DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-8 and 11-13 are pending. Claims 1, 3-4, 6-8, and 11 are amended. Claims 9-10 are cancelled. Claims 1-8 and 11-13 are currently under examination.
Response to Amendment
The Amendment filed 8/22/25 has been entered. Claims 1-8 and 11-13 are currently pending. Applicant’s amendments to claims 3-4 and 6-8 and the specification have overcome the 112(b) rejections and objections previously set forth in the Non-Final Office Action mailed 2/24/25. Applicant’s cancellation of claim 9 renders the 112(b) rejection in the Non-Final Office Action mailed 2/24/25 moot.
Response to Arguments
Applicant’s arguments, see pages 7-10, filed 8/22/25, with respect to the rejections of claims 1-8 and 11-13 under 35 USC 103 have been fully considered and are persuasive. Therefore, the rejections documented in the Non-Final mailed on 2/24/25 of claims 1-8 and 11-13 have been withdrawn. However, upon further consideration, new grounds of rejections necessitated by claim amendments are made in this Final Office Action.
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/PatentLegalFramework), 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 – Nucleotide and/or amino acid sequences appearing in the specification are not identified by sequence identifiers in accordance with 37 CFR 1.821(d).
Required response – Applicant must provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Claim Objections
Claims 1, 3, 7-8, and 12 objected to because of the following informalities:
periods may not be used elsewhere in the claims except for abbreviations (see MPEP 608.01(m). Parentheses and corrections of capitalization are recommended (e.g., for claim 1, “a) denaturing said…”).
Appropriate correction is required.
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.
Claims 1-3, 7, 11, and 13 are rejected under 35 U.S.C. 103 as being unpatentable over Stahl et al. (2015; US 2015/0337364 A1; USPat citation A in PTO-892 filed 2/24/25) in view of Hindson et al. (2019; US 10,323,279 B2; USPat citation B in PTO-892 filed 2/24/25).
This new 103 rejection is necessitated by claim amendments filed on 8/22/25.
(i) Stahl et al. teaches limitations relevant to claims 1-3, 7, 11, and 13.
Relevant to claim 1, Stahl et al. teaches a method wherein a “double-stranded or single-stranded nucleic acid… serves a substrate for nucleic acid synthesis, e.g., for a template-dependent extension or a transcription reaction” (page 11, paragraph 0066). Stahl et al. further teaches that the “template-dependent extension results in a partially or completely double-stranded nucleic acid” (page 11, paragraph 0067). The method of Stahl et al. does not include bisulfite treatment.
Relevant to claim 1 step a), Stahl et al. teaches “for a double-stranded DNA molecule, denaturation of at least a portion of its two strands may be performed prior to or in conjunction with nucleic acid synthesis” (page 11, paragraph 0066). This teaching reads on a) denaturing said at least one polynucleotide to produce single stranded polynucleotide.
Relevant to claim 1 step b), Stahl et al. teaches “In some embodiments, an oligonucleotide is single-stranded” (page 9, paragraph 0055). This teaching reads on a first single-stranded oligonucleotide.
Further relevant to claim 1 step b), Stahl et al. teaches “an oligonucleotide complementary to a portion of a nucleic acid template is hybridized via a hybridization sequence to a template, an appropriate polymerase may then synthesize a nucleic acid complementary to the template” (page 11, paragraph 0066). Stahl et al. teaches that “A oligonucleotide is capable of "hybridizing" with another nucleic acid… when a single stranded form of the nucleic acid molecule or hybridization sequence thereof can anneal to the other nucleic acid molecule under the appropriate conditions” (page 9, paragraph 0056). These teachings read on incubating the single stranded polynucleotide from step a) with a first-single stranded oligonucleotide… under conditions suitable for annealing.
Further relevant to claim 1 step b), Stahl et al. further teaches “the 3'-end of the hybridization sequence of an oligonucleotide that hybridizes with a nucleic acid template serves as a primer for a template-dependent extension that results in a new nucleic acid complementary to the nucleic acid template” (page 11, paragraph 0070). This teaching reads on a primer sequence… and then extending the primer with a polymerase to produce double-stranded polynucleotide.
Further relevant to claim 1 step b), Stahl et al. teaches that “an oligonucleotide having… an additional sequence 5' to the hybridization sequence…” that “may comprise a tag, barcode, index, adapter” (page 11, paragraph 0071). Stahl et al. teaches “an adapter sequence contains a sequencing primer hybridization sequence” (page 10, paragraph 0064). This teaching reads on oligonucleotide comprising a sequencing adaptor sequence.
Collectively, these teachings read on claim 1 step b).
Relevant to claim 1 step c), Stahl et al. teaches “for a double-stranded DNA molecule, denaturation of at least a portion of its two strands may be performed prior to or in conjunction with nucleic acid synthesis” (page 11, paragraph 0066). This teaching reads on c) denaturing the double-stranded polynucleotide of step b) to produce single stranded polynucleotide.
Relevant to claim 1 step d), Stahl et al. teaches “In some embodiments, the methods involve producing a synthetic RNA from a nucleic acid template that comprises a target region and an adjacent region; producing a double-stranded nucleic acid that comprises a first strand synthesized by a template-dependent extension using the synthetic RNA as a template and a second strand synthesized by a template-dependent extension using the first strand as a template, wherein the double-stranded nucleic acid is representative of the target…” (page 2, paragraph 0010). This teaching reads on d) incubating the single stranded polynucleotide from step c) with a second single-stranded oligonucleotide comprising a sequencing adaptor sequence and a primer sequence under conditions suitable for annealing of the second single-stranded oligonucleotide to the single stranded polynucleotide of step c), and then extending the primer with a polymerase to produce a population of double-stranded polynucleotide molecules.
Relevant to claim 2, Stahl et al. teaches “In some embodiments, methods provided herein are advantageous because they can be employed using RNA and/or DNA as a starting material” (page 1, paragraph 0002). This teaching reads on at least one polynucleotide in the sample is RNA or DNA. As seen in Fig. 1A, steps 101-102 display an RNA-DNA double-stranded hybrid molecule, and Fig. 1E step 119 displays a double-stranded DNA, reading on the population of double-stranded polynucleotide molecules is RNA or DNA.
Relevant to claim 3, Stahl et al. Fig. 3B steps 305-306 and Fig. 3C step 307 shows a RNA polynucleotide and reverse transcriptase (i.e., a polymerase) acting together to produce complementary DNA (cDNA), wherein the cDNA undergoes an extension reaction to produce double stranded cDNA molecules, as explained within Stahl et al. page 8, paragraph 0047.
Relevant to claim 7, Stahl et al. teaches that “In some embodiments, method disclosed herein involve removal or degradation of excess oligonucleotides and other nucleic acids in a reaction. In some embodiments, nucleic acids are degraded enzymatically. In some embodiments, E. coli Exonuclease I is used to degrade single stranded nucleic acids” (page 12, paragraph 0077). This teaching reads on both step b) and step d) removal of any remaining single stranded oligonucleotide with an exonuclease.
Further relevant to claim 7, Stahl et al. teaches that their methods “involve purification of nucleic acids from a reaction mixture in preparation for subsequent steps” and “any appropriate purification method may be used,” including an “AMPure kit that uses a bead-based solid-phase extraction” (page 12, paragraph 0080). This teaching reads on Step b. and Step d. further comprises purifying the single stranded polynucleotide… wherein either step optionally uses solid phase reversible immobilization (SPRI) beads.
Relevant to claim 11, Stahl et al. teaches that “synthesis of a complementary DNA strand from a template may be performed by a DNA polymerase enzyme,” including the “Klenow fragment” (page 11, paragraph 0068), reading on the polymerase is a Klenow DNA polymerase.
As discussed within claim 1 rejection, and further relevant to claim 13, Stahl et al. teaches “the 3'-end of the hybridization sequence of an oligonucleotide that hybridizes with a nucleic acid template serves as a primer for a template-dependent extension that results in a new nucleic acid complementary to the nucleic acid template” (page 11, paragraph 0070). This teaching reads on a sequence complementary to an amplification primer.
Further relevant to claim 13, Stahl et al. teaches that “an oligonucleotide having… an additional sequence 5' to the hybridization sequence…” that “may comprise a tag, barcode, index, adapter” (page 11, paragraph 0071). Stahl et al. teaches “an adapter sequence contains a sequencing primer hybridization sequence” (page 10, paragraph 0064). These teachings read on a sequence complementary to a sequencing primer; a barcode or index sequence.
Further relevant to claim 13, Stahl et al. teaches that the adapters can be used to “attach a nucleic acid… to a next generation sequencing platform or other substrate for purposes of immobilizing the nucleic acid” (page 10, paragraph 0064). This reads on a sequence to facilitate attachment to a solid surface.
(ii) Stahl et al. is silent to specifics regarding temperature increases during the extending of step b) and/or step d) relevant to claim 1 step d). However, these limitations were known in the prior art and taught by Hindson et al.
Hindson et al. teaches “Methods and systems for processing polynucleotides” (Title).
Relevant to claim 1 step d), Hindson et al. teaches “First, initial denaturation of the sample nucleic acid may be achieved at a denaturation temperature (e.g., 98° C., for 2 minutes) followed by priming of a random portion of the sample nucleic acid with the random N-mer sequence at a priming temperature (e.g., 30 seconds at 4°C.), FIG. 15A. The oligonucleotide sequence is hybridized with a blocking oligonucleotide (black dumbbell in FIGS. 15A-15G), to ensure that only the random N-mer primes the sample nucleic acid and not another portion of the oligonucleotide sequence. Subsequently, sequence extension (e.g., via polymerase that does not accept or process a uracil containing nucleotide as a template) may follow as the temperature ramps to higher temperature (e.g., at 0.1°C./second to 45°C. (held for 1 second)) (FIG. 15A)” (column 94, line 57 continued to column 95 line 3).
It is noted that there is not a limiting definition of the term “around” as used in claim 1 step d) extension step incubation. Instead, it is further noted that the last paragraph of page 17 continued through the first paragraph of page 18 of the instant specification allow for a range of preferred embodiment temperature ranges, reinforcing the lack of a limiting definition for this claim 1 step d) limitation.
Therefore, although the Hindson et al. teaching includes a ramping rate of 0.1°C/second (which converts to 6°C/minute) that exceeds the instant 4°C/minute, the skilled artisan would recognize that this difference is embraced by the non-limited around term.
(iii) It would have been prima facie obvious to include the Hindson et al. extension ramping temperature within the methodology rendered obvious by Stahl et al. It is noted that Stahl et al. and Hindson et al. are analogous disclosures to the instant method for generating a population of double-stranded polynucleotide molecules.
The skilled artisan would have been motivated to combine the analogous disclosures to include the Hindson et al. temperature ramping within the Stahl et al. methodology. Although Stahl et al. discloses isothermal amplification reactions within their examples, Stahl et al. teaches “In some embodiments, amplification is accomplished under isothermal conditions. In some embodiments, amplification is accomplished under conditions involving multiple thermal cycles, such as in a polymerase chain reaction” (paragraph 0065). This Stahl et al. teaching allows for the skilled artisan to recognize that the Stahl et al. methodology can employ thermal cycling PCR, and thus that the Hindson et al. invention contains analogous components.
The skilled artisan would have been motivated to combine the analogous disclosures. Hindson et al. teaches the motivation. Hindson et al. teaches that primer/temperature flexibility is advantageous within applications with modified N-mer primers. Hindson et al. teaches “For example, in some cases, it may be desirable to provide N-mer primers that have different melting/annealing profiles when subjected to thermal cycling, e.g., during amplification, in order to enhance the relative priming efficiency of the n-mer sequence” (column 43, lines 53-57). Hindson et al. further teaches that nucleotide analogoues may be incorporated into the N-mer primer sequences “in order to provide elevated temperature stability for the primers when hybridized to a template sequence, as well as provide generally enhanced duplex stability” (column 43, line 63 continued to column 44, line 1). Hindson et al. teaches “By providing enhanced hybridizing primer sequences, one may generate higher efficiency amplification processes using such primers, as well as be able to operate within different temperature regimes” (column 44, lines 4-7).
These Hindson et al. teachings render obvious amplification with ramping extension temperatures and motivations for the skilled artisan. The skilled artisan would recognize that the Hindson et al. temperature ramping enables primer sequence design flexibility and optimization of amplification efficiency. The skilled artisan would further recognize that the temperature ramping impacts amplification reaction products, as Hindson et al. teaches “Product length can be controlled by thermal cycling conditions (e.g., number of thermal cycles, temperatures utilized, cycle time, total run time, etc.)” (column 96, lines 17-19). The skilled artisan would thus be motivated to include the Hindson et al. temperature ramping within the Stahl et al. methodology in order to optimize upon the Hindson et al.-taught product length control, amplification efficiency, and primer designs.
The skilled artisan would have a reasonable expectation of success based on the disclosure of Stahl et al. in view of Hindson et al., as discussed in the preceding paragraphs.
Claims 4-6, 8, and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Stahl et al. (2015; US 2015/0337364 A1; USPat citation A in PTO-892 filed 2/24/25) in view of Hindson et al. (2019; US 10,323,279 B2; USPat citation B in PTO-892 filed 2/24/25), as applied to claims 1-3, 7, 11, and 13 above, and further in view of Sanches-Kuiper et al. (2015; WO 2015/057985 A1; FOR citation N in PTO-892 filed 2/24/25).
The teachings of Stahl et al. in view of Hindson et al. are applied to instantly rejected claims 4-6, 8, and 12 as they were previously applied to claims 1-3, 7, 11, and 13 as rendering obvious a method for generating a population of polynucleotide molecules.
Stahl et al. in view of Hindson et al. is silent to specifics regarding concentrations and qualities of input DNA (claim 4), sample material (claim 5), base excision repair (claim 6), PCR (claim 8), or random primers (claim 12). However, these limitations were known in the prior art and taught by Sanches-Kuiper et al.
Relevant to claim 4, Sanches-Kuiper et al. teaches that “In some embodiments, a sample includes low quality nucleic acids. Low quality nucleic acids include a population of nucleic acids that is a potentially poor substrate for typical methods of nucleic acid library preparation. Examples of poor substrates for nucleic acid library preparation include populations of nucleic acids in which a substantial fraction of the population is single-stranded, is nicked, is mutated, and/or is fragmented. A substantial fraction can include at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95%, and a range between any two of the foregoing parameters” (page 13, paragraph 0065).
Further relevant to claim 4, Sanches-Kuiper et al. teaches “The methods presented herein are surprisingly effective for generating sequencing libraries from low amounts of input material. For example, in some embodiments, the input material comprises no more than 100 ng, 80 ng, 70 ng, 60 ng, 50 ng, 40 ng, 30 ng, 20 ng, 10 ng, 5 ng, 2 ng, 1 ng or no more than 0.5 ng of input nucleic acid” (pages 13-14, paragraph 0065). These teachings read on the low quantity of DNA and/or low quality DNA limitations within claim 4.
Relevant to claim 5, Sanches-Kuiper et al. teaches that “In some embodiments, the sample is a formalin fixed paraffin-embedded sample” (page 7, paragraph 0043).
Relevant to claim 6, Sanches-Kuiper et al. teaches that “a population of nucleic acids from a fixed sample” are processed and that “nucleic acids are repaired… prior to library preparation” (page 17, paragraph 0074), reading on prior to the first denaturing step, the method comprises: extracting at least one polynucleotide from the sample. Sanches-Kuiper et al. teaches “In some embodiments, repairing nucleic acids can include contacting the nucleic acids with a DNA glycosylase. DNA glycosylases are a family of enzymes involved in base excision repair by which damaged bases in DNA are removed and replaced” (page 16, paragraph 0073). Sanches-Kuiper et al. further teaches “Persons skilled in the art would appreciate that any suitable DNA glycosylase… may be used” and include an example of “formamidopyrimidine-DNA glycosylase (FPG)” (page 17, paragraph 0073).
Relevant to claim 8, Sanches-Kuiper et al. teaches “the PCR TruSeq™ Nano program was run for 8 cycles,” specifying the temperature conditions used within the 8 cycle-program and that “the library was sequenced” (page 31, paragraph 0101).
Relevant to claim 12, Sanches-Kuiper et al. teaches “In certain other embodiments, the set of random amplification primers are about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or greater than about 30 nucleotides in length, or a combination thereof” (page 20, paragraph 0078).
Although Stahl et al. in view of Hindson et al. is silent to the Sanches-Kuiper et al. limitations, it would have been prima facie to the skilled artisan to include the Sanches-Kuiper et al. concentrations and qualities of input DNA, sample material, base excision repair, PCR, and random primers within the methodology rendered obvious by Stahl et al. in view of Hindson et al. It is noted that Stahl et al., Hindson et al., and Sanches-Kuiper et al. are all analogous disclosures to the instant method for generating a population of double-stranded polynucleotide molecules.
The skilled artisan would have been motivated to combine the analogous art. Stahl et al. teaches their methods “can be employed using nucleic acids extracted from a variety of different types of samples” (page 1, paragraph 0002), including “biological samples (e.g., formalin fixed tissue sections…)” (page 3, paragraph 0017). Sanches-Kuiper et al. teaches methods for formalin-fixed and paraffin-embedded samples, which are a type of Stahl et al.’s formalin fixed samples, in which Sanches-Kuiper et al. teaches that these nucleic acids “are often of low quality with significant fragmentation, an increased proportion of single stranded DNA, and a variety of chemically induced DNA lesions including strand breakage, abasic sites and chemically modified bases. Often the amount of DNA that can be extracted from FFPE samples and then analyzed is small” (pages 1-2, paragraph 0004). Thus, the skilled artisan would be motivated to include the methodologies of Sanches-Kuiper et al. to improve the methodologies of Stahl et al. in view of Hindson et al. because Sanches-Kuiper et al. teaches “The quality and small amounts of DNA that may be prepared from low quality nucleic acid samples make such samples difficult to use in preparing sequencing libraries of sufficient yield, complexity and genomic coverage. Thus, methods and compositions are desirable [such as the ones taught by Sanches-Kuiper et al.] for the enrichment of nucleic acids obtained from low quality nucleic acid samples that are suitable for nucleic acid sequence analysis” (page 2, paragraph 0005).
Although Stahl et al. teaches nucleic acid syntheses of template-dependent polymerase extension and oligonucleotides with sequencing adapter sequences, they are silent to specifics regarding PCR amplification and subsequent sequencing. However, a skilled artisan would be motivated to include the PCR amplification and subsequent sequencing of Sanches-Kuiper et al. because Sanches-Kuiper et al. teaches that “[Next Generation Sequencing] NGS methods can be broadly divided into those that typically use template amplification and those that do not” (page 22, paragraph 0082). The skilled artisan would want to ensure the nucleic acids and workflows would be compatible with any number of available sequencing platforms.
Although Stahl et al. teaches oligonucleotides with “random or pseudorandom sequences” (page 8, paragraph 0051), Stahl et al. does not provide specifics or guidance on random primer sequence length. However, a skilled artisan would be motivated to further include the random amplification primers and lengths of Sanches-Kuiper et al. because Sanches-Kuiper et al. teaches that “the length of the primers in the primer mixture can be selected to optimize amplification across the sample nucleic acids” and “that the exact length, composition of each base in a mixture of n-mers can be adjusted as needed to generate a desired level of amplification uniformity across a nucleic acid target” (page 20, paragraph 0078).
The skilled artisan would have a reasonable expectation of success based on the disclosures of Stahl et al. in view of Hindson et al., and further in view of Sanches-Kuiper et al.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/SARAH JANE KENNEDY/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682