises two nucleic acid strands forming a duplex (Fig 1).
Regarding claim 3, Schatz teaches the first polynucleotide is capable of forming one or more hairpins (Fig. 1).
Regarding claim 4, Schatz teaches the first and/or second polynucleotide comprises a tag, a barcode, an amplification site, a unique molecular identifier (UMI), or any combination thereof (Page 3, Second Paragraph, Page 12, Last Paragraph, Page 14, First Paragraph).
Regarding claim 25, Schatz teaches the 5' end of the second polynucleotide is blocked from ligation, extension, and/or hybridization (Page 6, Second Paragraph).
Regarding claim 41, Schatz teaches the 3' overhang is between about 1 and about 100 nucleotides in length (Page 3, Second Paragraph and Page 6, Second to Last Paragraph).
Regarding claim 42, Schatz teaches the 3' overhang is between about 2 and about 20 nucleotides in length (Page 3, Second Paragraph and Page 6, Second to Last Paragraph).
Regarding claim 45, Schatz teaches the contained reaction volume comprises one or more Type IIS restriction enzymes, one or more polymerases, one or more ligases, and/or one or more nucleases other than a Type IIS restriction enzyme (Page 3, Second and Fifth Paragraph, Page 6, Second to Last Paragraph, Page 2, Fifth Paragraph and Page 9, First Paragraph).
Regarding claim 49, Schatz teaches the second polynucleotide forms the hairpin molecule, and all or a portion of the 3' overhang hybridizes to all or a portion of the single-stranded 3' end sequence of the first subsequence to form a hybridization complex (Fig 1.).
Regarding claim 55, Schatz teaches a double-stranded polynucleotide comprising the first subsequence, the second subsequence, and the Type IIS restriction enzyme recognition sequence is generated by a polymerase that extends the 3' end sequence of the first subsequence using the second polynucleotide as template (Page 5, Second Paragraph and Page 12, First Paragraph).
Regarding claim 56, Schatz teaches a Type IIS restriction enzyme recognizes the Type IIS restriction enzyme recognition sequence and cleaves the double-stranded polynucleotide, thereby generating a cleaved double-stranded polynucleotide comprising the first subsequence connected to the second subsequence (Fig. 1).
Regarding claim 57, Schatz teaches the cleaved double-stranded polynucleotide comprises a single-stranded 3' end sequence (Page 3, Second Paragraph, Page 6, Second Paragraph and Fig. 1).
Regarding claim 58, Schatz teaches the single-stranded 3' end sequence of the cleaved double-stranded polynucleotide is between about 2 and about 10 nucleotides in length (Page 6, Second to last Paragraph).
Regarding claim 61, Schatz teaches the support comprises a particle, a bead, a solid substrate, a plate, a well, an array, a membrane, or a combination thereof (Page 3, Second Paragraph).
Regarding claim 62, Schatz teaches the target polynucleotide is at least about 100 nucleotides in length (Page 2, Fourth Paragraph, Page 4, Last Paragraph and Page 7, Third Paragraph).
Regarding claim 64, Schatz teaches the target polynucleotide is a DNA (Page 2, Fourth Paragraph and Title)
Regarding claim 66, Schatz teaches a method of assembling a target polynucleotide (Abstract, Page 4, Seventh Paragraph). Schatz teaches partitioning a plurality of polynucleotides into a contained reaction volume (Page 3, Second Paragraph and Page 6, Ninth Paragraph). Schatz teaches the plurality of polynucleotides comprise a first polynucleotide and a second polynucleotide and the second polynucleotide is attached to a support (Splinker Oligo and Anchor oligo attached to a solid support, Fig. 1). Schatz teaches the first polynucleotide comprises a first subsequence of a target polynucleotide, wherein the first polynucleotide comprises a single-stranded 3' end sequence (Splinker oligo ligated to an anchor oligo to form a larger nucleic acid (Target Polynucleotide), Page 3, Second Paragraph, Page 6, Second and Seventh Paragraphs, Page 7, Last Two Paragraphs and Fig. 1). Schatz teaches the second polynucleotide comprises, in the 3' to 5' direction, a single-stranded 3' end sequence, a second subsequence of the target polynucleotide (Splinker oligo ligated to an anchor oligo to form a larger nucleic acid (Target Polynucleotide)) a Type IIS restriction enzyme recognition sequence, and a complementary sequence capable of hybridizing to all or a portion of the second subsequence (Page 2, Fifth Paragraph, Page 3, Second Paragraph, Page 4, Last Paragraph, Page 6, Second, Seventh and Ninth Paragraphs, Page 7, Last Two Paragraphs, Page 13, Second Paragraph and Fig.1). Schatz teaches the second polynucleotide is capable of forming a hairpin molecule comprising a 3' overhang, a stem formed by intramolecular nucleotide base pairing between all or a portion of the second subsequence and the complementary sequence, and a loop, wherein the hairpin molecule is in a configuration that is not cleaved by a Type IIS restriction enzyme (Page 3, Second Paragraph and Fig. 1). Schatz teaches the first and second polynucleotides are connected within the contained reaction volume, thereby assembling the first and second subsequences (Splinker oligo ligated to an anchor oligo to form a larger nucleic acid (Target Polynucleotide), Page 3, Second Paragraph, Page 6, Second, Seventh and Ninth Paragraphs, Page 7, Last Two Paragraphs and Fig.1). Schatz teaches the assembly of subsequences of each target polynucleotide is carried out in parallel (Abstract, Page 2, Fourth Paragraph and Page 5, Third Paragraph).
Regarding claim 68, Schatz teaches the subsequences in the plurality of polynucleotides for each target polynucleotide are between about 20 and about 200 nucleotides in length. (Page 2, Fourth Paragraph, Page 4, Last Paragraph and Page 7, Third Paragraph).
Regarding claim 69, Schatz teaches the plurality of polynucleotides for each target polynucleotide are synthesized, and the synthesis comprises base-by-base synthesis (Page 2, Fourth Paragraph).
Regarding claim 71, Schatz teaches the partitioning comprises capturing all or a subset of the plurality of polynucleotides for each target polynucleotide on a bead that is specific for the target polynucleotide (Page 3, Second Paragraph, Page 5, Last Paragraph, Page 6, Second to Last Paragraph, Page 9, First Paragraph and Page 11, Second Paragraph).
Regarding claim 72, Schatz teaches the bead comprises a capture probe that specifically binds to a capture tag that is unique for the target polynucleotide and the capture tag is common in all or a subset of the plurality of polynucleotides comprising subsequences of the target polynucleotide (Page 3, Second Paragraph, Page 9, Fourth Paragraph, Page 17, Sixth Paragraph).
Schatz teaches each and every limitation of claims 1-4, 25, 41-42, 45, 49, 55-58, 61-62, 64, 66, 68-69, 71-72, and therefore Schatz anticipates claims 1-4, 25, 41-42, 45, 49, 55-58, 61-62, 64, 66, 68-69, 71-72.
Claim Rejections - 35 USC § 103
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 44, 60, 73, 75-77, 80 and 100 are rejected under 35 U.S.C. 103 as being unpatentable over Schatz et al. (WIPO International Patent Application Publication WO 00/75368 A2, published December 14, 2000), cited on the IDS filed May 02, 2023, as applied to claims 1-4, 25, 41-42, 45, 49, 55-58, 61-62, 64, 66, 68-69, 71-72 above, in view of Hindson et al. (United States Patent US 10,323,279 B2, patented June 18, 2019, effectively filed August 1, 2018).
Regarding claim 44, Schatz teaches a contained reaction volume as discussed above.
Regarding claim 60, Schatz teaches the first and Second polynucleotides as discussed above.
Regarding claim 73, Schatz teaches partitioning as discussed above.
Regarding claims 75, Schatz teaches parallel assembly as discussed above.
Regarding claim 76, Schatz teaches the one or more concerted reaction cycles comprise an isothermal reaction (Page 15, Fourth and Sixth Paragraph).
Regarding claim 77, Schatz teaches the one or more concerted reaction cycles comprise ligation by a ligase, primer extension by a polymerase, and cleavage by a Type IIS restriction enzyme (Page 4, Last Paragraph, Page 6, Last Paragraph—Page 7, First Two Paragraphs, Page 15, Sixth Paragraph, Page 3, Second and Fifth Paragraph, Page 6, Second to Last Paragraph, Page 2, Fifth Paragraph and Page 9, First Paragraph).
Regarding claim 80, Schatz teaches a method of assembling a target polynucleotide (Abstract, Page 4, Seventh Paragraph). Schatz teaches the plurality of polynucleotides comprise a first polynucleotide and a second polynucleotide attached to a the bead (Splinker Oligo and Anchor oligo attached to a solid support/bead, Page 3, Second Paragraph and Fig. 1). Schatz teaches the first polynucleotide comprises a first subsequence of a target polynucleotide and the first polynucleotide comprises a single-stranded 3' end sequence (Splinker oligo ligated to an anchor oligo to form a larger nucleic acid (Target Polynucleotide), Page 3, Second Paragraph, Page 6, Second and Seventh Paragraphs, Page 7, Last Two Paragraphs and Fig. 1). Schatz teaches the second polynucleotide comprises, in the 3' to 5' direction a single-stranded 3' end sequence capable of hybridizing to the single-stranded 3' end sequence of the first polynucleotide, a second subsequence of the target polynucleotide (Splinker oligo ligated to an anchor oligo to form a larger nucleic acid (Target Polynucleotide)), a Type IIS restriction enzyme recognition sequence, and a complementary sequence capable of hybridizing to all or a portion of the second subsequence (Page 2, Fifth Paragraph, Page 3, Second Paragraph, Page 4, Last Paragraph, Page 6, Second, Seventh and Ninth Paragraphs, Page 7, Last Two Paragraphs, Page 13, Second Paragraph and Fig.1). Schatz teaches the second polynucleotide further comprises a tag sequence and/or a barcode sequence 5' to the Type IIS restriction enzyme recognition sequence (Page 3, Second Paragraph, Page 12, Last Paragraph, Page 14, First Paragraph). Schatz teaches the second polynucleotide forms a hairpin molecule comprising a 3' overhang, a stem formed by intramolecular nucleotide base pairing between all or a portion of the second subsequence and the complementary sequence, and a loop, wherein the hairpin molecule is in a configuration that is not cleaved by a Type IIS restriction enzyme (Page 3, Second Paragraph and Fig. 1). Schatz teaches allowing the 3' overhang of the hairpin molecule to hybridize to the single-stranded 3' end sequence of the first polynucleotide (Fig. 1). Schatz teaches extending the 3' end sequence of the first polynucleotide using the second polynucleotide as template, thereby generating a double-stranded polynucleotide comprising the first subsequence, the second subsequence, and the Type IIS restriction enzyme recognition sequence (Page 5, Second Paragraph and Page 12, First Paragraph and Fig. 1). Schatz teaches cleaving the double-stranded polynucleotide using a Type IIS restriction enzyme, thereby generating a cleaved double-stranded polynucleotide comprising the first subsequence and the second subsequence and the cleaved double-stranded polynucleotide comprises a single- stranded 3' end sequence, thereby assembling the first and second subsequences (Fig. 1).
Regarding claim 100, Schatz teaches a method of assembling a target polynucleotide (Abstract, Page 4, Seventh Paragraph). Schatz teaches partitioning a plurality of polynucleotides (Page 3, Second Paragraph and Page 6, Ninth Paragraph). Schatz teaches the plurality of polynucleotides comprise a first polynucleotide and a second polynucleotide attached to a bead (Splinker Oligo and Anchor oligo attached to a bead, Fig. 1). Schatz teaches the second polynucleotide comprises, in the 3' to 5' direction, a single-stranded 3' end sequence capable of hybridizing to the top strand single- stranded 3' end sequence of the first polynucleotide, a second subsequence of the target polynucleotide (Splinker oligo ligated to an anchor oligo to form a larger nucleic acid (Target Polynucleotide)), a Type IIS restriction enzyme recognition sequence, and a complementary sequence capable of hybridizing to all or a portion of the second subsequence (Page 2, Fifth Paragraph, Page 3, Second Paragraph, Page 4, Last Paragraph, Page 6, Second, Seventh and Ninth Paragraphs, Page 7, Last Two Paragraphs, Page 13, Second Paragraph and Fig.1). Schatz teaches the second polynucleotide forms a hairpin molecule comprising a 3' overhang, a stem formed by intramolecular nucleotide base pairing between all or a portion of the second subsequence and the complementary sequence, and a loop, wherein the hairpin molecule is in a configuration that is not cleaved by a Type IIS restriction enzyme (Page 3, Second Paragraph and Fig. 1). Schatz teaches the 5' ends of the hairpin molecules are blocked from ligation to the 3' ends of the first polynucleotide after hybridization (Page 6, Second Paragraph). Schatz teaches ligating the 3' ends of the hairpin molecules to the 5' ends of the first polynucleotide (Fig. 1). Schatz teaches cleaving the double-stranded polynucleotide using a Type IIS restriction enzyme, thereby generating a cleaved double-stranded polynucleotide comprising the first subsequence flanked by the second subsequence on one side (Fig. 1).
Schatz does not teach or suggest the contained reaction volume is an emulsion droplet or partitioning the polynucleotides in an emulsion droplet. Schatz does not teach or suggest the first polynucleotide comprises a first subsequence of a target polynucleotide and is double-stranded, comprising a single-stranded 3' end sequence in the top strand and a single- stranded 3' end sequence in the bottom strand. Schatz does not teach or suggest in the emulsion droplet, releasing the second and third polynucleotide from the bead. Schatz does not teach or suggest a single-stranded 3' end sequence capable of hybridizing to the bottom strand single-stranded 3' end sequence of the first polynucleotide. Schatz does not teach or suggest Schatz does not teach or suggest a third polynucleotides that is attached to the support (or bead) and comprises, in the 3' to 5' direction, a single-stranded 3' end sequence, a third subsequence of the target polynucleotide, a Type IIS restriction enzyme recognition sequence, and a complementary sequence capable of hybridizing to all or a portion of the third subsequence. Schatz does not teach or suggest the third polynucleotide is capable of forming a hairpin molecule comprising a 3' overhang, a stem formed by intramolecular nucleotide base pairing between all or a portion of the third subsequence and the complementary sequence, and a loop. Schatz does not teach or suggest allowing the 3' overhangs of the hairpin molecules formed by the second and third polynucleotides to hybridize to the top strand single-stranded 3' end sequence and the bottom strand single-stranded 3' end sequence, respectively, of the first polynucleotide. Schatz does not teach or suggest extending the 3' end sequences of the first polynucleotide using the second and third polynucleotides as template, thereby generating a double-stranded polynucleotide comprising the first subsequence flanked by the second subsequence on one side and the third subsequence on the other side. Schatz does not teach or suggest generating a cleaved double-stranded polynucleotide comprising the first subsequence flanked by the second subsequence on one side and the third subsequence on the other side wherein the cleaved double-stranded polynucleotide comprises a single-stranded 3' end sequence in the top strand and a single-stranded 3' end sequence in the bottom strand, thereby assembling the first, second, and third subsequences. Schatz does not teach or suggest the first, second, and third polynucleotides are connected sequentially within the contained reaction volume, thereby assembling the first, second, and third subsequences. Schatz does not teach or suggest the partitioning comprises encapsulating the bead in an emulsion droplet, thereby generating a plurality of emulsion droplets for parallel assembly of the plurality of target polynucleotides. Schatz does not teach or suggest the parallel assembly of the plurality of target polynucleotides is carried out in each emulsion droplet by one or more concerted reaction cycles.
Hindson teaches assembly of polynucleotides (Column 13, Lines 47-60). Hindson teaches a first and Second polynucleotide contained in a reaction volume that is an emulsion droplet (Column 12, Lines 42-67, Column 22, Lines 8-45 and Column 14, Lines 10-23). Hindson teaches in the emulsion droplet, releasing the second and third polynucleotide from the bead (Column 4, Lines 50-61, Column 7, Lines 64-67 and Column 11, Line 44—Col 12, Lines 4). Hindson teaches a third polynucleotides that is attached to the support (Column 54, Lines 1-39, column 7, Lines 7-24). Hindson teaches a single-stranded 3' end sequence capable of hybridizing to the bottom strand single-stranded 3' end sequence of the first polynucleotide (Figs. 4 and 15). Hindson teaches the first polynucleotide comprises a first subsequence of a target polynucleotide and is double-stranded, comprising a single-stranded 3' end sequence in the top strand and a single- stranded 3' end sequence in the bottom strand (Figs 4 and 15). Hindson teaches a single-stranded 3' end sequence, a third subsequence of the target polynucleotide, a restriction enzyme recognition sequence, and a complementary sequence capable of hybridizing to all or a portion of the third subsequence (Column 3, Lines 38-55, Column 10, Lines 15-20, Column 22, Lines 16-19, Column 25, Lines 48-67, Column 30, Lines 54-67, Column 45, Lines 48-65, Column 54, Lines 1-11, Column 96, Lines 56-59, Column 118, Lines 15-45 and Fig. 38). Hindson teaches the third polynucleotide is capable of forming a hairpin molecule comprising a 3' overhang, a stem formed by intramolecular nucleotide base pairing between all or a portion of the third subsequence and the complementary sequence, and a loop (Column 85, Lines 1-22, Figs. 34 and 38). Hindson teaches allowing the 3' overhangs of the hairpin molecules formed by the second and third polynucleotides to hybridize to the top strand single-stranded 3' end sequence and the bottom strand single-stranded 3' end sequence (Fig. 15). Hindson teaches extending the 3' end sequences of the first polynucleotide using the second and third polynucleotides as template, thereby generating a double-stranded polynucleotide comprising the first subsequence flanked by the second subsequence on one side and the third subsequence on the other side (Column 77, Line 63—Column 12, Line31 and Fig. 15). Hindson teaches generating a cleaved double-stranded polynucleotide comprising the first subsequence flanked by the second subsequence on one side and the third subsequence on the other side and the cleaved double-stranded polynucleotide comprises a single-stranded 3' end sequence in the top strand and a single-stranded 3' end sequence in the bottom strand, thereby assembling the first, second, and third subsequences Column 36, Lines 33-40, Column 37, Lines 10-13, Column 54, Lines 1-7 and Column 118, Lines 15-31. Hindson teaches the first, second, and third polynucleotides are connected sequentially within the contained reaction volume, thereby assembling the first, second, and third subsequences (Column 36, Lines 33-40, Column 37, Lines 10-13, Column 54, Lines 1-7 and Column 118, Lines 15-31). Hindson teaches the partitioning comprises encapsulating the bead in an emulsion droplet, thereby generating a plurality of emulsion droplets for parallel assembly of the plurality of target polynucleotides (Column 4, Lines 20-25, Column 6, Lines 53-60, Column 37, Line 13 and Column 82, 13-15). Hindson teaches the parallel assembly of the plurality of target polynucleotides is carried out in each emulsion droplet by one or more concerted reaction cycles (Column 78, Lines 32-67 and Column 79, Lines 32-67). Hindson teaches using these methods allows for extremely high multiplexing capabilities as well as reduces sequencing errors, sample preparation time and cost (Column 109, Lines 37-42, Column 96, Lines 2-38 and Column 111, Lines 13-32).
It would have been obvious to one having ordinary skill in the art before the effective filing date of the invention to modify the teachings of Schatz with the teachings of Hindon, partitioning the plurality of polynucleotides and beads into an emulsion droplet as well as using a third polynucleotide sequence. This would allow for extremely high multiplexing capabilities as well as reduces sequencing errors, sample preparation time and cost as taught by Hindson (Column 109, Lines 37-42, Column 96, Lines 2-38 and Column 111, Lines 13-32).
Claims 107 and 109 are rejected under 35 U.S.C. 103 as being unpatentable over Schatz et al. (WIPO International Patent Application Publication WO 00/75368 A2, published December 14, 2000), cited on the IDS filed May 02, 2023, as applied to claims 1-4, 25, 41-42, 45, 49, 55-58, 61-62, 64, 66, 68-69, 71-72 above, in view da Veiga Beltrame et al. (U.S. Patent Application Publication US 2020/0102556 A1, published April 02, 2020).
Regarding claim 107, Schatz teaches a method of assembling a target polynucleotide (Abstract, Page 4, Seventh Paragraph). Schatz teaches partitioning a plurality of polynucleotides (Page 3, Second Paragraph and Page 6, Ninth Paragraph). Schatz teaches the plurality of polynucleotides comprise a first polynucleotide and a second polynucleotide and the second polynucleotide is attached to a support (Splinker Oligo and Anchor oligo attached to a bead, Fig. 1). Schatz teaches the first polynucleotide comprises a first subsequence of a target polynucleotide, wherein the first polynucleotide comprises a single-stranded 3' end sequence (Splinker oligo ligated to an anchor oligo to form a larger nucleic acid (Target Polynucleotide), Page 3, Second Paragraph, Page 6, Second and Seventh Paragraphs, Page 7, Last Two Paragraphs and Fig. 1). Schatz teaches a second subsequence of the target polynucleotide, a Type IIS restriction enzyme recognition sequence, and a complementary sequence capable of hybridizing to all or a portion of the second subsequence, and the second polynucleotide further comprises a tag sequence and/or a barcode sequence 5' to the Type IIS restriction enzyme recognition sequence (Page 2, Fifth Paragraph, Page 3, Second Paragraph, Page 4, Last Paragraph, Page 6, Second, Seventh and Ninth Paragraphs, Page 7, Last Two Paragraphs, Page 13, Second Paragraph, Page 12, Last Paragraph, Page 14, First Paragraph and Fig.1). Schatz teaches the second polynucleotide forms a hairpin molecule comprising a 3' overhang, a stem formed by intramolecular nucleotide base pairing between all or a portion of the second subsequence and the complementary sequence, and a loop, and the hairpin molecule is in a configuration that is not cleaved by a Type IIS restriction enzyme (Page 3, Second Paragraph and Fig. 1). Schatz teaches a single-stranded 3' end sequence capable of hybridizing to the single-stranded 3' end sequence of the first polynucleotide (Fig. 1). Schatz teaches allowing the 3' overhang of the hairpin molecule to hybridize to the single-stranded 3' end sequence of the first polynucleotide to form a hybridization complex (Fig. 1). Schatz teaches the 5' end of the hairpin molecule is blocked from ligation to the 3' end of the first polynucleotide after hybridization (Page 6, Second Paragraph). Schatz teaches a nick or gap between the 3' end of the first polynucleotide and the 5' end of the second polynucleotide, and a nick or gap between the 5' end of the first polynucleotide and the 3' end of the second polynucleotide (Fig. 1). Schatz teaches extending the 3' end sequence of the first polynucleotide using the second polynucleotide as template, thereby generating a double-stranded polynucleotide comprising the first subsequence, the second subsequence, and the Type IIS restriction enzyme recognition sequence (Page 5, Second Paragraph and Page 12, First Paragraph and Fig. 1). Schatz teaches cleaving the double-stranded polynucleotide using a Type IIS restriction enzyme, thereby generating a cleaved double-stranded polynucleotide comprising the first subsequence and the second subsequence and the cleaved double-stranded polynucleotide comprises a single- stranded 3' end sequence, thereby assembling the first and second subsequences (Fig. 1).
Regarding claim 109, Schatz teaches Schatz teaches a method, comprising contacting a at least a first and second polynucleotides with beads (Fig. 1). Schatz teaches a first and second set of polynucleotides with first and second subsequences (Splinker oligo ligated to an anchor oligo to form a larger nucleic acid (Target Polynucleotide), Page 3, Second Paragraph, Page 6, Second and Seventh Paragraphs, Page 7, Last Two Paragraphs and Fig. 1). Schatz teaches the first polynucleotide comprises a first subsequence of a target polynucleotide, wherein the first polynucleotide comprises a single-stranded 3' end sequence (Splinker oligo ligated to an anchor oligo to form a larger nucleic acid (Target Polynucleotide), Page 3, Second Paragraph, Page 6, Second and Seventh Paragraphs, Page 7, Last Two Paragraphs and Fig. 1). Schatz teaches the second polynucleotide comprises, in the 3' to 5' direction, a single-stranded 3' end sequence, a second subsequence of the target polynucleotide (Splinker oligo ligated to an anchor oligo to form a larger nucleic acid (Target Polynucleotide)) a Type IIS restriction enzyme recognition sequence, and a complementary sequence capable of hybridizing to all or a portion of the second subsequence (Page 2, Fifth Paragraph, Page 3, Second Paragraph, Page 4, Last Paragraph, Page 6, Second, Seventh and Ninth Paragraphs, Page 7, Last Two Paragraphs, Page 13, Second Paragraph and Fig.1). Schatz teaches the second polynucleotide is capable of forming a hairpin molecule comprising a 3' overhang, a stem formed by intramolecular nucleotide base pairing between all or a portion of the second subsequence and the complementary sequence, and a loop, wherein the hairpin molecule is in a configuration that is not cleaved by a Type IIS restriction enzyme (Page 3, Second Paragraph and Fig. 1).
Schatz does not teach or suggest partitioning a plurality of polynucleotides into an emulsion droplet. Schatz does not teach or suggest in the emulsion droplet, releasing the second polynucleotide from the bead. Schatz does not teach or suggest a library or beads. Schatz does not teach a pool of polynucleotides comprising at least 6 polynucleotide targets and subsequences. Schatz does not teach or suggest at least 6 polynucleotide sets comprise a tag in all or a subset of the targets. Schatz does not teach or suggest at least 3 different beads with at least 3 different capture moieties that specifically bind to at least 3 different tags, thereby capturing at least one polynucleotide on one of the beads in the library.
Da Veiga Beltrame teaches assembling particle associated sets of polynucleotides and preparing a pool of library barcoded beads (Abstract and Page 3, [0018]). Da Veiga Beltrame teaches partitioning the sets of polynucleotides on beads within a droplet emulsion (Page 4, [0021] and Page 7, [0039]). Da Veiga Beltrame teaches a first polynucleotide a second polynucleotide attached to a bead, the first polynucleotide comprises a first subsequence of a target polynucleotide (Figs. 1A-1B). Da Veiga Beltrame teaches the first polynucleotide comprises a single-stranded 3' end sequence and the second polynucleotide comprises, in the 3' to 5' direction, a single-stranded 3' end sequence capable of hybridizing to the single-stranded 3' end sequence of the first polynucleotide (Page 1, [0005]-[0008] and Figs. 1A-1B). Da Veiga Beltrame teaches in the emulsion droplet, releasing the second polynucleotide from the bead (Page 22, [0129] and Page 24, [0146]). Da Veiga Beltrame teaches a first and second polynucleotide each with a 3’ single-stranded overhang on a double-stranded hairpin molecule (Figs. 1 and 2). Da Veiga Beltrame teaches the beads may contain one or more attaches barcodes and UMIs (Page 21, [0124]-[0125]). Da Veiga Beltrame teaches a pool of polynucleotides comprising at least 6 polynucleotide targets and subsequences and the at least 6 polynucleotide sets comprise a tag in all or a subset of the targets (Page 2, [0013] Page 3, [0019] Page , [0028] and Page 18, [0107]). Da Veiga Beltrame teaches at least 6 different beads with at least 6 different capture moieties that specifically bind to at least 6 different tags, thereby capturing at least one polynucleotide on one of the beads in the library (Page 2, [0013] Page 3, [0019] Page , [0028] and Page 18, [0107]). Da Veiga Beltrame teaches using these methods allows for a at least a ten-fold reduction in cost (Pages 8-9, [0056]-[0057]).
It would have been obvious to one having ordinary skill in the art before the effective filing date of the invention to modify the teachings of Schatz with the teachings of Da Veiga Beltrame, using a pool of polynucleotides comprising at least 6 polynucleotide targets and subsequences. Schatz does not teach or suggest at least 6 polynucleotide sets comprise a tag in all or a subset of the targets as well as at least 3 different beads with at least 3 different capture moieties that specifically bind to at least 3 different tags, thereby capturing at least one polynucleotide on one of the beads in the library. Using these methods would allow for a ten-fold reduction in cost, as taught by Da Veiga Beltrame (Pages 8-9, [0056]-[0057]).
Conclusion
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/JESSICA D PARISI/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684