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 2, 5, 10, 12-13, 15-16, 21-29, 31-33, 38-44, 46 and 48-50. Claims 1, 3-4, 6-9, 11, 14, 17-20, 30, 34-37, 45 and 47 are currently pending. Claims 35-37, 45 and 47 are withdrawn as being drawn to a nonelected invention. Claims 1, 3-4, 6-9, 11, 14, 17-20, 30 and 34 are currently under examination.
Election/Restrictions
Applicant’s election without traverse of Group I: claims 1, 3-4, 6-9, 11, 14, 17-20, 30 and 34 drawn to a method of preparing a library of cell-free DNA (cfDNA) for sequencing and the library prepared by the method in the reply filed on May 15, 2025 is acknowledged.
Claims are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on May 15, 2025.
Information Disclosure Statement
The Information Disclosure Statement filed June 21, 2023 has 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:
Include a Sequence Listing Incorporation by Reference paragraph at page 1 of the
specification. The Sequence Listing Incorporation by Reference paragraph should list the size of the ASCII text file as 1840 bytes.
Specification
The use of the term User©, NextSeq®, Illumina®, NEBNext® Ultra™ II Q5® (see Page 8, Line 27 and Line 29, Page 24, Line 30, Page 37, Line 25, Page 38, Line 1, Page 44, Line 6 and Line 31, Page 45, Line 16, Page 46, Lines 24-23, Page 47, Line 13 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 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, 3-4, 6, 8, 11, 14, 17-20, 30 and 34 are rejected under 35 U.S.C. 103 as being unpatentable over Azab et al. (WIPO International Application Publication WO 2018/136888 A1, published July 26, 2018, cited on the IDS filed June 21, 2023, in view of Galvin et al. (U.S. Patent Application Publication US 2018/0223350 A1, published August 09, 2018).
Regarding claim 1, Azab teaches a method of preparing a library of cell-free DNA (cfDNA) for sequencing, the method comprising obtaining a sample comprising a plurality of cfDNA and performing end-repair and A-tailing reactions of the plurality of cfDNA in a reaction having a first reaction volume (Page 166, Last Two Paragraphs and Page 167, First Paragraph). Azab teaches contacting cfDNA with a population of stem-loop adaptors and a ligase in a second reaction volume that is about equal to the first reaction volume, thereby ligating a stem-loop adaptor to each end of the plurality of cfDNA to produce adaptor-ligated cfDNA (Page 166, Second to Last Paragraph, Page 56, Lat Paragraph and Page 75, First Paragraph). Azab teaches linearizing the adaptor-ligated cfDNA by cleaving the cleavable base (Page 75, First Paragraph and Page 47, Last Two Paragraphs). Azab teaches amplifying the linearized adaptor-ligated cfDNA to produce amplified adaptor-ligated cfDNA, wherein the amplification uses forward and reverse primers complementary to known sequences in the stem-loop adaptors (Page 47, Last Two Paragraphs, Page 166, Second to Last Paragraph, Page 167, First Paragraph and Page 50, Last Paragraph). Azab teaches contacting the amplified adaptor-ligated cfDNA with RNA baits that hybridize to selected molecules of the plurality of cfDNA (Page 50, Last Paragraph, Page 62, First Paragraph and Page 168, Second Paragraph). Azab teaches isolating the molecules of the plurality of cfDNA having a hybridized RNA bait, thereby producing enriched cfDNA (Page 14, Second Paragraph, Page 42, First Paragraph and Page 74, Second Paragraph). Azab teaches amplifying the enriched cfDNA with indexing primers, thereby producing a library of cfDNA for sequencing (Page 19, Second Paragraph, Page 50, Second Paragraph, Page 58, First—Second Paragraph and Page 167, First Paragraph).
Regarding claim 3, Azab teaches the cfDNA comprises double-stranded DNA molecules (Page 40, Last Paragraph).
Regarding claim 4, Azab teaches the cfDNA is obtained from a body fluid (Page 42, First Paragraph).
Regarding claim 6, Azab teaches the cfDNA is obtained from an individual having a cancer (Page 35, Last Paragraph).
Regarding claim 8, Azab teaches the stem- loop adaptors comprise a 3' T overhang (Page 57, Third Paragraph and 167, First Paragraph).
Regarding claim 11, Azab teaches the stem-loop adaptors each comprise a constant region having a known sequence that is constant among the population of stem-loop adaptors and a barcode region having a sequence that is degenerate among the population of stem-loop adaptors
(Page 56, Last Paragraph—Page 57, First Paragraph, Page 16, First Paragraph and Page 12, Second Paragraph).
Regarding claim 14, Azab teaches the barcode as discussed above.
Regarding claims 17-18, Azab teaches a cleavable base as discussed above.
Regarding claim 19, Azab teaches contacting the amplified adaptor-ligated cfDNA with adaptor blockers (Page 168, Last Paragraph).
Regarding claim 20, Azab teaches the RNA baits hybridize to selected genomic loci in a reference genome and the selected genomic loci comprise disease-associated genetic loci (Page 50, Second Paragraph, Page 11, Last Paragraph and Page 137, Second Paragraph).
Regarding claim 30, Azab teaches comprises contacting the hybridized molecules from step (f) with a molecule or particle that binds to the RNA baits and isolating the RNA bait sequences, thereby isolating the subgroup of cfDNA molecules that hybridized to the RNA baits (Page 14, Second Paragraph, Page 42, First Paragraph, Page 74, Second Paragraph, Page 50, Last Paragraph, Page 62, First Paragraph and Page 168, Second Paragraph).
Regarding claim 34, Azab teaches a library of cfDNA molecules generated by the method of claim 1 (Page 19, Last Paragraph and Page 58, Last Paragraph).
Azab does not explicitly teach or suggest between about 5 ng and 30 ng of the cfDNA. Azab does not explicitly teach or suggest between about 2.5 ng and about 15 ng of cfDNA. Azab does not teach or suggest explicitly the adaptor loop comprises at least on cleavable base. Azab does not teach or suggest the stem-loop adaptors comprise an inverted repeat. Azab does not explicitly teach or suggest the weight ratio of RNA baits : amplified adaptor- ligated cfDNA is between about 1:25 and about 1:250. Azab does not teach or suggest the cleavable base is deoxyuridine. Azab does not teach or suggest wherein the cleavable base is cleaved prior to amplification (step (e)).
Galvin teaches methods of NGS sequencing library preparation using barcoded adapters and cfDNA (Abstract, Page 8, [0104] and Example 5). Galvin teaches performing end-repair and a-tailing reactions on between about 5 ng and about 30 ng of the plurality of cfDNA (Page 3, [0030], Pages 6-7, [0078]-[0080], Page 8, [0092]-[0093], [0096]-[0097] and [0100]-[0101], Page 12, [0176] and [0182] and Fig. 3). Galvin teaches contacting between about 2.5 ng and about 15 ng of the plurality of cfDNA with a population of stem-loop adaptors and a ligase (Page 3, [0021]-[0022], Page 8, [0096]-[0097] and [0100]-[0101], Page 12, [0176] and [0182]). Galvin teaches stem-loop adaptors each comprise an inverted repeat and a loop (Page 3, [0025]). Galvin teaches the loop comprises at least one cleavable base, that may be deoxyuridine (Page 2, [0014] and Page 3, [0023]). Galvin teaches capture probes, forward/reverse primers and enriched cfDNA (Page 9, [0124], Pages 10-11, [0146] and Page 12, [0176]). Galvin teaches the cleavable base is cleaved prior to amplification (Page 3, [0023]-[0024]). Galvin teaches the cfDNA comprises double-stranded DNA molecules (Page 9, [0112]). Galvin teaches amplifying the enriched cfDNA using indexing primers (Pages 10-11, [0146]). Galvin teaches using degenerate barcode sequences (Page 3, [0033], Page 5, [0070] and Page 13, [0201]). Galvin teaches the barcode region is in the inverted repeat (Page 24, [0222]). Galvin teaches the methods disclosed reduce and/or eliminate artifactual mutations which would be useful for rare mutation detection, sequencing cfDNA, in sequencing FFPE samples, in single cell sequencing, or in sequencing liquid biopsies or ctDNA (Pages 1-2, [0011]). Galvin teaches using duplexed adapters with unique molecular identifiers increases the sensitivity of NGS (Pages 1-2, [0011]).
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 Azab with the teachings of Galvin, to performing end-repair and a-tailing reactions on between about 5 ng and about 30 ng of the plurality of cfDNA as well as a stem-looped adapter with a barcoded inverted repeat and cleavable base. This would allow for reducing and/or eliminating artifactual mutations which would be useful for rare mutation detection, sequencing cfDNA, in sequencing FFPE samples, in single cell sequencing, or in sequencing liquid biopsies or ctDNA as taught by Galvin (Pages 1-2, [0011]). Additionally, using duplexed adapters with unique molecular identifiers increases the sensitivity of NGS as taught by Galvin (Pages 1-2, [0011]).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the invention to modify the method of Azab to include a weight ratio between about 1:25 to about 1:250, RNA baits : amplified adaptor-ligated CfDNA, since it has been held that where the general conditions of a claim are disclosed in the prior art, discovering the optimum working ranges involves only routine skill in the art. In re Aller, 105 USPQ 233 (CCPA 1955). Additional motivation for doing so would be to use sufficient levels of cfDNA from a subject to generate a library that accurately reflects the variant allelic frequency within the sample while not requiring so much cfDNA that an excessively large biological sample would be required to recover said cfDNA as well as to use a sufficient quantity of RNA baits to capture adaptor-ligated cfDNA for amplification for subsequent use such as sequencing and quantification.
Claims 7 and 9 are rejected under 35 U.S.C. 103 as being unpatentable over Azab et al. (WIPO International Application Publication WO 2018/136888 A1, published July 26, 2018, cited on the IDS filed June 21, 2023, and Galvin et al. (U.S. Patent Application Publication US 2018/0223350 A1, published August 09, 2018), as applied to claims 1, 3-4, 6, 8, 11, 14, 17-20, 30 and 34 above, in view of Sun et al. (WIPO International Application Publication WO 2018/031588 A1, published February 15, 2018).
Regarding claim 7, Azab teaches end repair and A-tailing (adding adenine deoxyribonucleotide) as discussed above.
Regarding claim 9, Azab teaches the stem-loop adaptors as discussed above.
Azab and Galvin do not explicitly teach or suggest where the end-repair comprises exposing the plurality of cfDNA to a terminal deoxynucleotidyl transferase. Azab and Galvin do not teach or suggest the stem- loop adaptors comprise a 3' hydroxyl and a 5' phosphate.
Sun teaches using stem-loop adaptors to generate nucleic acid libraries, specifically cfDNA (Abstract Page 25, [0094] and Page 33, [00122]). Sun teaches performing end repair and A-tailing using terminal deoxyribonucleotidyl transferase (Page 12, [0050]). Sun the stem-loop adaptors each comprise an inverted repeat (stem) with a barcode and a loop, wherein the loop comprises at least one cleavable base that may be deoxyuridine (Pages 3-4, [0017], Page 10-11, [0045]-[0046], Page 17, [0066], Pages 17-18, [0068] and Page 23, [0087]). Sun teaches using forward and reverse index primers (Page 20, [0078]). Sun teaches the stem-loop adaptors have a constant sequence (Pages 11-12, [0049]) Sun teaches that the disclosed created libraries are useful in capture target enrichment platforms (Page 11, [0048]). Sun teaches using these methods allows for identify and quantify different molecules in the same input sample that otherwise would be indistinguishable on the basis of sequence or other properties as well as multiple sequences that have been amplified with the same molecular tag can be grouped together to remove artifacts that are created during the library amplification and sequencing processes (Page 1, [0003]).
As a common field of endeavor, Azab, Galvin and Sun all disclose methods for preparing cfDNA libraries using stem-loop/hairpin adaptors.
It would have been obvious to one having ordinary skill in the art before the effective filing date of the invention to modify the combined teachings of Azab and Galvin, with the teachings of Sun wherein end repair comprises exposing the plurality of cfDNA to a terminal deoxynucleotidyl transferase and the stem-loop adaptors comprising a 3' hydroxyl and a 5' phosphate. Using these methods would allow for identify and quantify different molecules in the same input sample that otherwise would be indistinguishable on the basis of sequence or other properties as well as multiple sequences that have been amplified with the same molecular tag can be grouped together to remove artifacts that are created during the library amplification and sequencing processes as taught by Sun (Page 1, [0003]). Additionally, it would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the invention to modify the combined teachings of Azab and Galvin, with the teachings of Sun wherein end repair comprises exposing the plurality of cfDNA to a terminal deoxynucleotidyl transferase and the stem-loop adaptors comprising a 3' hydroxyl and a 5' phosphate, since it has been held that the simple substitution of one known element for another to obtain predictable results is obvious. In re Fout, 213 USPQ 532 (CCPA 1982), In re O'Farrell, 7 USPQ2d 1673 (Fed. Cir. 1988). Sun’s adaptors are well suited for the system of Azab without unexpected results because both Sun and Azab disclose using stem-loop adaptors for cfDNA library preparation and simply substituting Azab’s stem-loop adaptors with Sun’s stem-loop adaptors would obtain predictable results.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JESSICA DANIELLE PARISI whose telephone number is (571)272-8025. The examiner can normally be reached Mon - Friday 7:30-5:00 Eastern with alternate Fridays off.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached at 571-272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/JESSICA D PARISI/ Examiner, Art Unit 1684
/HEATHER CALAMITA/ Supervisory Patent Examiner, Art Unit 1684