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 .
Office Action: Notice
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 1/27/2026 has been entered.
Claim Status
Claims 1 and 11 have been amended (1/27/2026). Claims 5 and 10 have been canceled (1/27/2026). No new matter was added. Thus, claims 1-4, 6-9 and 11-28 are under examination.
Rejections Withdrawn
Claim Rejections - 35 USC § 102
The rejection of claims 1-4, 6-9 and 12-28 under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Blauwkamp et al., (WO 2016/187234 A1, published 11/24/2016) is withdrawn in view of Applicant’s amendment of independent claim 1, with limitations from now cancelled claim 10 (1/27/2026), which was previously rejected under 35 U.S.C. 103 as being unpatentable over Blauwkamp et al., (WO 2016/187234 A1, published 11/24/2016), in view of Langevin et al. (“Peregrine: A rapid and unbiased method to produce strand-specific RNA-Seq libraries from small quantities of starting material”, RNA Biol., published 4/2013, from IDS 12/11/2023).
Claim Rejections - 35 USC § 103
The rejection of claim 10 under 35 U.S.C. 103 as being unpatentable over Blauwkamp et al., (WO 2016/187234 A1, published 11/24/2016), in view of Langevin et al. (“Peregrine: A rapid and unbiased method to produce strand-specific RNA-Seq libraries from small quantities of starting material”, RNA Biol., published 4/2013, from IDS 12/11/2023), is withdrawn due to the Applicant’s cancellation of instant claim 10.
New Rejections
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 1-4, 6-9 and 11-28 are rejected under 35 U.S.C. 103 as being unpatentable over Blauwkamp et al., (WO 2016/187234 A1, published 11/24/2016), in view of Langevin et al. (“Peregrine: A rapid and unbiased method to produce strand-specific RNA-Seq libraries from small quantities of starting material”, RNA Biol., published 4/2013, from IDS 12/11/2023).
Regarding claim 1, Blauwkamp teaches a method for enriching a particular population of nucleic or a nucleic acid sample within a complex mixture of nucleic acids, where the population of interest may make up a minor portion of the complex mixture (Figure 1; Abstract). Blauwkamp further teaches that purification occurs chemically (Figure 12) in order to create a sequencing-ready library of nucleic acids (Figure 2; Paragraph 125, lines 1-5; Paragraph 129, lines 10-15) via polymerase chain reaction, reverse transcription, transcription-mediated amplification, or ligase chain reaction (Paragraph 23, lines 1-5). Additionally, Blauwkamp teaches that the purified sample may be further subjected to conditions to remove double-stranded DNA (i.e., duplex-specific nuclease) to preferentially remove the background population DNA in the sample of interest (i.e., RNA combined with background population)
(Paragraph 140, lines 10-15) to create a library of domains of nucleotides with sequences that may match (either completely or substantially) or may be complementary (either completely or substantially) and individualized or free of repetitive sequences to analyze certain population of interest sequences (Paragraph 56, lines 10-15). Blauwkamp teaches that these populations can include DNA, RNA, cDNA, dsDNA, ssDNA, mRNA, or cRNA (a class of ncRNA) (Paragraph 66, lines1-5) to be further primed or captured and then sequenced by via a sequencing assay (i.e., a Next Generation sequencing assay, (Paragraph 132, lines 1-5).
Blauwkamp teaches a method for enriching targeted nucleic acids from a complex mixture containing host nucleic acids, via creating sequencing-ready libraries using various techniques including reverse transcription, PCR, and ligase chain reaction (Figure 2; Paragraph 125, lines 1-5; Paragraph 129 lines 10-15). Specifically, Blauwkamp teaches that oligonucleotides can be used as primers in reverse-transcription reactions (Paragraph 49, lines 1-10).
Regarding claim 2, Blauwkamp teaches that a digestion step could be employed after adapter ligation to sequence fragments, in order to pre-digest and increase efficiency of the host derived library elements or DNA-RNA hybrids (Figure 10B; Paragraph 177, lines 10-15), at various degrees of degeneracy (Paragraph 123, lines 1-5), and occurring at specified temperatures (i.e., room temperature) (Paragraph 138, lines 10-15).
Regarding claim 3, Blauwkamp teaches that a chemical denaturant (i.e., acid, base, solvent, chaotropic agent, salt) can be used in combination with rehybridization in order to minimize mismatches to one or more background population nucleic acids (Paragraph 74, lines 5-10; Paragraph 111, lines 1-5).
Regarding claim 4, Blauwkamp teaches those abundant nucleic acids (i.e., background population) hybridize faster targeted nucleic acid within the sample, and as a result of this concentration dependence, a portion of double-stranded nucleic acids or alternatively via isolation of a portion of single-stranded nucleic acids following partial hybridization of a population of nucleic acids can be performed to enrich the overall library complex (Paragraph 139, lines 1-8). Further, Blauwkamp teaches that these alterations can include construction of a library or background population comprising RNA, double-stranded or single-stranded populations or a mixture of both (i.e., human exome sequences) (Paragraph 140, lines 10-15).
Regarding claims 6-9, Blauwkamp teaches that the nucleic acid sample can include RNA (Paragraph 144, lines 10-15; Paragraph 145, lines 1-5; Paragraph 172, lines 1-5) and is extracted via a specialized nucleic acid kit from a human biological sample (Paragraph 172, lines 1-15) (i.e., blood, plasma, serum, whole blood, mucus, saliva, cerebrospinal fluid, synovial fluid, lavage, urine, tissue biopsies, cellular samples, skin samples, and stool (Paragraph 189, lines 1-5).
Regarding claim 11, Blauwkamp teaches a method for enriching targeted nucleic acids from a complex mixture containing host nucleic acids, via creating sequencing-ready libraries using various techniques including reverse transcription, PCR, and ligase chain reaction (Figure 2; Paragraph 125, lines 1-5; Paragraph 129 lines 10-15). Specifically, Blauwkamp teaches that oligonucleotides can be used as primers in reverse-transcription reactions (Paragraph 49, lines 1-10).
Regarding claims 12-16, Blauwkamp teaches that prior to subjecting the purified sample to conditions to remove double-stranded DNA (i.e., duplex-specific nuclease) to preferentially remove the background population DNA in the sample of interest (i.e., RNA combined with background population) (Paragraph 140, lines 10-15), in order to create a library of domains of nucleotides, denaturation occurs for at least a portion of the nucleic acids in the sample of nucleic acids using heat, at levels including 80-90°C, allowing the background population nucleic acids to specified domains within the collection of oligonucleotides (Figure 3; Paragraph 13, lines 15-20; Paragraph 105, lines 15-25). Further, Blauwkamp teaches that when high heat is used, the sample is subjected to tiered heating (i.e., 65 °C for 3 min) followed by a period of incubation (i.e., 36 °C) to prevent reannealing (Paragraph 144, lines 1-5). Blauwkamp teaches that following the previously explained steps, further renaturation can occur via single-stranded nucleic acids can hybridize or reanneal into double-stranded nucleic acids (i.e., DNA, double-stranded RNA, or a DNA-RNA duplex) (Paragraph 143, lines 1-5; Paragraph 144, lines 10-20; Paragraph 145, lines 10-15). Additionally, Blauwkamp teaches that the formed duplex DNA/RNA library is a human, dog, cat, rodent, mouse, hamster, cow, bird, chicken, pig, horse, goat, sheep, rabbit, microbe, pathogen, bacteria, virus, fungus, or parasite (Paragraph 77, lines 1-5).
Regarding claims 17-23, Blauwkamp teaches that depletion of background population nucleic acids may occur from 5-100% (Paragraph 188, lines 1-5), optimized by temperature, buffer composition, reaction time, concentration, and nuclease to substrate ratio (Paragraph 144, lines 10-20). For example, the substrate preference of the double-strand specific DNase from Northern shrimps has a reaction time of 30 minutes and is performed in the presence of magnesium (Paragraph 144, lines 15-25). Specifically, Blauwkamp teaches that a binding buffer (i.e., EDTA) can be used to cease depletion and begin adsorption (Paragraph 158, lines 1-5). Further, Blauwkamp teaches that one may target specific sequence motifs, structures, base modifications, etc. as a mechanism for binding, pulling out, precipitating, digesting, or otherwise removing or depleting for one of the host or non-host nucleic acids within the DNA/RNA duplexes via a porous filter (Paragraph 15, lines 10-20; Paragraph 176, lines 5-10). Blauwkamp teaches that the unwanted or non-host nucleic acids, specifically found within exosomes, can be isolated via a circulating, yielding ribosomal RNA kit that can be extracted via exosome isolation (Paragraph 172, lines 5-10). Following extraction of unwanted or non-population of interest, Blauwkamp teaches that the purified sample may be compared to the absolute number of recovered normalization oligonucleotide reads to normalize for differences in molecular manipulation efficiencies between samples (Paragraph 186, lines 10-20), and thus limiting human cell-free mRNA or background from the sample to <1000 (Figure 8A; Paragraph 172, lines 10-15) or a range of 5-99% removal (Paragraph 168, lines 1-10).
Regarding claims 24-28, Blauwkamp teaches that the purified sample may be compared to the absolute number of recovered normalization oligonucleotide reads to normalize for differences in molecular manipulation efficiencies between samples (Paragraph 186, lines 10-20). Specifically, Blauwkamp teaches that the fragments reflected in an enriched sample will include fragments that are within the range of 5%-30% of original size range, where the host to non-host DNA ratio is at the lowest point (Paragraph 154, lines 10-20). Further, Blauwkamp teaches that the true abundance versus measured abundancies or experimental variability (or within statistical experimental error), and the number or numerical range may vary from 1% to 15% (Paragraph 207, lines 1-5). Blauwkamp teaches that through the utilization of the above purified samples, following DSN depletion (Paragraph 144, lines 10-20), the method further comprises preferentially amplifying the primed or captured non-host nucleic acids in a reaction by conducting a sequencing assay, such as a Next Generation sequencing assay, a high-throughput sequencing assay, a massively parallel sequencing assay, a Nanopore sequencing assay, or a Sanger sequencing assay (Paragraph 7, lines 1-5; Paragraph 133: lines 1-5) on the nucleic acid sequencing-ready library (Paragraph 9, lines 35-45).
Blauwkamp does not teach or suggest using random hexamers with Moloney murine leukemia virus (MMLV) reverse transcriptase.
Langevin teaches an improved RNA-Seq library preparation method that uses MMLV reverse transcriptase with random hexamer primers (Abstract; Peregrine RNA-Seq library preparation method: Design considerations and mechanics: Paragraph 1). Further, Langevin teaches that this method is specifically designed to minimize cost, time and sample manipulation while producing representative libraries, showcasing specific technical details highlighting that MMLV reverse transcriptase effectively polymerizes first stranded cDNA from the random hexamer-primed RNA (Figure 1; Discussion: Paragraph 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Blauwkamp’s reverse transcription method by specifically using Langevin’s MMLV reverse transcriptase and random hexamers to generate representative first-strand cDNA suitable for sequencing-library preparation.
Specifically, Blauwkamp teaches enriching a population of nucleic acids from a complex biological mixture through reverse transcription, duplex-specific nuclease (DSN) depletion of abundant background nucleic acids, normalization, and preparation of sequencing-ready libraries for downstream next-generation sequencing analysis. Although Blauwkamp does not specify the particular reverse transcriptase enzyme or primer strategy of the amended independent claims, Langevin teaches RNA-seq library prep0reation using MMLV reverse transcriptase with random hexamer priming, demonstrating that this combination efficiently polymerizes first-strand cDNA from diverse RNA templates while minimizing bias, reducing handling time, and enabling representative transcript capture from limited or complex samples. Because Langevin shows that MMLV + random hexamers constitute a well-established, reliable reverse-transcription approach for sequencing-library construction, a skilled artisan for sequencing-library construction, a skilled artisan would have recognized this technique as a predictable substitute for the unspecified reverse-transcription conditions of Blauwkamp.
One would have been motivated to do so because this represents use of known techniques (MMLV reverse transcriptase coupled with random hexamers) to improve efficiency and representativeness of cDNA synthesis from complex RNA populations, reduce sequence-dependent priming bias relative to more selective primers, and facilitate downstream DSN depletion and sequencing-library normalization using known, compatible RNA-seq preparation chemistries. Such substitution merely applies a known technique to a known method to obtain predictable results, consistent with established RNA-Seq practices. Blauwkamp does not teach away from the use of MMLV reverse transcriptase or random hexamers, and Langevin further confirms their suitability specifically for sequencing-library generation from complex RNA samples, in the same technological context addressed by Blauwkamp.
Therefore, one of ordinary skill in the art would have had a reasonable expectation of success because Langevin demonstrates successful use of the MMLV combination for RNA-Seq library preparation, as such that coupling with random hexamers is an established tool for reverse transcription and represents routine optimization of reverse-transcription conditions within a known nucleic-acid enrichment workflow.
Applicant’s Response: The Applicant asserts that the claimed method is not obvious over Blauwkamp in view of Langevin because the claims require performing DSN depletion immediately after reverse transcription and prior to double-stranded library preparation, without the use of pull-down techniques or an added exome DNA library as in Blauwkamp, resulting in a streamlined workflow and superior enrichment of target sequences. The Applicant further argues that Langevin’s RNA-Seq approach, even using MMLV reverse transcriptase with random hexamers, fails to achieve the level or enrichment demonstrated by the claimed method and therefore does not render the claimed improvements predictable. Accordingly, the Applicant asserts that the enhanced enrichment and workflow simplification represent unexpected results and substantive methodological distinctions.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully considered but are only found partially persuasive, as discussed below.
As of note, the rejection of claims 1-4, 6-9 and 12-28 under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Blauwkamp et al., (WO 2016/187234 A1, published 11/24/2016) was withdrawn in view of Applicant’s amendment of independent claim 1. However, claims 1-4, 6-9 and 11-28 are still subjected to an obviousness rejection and are therefore rejected under 35 U.S.C. 103 as being unpatentable over Blauwkamp et al., (WO 2016/187234 A1, published 11/24/2016), in view of Langevin et al. (“Peregrine: A rapid and unbiased method to produce strand-specific RNA-Seq libraries from small quantities of starting material”, RNA Biol., published 4/2013, from IDS 12/11/2023).
Although the Applicant contends that the claimed methods are not obvious over Blauwkamp in view of Langevin because the claims allegedly require duplex-specific nuclease (DSN) depletion to be performed immediately following reverse transcription and prior to double-stranded library preparation, without the use of pull-down techniques or an added exome DNA library as described in Blauwkamp, and that this ordering purportedly results in a streamlined workflow and unexpectedly superior enrichment of target sequences. The Applicant further argues that Langevin’s RNA-Seq approach, even when using MMLV reverse transcriptase with random hexamers, does not achieve the level or enrichment demonstrated by the presently claimed method and therefore would not have rendered the claimed invention predictable.
These arguments are not found persuasive because the actual language of amended independent claim 1 merely requires reverse transcription using random hexamers and a reverse transcriptase, formation of a DNA/RNA library and incubation with DSN to obtain a purified sample enriched for coding and non-coding RNA and depleted of ribosomal RNA prior to next-generation sequencing library preparation. The claim does not exclude the presence of additional depletion, capture, normalization, or enrichment techniques, nor does it prohibit the use of exome-based depletion or pull-down procedures. Because the claim employs the open transitional phrase “comprising”, the method encompasses workflows that include the additional steps taught by Blauwkamp (see MPEP 2111.03). Accordingly, the asserted distinction based on the absence of exome-library pull-down or other preparatory steps is not commensurate with the scope of the claims.
Further, the Applicant’s reliance on allegedly superior enrichment or unexpected results is unpersuasive. The claims do not recite any quantitative enrichment threshold, correlation coefficient, or magnitude of improvement, and therefore evidence of improved enrichment relative to Langevin is not commensurate in scope with the claimed subject matter (see MPEP 716.02(d)). Moreover, applying a known reverse-transcription system such as Langevin’s MMLV/random hexamer strategy within the known DSN-based enrichment workflow of Blauwkamp would have yielded predictable improvements in cDNA representativeness and sequence readiness, which constitutes no more than the expected result of combining familiar RNA-Seq preparation techniques (see KSR v. Teleflex, Inc., 50 US 398 (2007), MPEP 2143).
The Applicant also argues that Blauwkamp requires the addition of exome DNA libraries and therefore differs fundamentally from the claimed approach. However, even assuming Blauwkamp discloses additional optional steps, the presence or absence of such steps represents routine variation or simplification of a known workflow, which would have been within the ordinary skill in the art and does not confer patentable distinction where the underlying enrichment, reverse transcription, and DSN depletion steps remain taught or suggested by the prior art (see MPEP 2144.04 (VI)). Rearranging or optimizing the order of known process steps to improve efficiency likewise constitutes routine optimization of result-effective variables, which is ordinarily obvious (see MPEP 2144.05; In re Aller, 220 F. 2d 454 (CCPA 1955)).
For these reasons, the combination of Blauwkamp and Langevin continues to teach or suggest all limitations of independent claim 1, and the Applicants’ arguments do not demonstrate reversible error in the rejection. Because dependent claims 2-4, 6-9, and 11-28 recite only routine experimental parameters, sample sources, sequencing conditions, normalization features, or expected properties of RNA-Seq enrichment workflows, they likewise would have been obvious for the same reasons set forth above. As of note, to potentially overcome this rejection, the Applicant may amend the independent claim to recite a specific structural or process limitation, such as a narrowly defined DSN treatment condition, shown to produce unexpected results commensurate in scope with the claims.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ELIZABETH ROSE LAFAVE whose telephone number is (703)756-4747. The examiner can normally be reached Compressed Bi-Week: M-F 7:30-4:30.
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/ELIZABETH ROSE LAFAVE/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684