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 .
Continued Examination Under 37 CFR 1.114
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 August 20, 2026 has been entered.
Allowable Subject Matter
Claim 7 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
The following subject matter is not taught or suggested in the prior art:
Claim 7 recites:
"wherein the pool of nucleic acid precursors comprises 978 different nucleic acid precursors, wherein each one of the nucleic acid precursors has a sequence selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 978."
No prior art teach or suggest a specific pool of 978 different nucleic acids, wherein each of the nucleic acid in the pool comprise a sequence selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 978.
A comprehensive sequence search, including databases such as Geneseq, NCBI GenBank, Issued Patents, Pending Patents, and Published Applications, did not reveal any full match for SEQ ID NOs:1-10, as required by the claim. Each of these sequences is non-naturally occurring, as they contain synthetic sequence structures, including primer binding sites, which do not correspond to any naturally occurring sequences. For instance, the nucleotide bases near the 5' end and 3'end of SEQ ID NO:1 do not map to any naturally occurring sequences in GenBank. Given the breadth of the search covering SEQ ID NOs: 1-10 and the unique nature of these synthetic sequences, the search is considered sufficiently exhaustive for the large number of sequences claimed. In addition, the Examiner could not find any of these sequences in the prior art. Therefore, the nucleic acid precursor sequences SEQ ID Nos: 1-978 are considered to be allowable.
Status of Claims / Response to Amendment
This office action is in response to an amendment filed on August 20, 2026
Claims 1-10, 12-16, 18-19 and 26-29 were previously pending. Applicant amended claim 1; claim 30 is newly added.
Claims 1-10, 12-16, 18-19 and 26-30 are currently pending, with claim 28 withdrawn.
Claims 1-10, 12-16, 18-19 and 26-27 and 29-30 are under consideration.
No rejection has been overcome by amendment. Applicant' s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
This office action contains new grounds for rejection necessitated by amendment.
Claim Objections
Claims 16 and 30 are objected to because of the following informalities:
In claim 16, lines 1-2, it should read "wherein the affinity-tag is biotin and the solid support comprises streptavidin," to properly reference the term first introduced in claim 1, part (b), line 4.
In claim 30, lines 1-2, it should read "wherein the amplified double-stranded DNA precursors consist of double-stranded deoxyribonucleic acid (DNA)" to properly reference the term first introduced in claim 1, part (b), lines 4-5.
Priority -- updated in view of amendment
The priority date of the instant claims 1-10, 12-16, 18-19 and 26-27 and 29-30 is June 12, 2018, filling date of the European Patent Application Number 18177178.3, to which the present application claims priority.
Claim Interpretation
In evaluating the patentability of the claims presented in this application, claim terms have been given their broadest reasonable interpretation (BRI) consistent with the specification, as understood by one of ordinary skill in the art, as outlined in MPEP§ 2111.
For the purpose of applying prior art, claim 1 has been amended in step (f) to recite "discarding the solid support." In light of the specification and under BRI, this step is interpreted as separating or removing the solid support from a mixture, including by magnetic separation.
The specification does not provide an express definition or description of any step of "discarding the solid support." The most relevant description in the specification involves separating the solid support from single-stranded oligonucleotides. For example, where the solid support comprises magnetic beads, the solid support may be removed by magnetic separation, e.g. by placing a magnet in close vicinity of the solid support.
"Separation
The preferred method of the invention wherein the second strand or part thereof comprising the reverse complement of the sequence of interest is separated from the first strand or part thereof comprising the sequence of interest, comprises a step of removing the solid support to obtain a single-stranded oligonucleotide having the sequence of interest.
The solid support comprises the capture agent. In the method of the invention, the capture agent (e.g. streptavidin) has captured the affinity tag (e.g. biotin) and the affinity tag is preferably coupled to the complementary (second) strand of the nucleic acid precursor. Hence, separating the solid support from the single-stranded oligonucleotide also entails separating the (tagged) complementary strand from the single-stranded oligonucleotide.
Separating the solid support from the single-stranded oligonucleotide can be done using any conventional method known in the art and the method will be dependent on the type of solid support that is used. E.g. in case the solid support comprises small particles, these particles may be spun down and preferably the supernatant comprising the oligonucleotide may be transferred to another vial.
In case the solid support comprises magnetic or paramagnetic beads, the solid support may be removed by magnetic separation, e.g. by placing a magnet in close vicinity of the solid support. " (specification, p. 35, lines 23-40)
Accordingly, in the context of the disclosed invention, the discarding step is understood as having the same meaning as removing the solid support from a mixture comprising the solid support and nucleic acids, and encompasses magnetic separation.
Claim 1 recites "optionally a second strand that is complementary to the first strand." The term "optional" is interpreted according to its ordinary meaning as identifying a feature that is permitted but not required.
Claim 5 recites the term "about," which is defined in the specification as follows:
"As used herein, the term “about” is used to describe and account for small variations. For example, the term can refer to less than or equal to ±(+ or −) 10%, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%." (page 7, lines 15-19)
Accordingly, under BRI and in light of the specification, "about" is interpreted as +/- 10% of the reported numerical value.
Response to Arguments
Applicant's amendments and arguments filed on August 20, 2025 have been fully considered.
35 U.S.C. § 103 rejection
In the prior Office Action (Final Office Action - 05/21/2026):
Claims 1-4, 6, 8-10, 13-16, 18-19, 26 and 29 were rejected under 35 U.S.C. 103 as being unpatentable over Wang1, as evidenced by Eijk; Pound; Yuce; and Wilson;
Claim 5 was rejected under 35 U.S.C. 103 as being unpatentable over Wang1, as applied to claims 1 and 4 above and further in view of Wang2;
Claims 12 and 27 were rejected under 35 U.S.C. 103 as being unpatentable over Wang1, as applied to claim 1 above and further in view of Lobato.
These rejections are maintained in this Office Action for reasons below.
As an initial matter, claim 1 has been amended to recite "discarding the solid support." Applicant appears to argue that "discarding" requires actively disposing of the solid support (Remarks, p. 10, para 2). However, this is not consistent with the broadest reasonable interpretation of the claim in view of the specification.
The specification's description of separation of the solid support appears to be the closest support for the amendment (specification, p. 35, lines 23-40). This is consistent with Applicant's remarks citing para. [0287] as support for the amendment (Remarks, p. 6). Although the specification as filed does not include paragraph numbers, this citation appears to refer to para. [0287] of the published application (US20210164021A1), which describes obtaining single-stranded oligonucleotide after removing the solid support. The preceding para. [0286], which corresponds to p. 35, lines 38-40 in the instant specification, describes removing or separating the solid support by magnetic separation:
“[0286] In case the solid support comprises magnetic or paramagnetic beads, the solid support may be removed by magnetic separation, e.g. by placing a magnet in close vicinity of the solid support.”
Nowhere does the disclosure describe a separate step of actively disposing of, or throwing away the solid support after separation.
Accordingly, in light of the specification and under BRI, "discarding the solid support" is interpreted as encompassing separating or removing the solid support from a reaction mixture, including by magnetic separation. The term is not limited to active disposal of the solid support.
To the extend Applicant intends the term "discarding" to require a separate step of actively disposing of the solid support, it is noted that such feature is not supported by the disclosure because the specification only describes removal of solid support by magnetic separation, not active disposal.
First, Applicant argues that the cited references, in combination, do not teach or suggest the claimed combination of steps, specifically highlighting amended step (f), which recites "discarding the solid support."
This argument has been fully considered but is not persuasive. Under the current claim interpretation under BRI, and as detailed in the maintained rejection below, the combined teachings of the references fully teach and suggest the method of claim 1.
Specifically, "discarding the solid support" is interpreted as encompassing separating or removing the solid support from a reaction mixture, including by magnetic separation. This is taught by Wang1.
Wang1 teaches magnetic separation of tagged strand and untagged strands in FIG. 2 ([0135]). Specifically, Wang1 teaches separating the solid support from single-stranded oligonucleotides, thereby to obtain the pool of multiple single-stranded oligonucleotide having the sequence of interest ([0135] the MIPs (which are not biotinylated) are released from the streptavidin beads and recovered").
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Additionally, magnetic separation is well-known in the art, this is supported by Eijk (col 33-35, Example VI), Pound (Figure 1), Yuce (Scheme 1, Abstract), Kao (Fig. 1), and Wilson (FIG. 2), each teach the use of streptavidin beads as solid support to remove biotin-tagged antisense strand from solution, thereby isolating the target sense strand.
Example VI of Eijk teaches generating single-stranded oligonucleotides for further analysis by biotin-streptavidin magnetic bead separation (Eijk, col 34, lines 60-63), wherein a precursor comprising a biotin tag on the complementary strand is double restriction digested (Eijk, col 34, lines 50-67) and denatured by alkali or heat treatment to separate the untagged strand from the tagged complementary strand. Because the biotin-tagged strand is resistance to nuclease, only the untagged strand are released from the beads for downstream analysis (Eijk, col 35, lines 1-20).
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Pound (Figure 1) teaches magnetic separation of tagged and untagged strands, and collecting the untagged strand as the product of interest.
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Yuce (Scheme 1, Abstract) also teaches a method of single-stranded DNA production, using magnetic separation of tagged and untagged strands, and collecting the untagged strand as the product of interest.
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Kao (Fig. 1; p. 1070, left-hand col, para. 1) also teaches magnetic separation of biotin-tagged and untagged strands, and collecting the untagged strand as the product of interest.
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Wilson (FIG. 2, Abstract) teaches methods for the preparation of single-stranded DNA, based on streptavidin magnetic beads separation and alkaline denaturation with NaOH, which produces high yields of single-stranded DNA.
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Accordingly, the amended "discarding the solid support" is still obvious in view of the cited prior art references. The "thereby removing only the second strand, or only a part thereof comprising the reverse complement of the sequence of interest to obtain the pool of multiple single-stranded oligonucleotides having the sequence of interest" clause in the step is obvious in view of Wang1 because it merely states the intended result of the process taught and suggested by Wang1 and the knowledge in the art as discussed above. Specifically, in affinity separation, the tagged strand will be affinity-bound to the solid support and removed, whereas the untagged strand will remain in solution.
Second, Applicant argues that the rejection lacks motivation to combine (Remarks, page 10), specifically asserting that "[t]he Office Action has not articulated any motivation to reverse the purpose of the affinity tag from positive selection (retaining the tagged strand for further processing) as in the prior art to negative selection ( discarding the tagged strand)."
This argument is not persuasive. As discussed above, under BRI, "discarding the solid support" is interpreted as encompassing separating or removing the solid support from a reaction mixture. The term is not limited to an active disposal step, because there is no basis in the specification for such a narrow interpretation.
Further, the application's disclosure does not support the asserted distinction between negative selection and a method in which the tagged strand is retained. The specification describes removing or separating the solid support via magnetic separation (see para. [0286] of the published application US20210164021A1; same disclosure in specification, p. 35, lines 38-40), but does not further describe how the solid support, such as magnetic beads, must be treated after separation, including whether it is retained or thrown away.
USPTO's Updated Guidance for Making a Proper Determination of Obviousness (effective on February 27, 2024) provides the following guidance regarding providing a reason To modify the prior art:
"Consistent with KSR, the Federal Circuit makes it clear that the obviousness analysis is not ‘‘confined by a formalistic conception of the words teaching, suggestion, and motivation.’’ Intel Corp. v. Qualcomm Inc., 21 F.4th 784, 795 (Fed. Cir. 2021), quoting KSR, 550 U.S. at 419, 127 S. Ct. at 1741. To be sure, the Federal Circuit continues to use the word ‘‘motivation’’ in its obviousness jurisprudence. However, it is evident that the term is no longer understood in a rigid or formalistic way. See, for example, Norgren Inc. v. Int’l Trade Comm’n, 699 F.3d 1317, 1322 (Fed. Cir. 2012) (‘‘A flexible teaching, suggestion, or motivation test can be useful to prevent hindsight when determining whether a combination of elements known in the art would have been obvious.’’); Outdry Techs. Corp. v. Geox S.p.A., 859 F.3d 1364, 1370–71 (Fed. Cir. 2017) (‘‘Any motivation to combine references, whether articulated in the references themselves or supported by evidence of the knowledge of a skilled artisan, is sufficient to combine those references to arrive at the claimed process.’’)
In keeping with this flexible approach to providing a rationale for obviousness, the Federal Circuit has echoed KSR in identifying numerous possible sources that may, either implicitly or explicitly, provide reasons to combine or modify the prior art to determine that a claimed invention would have been obvious.
These include ‘‘market forces; design incentives; the ‘interrelated teachings of multiple patents’; ‘any need or problem known in the field of endeavor at the time of invention and addressed by the patent’; and the background knowledge, creativity, and common sense of the person of ordinary skill.’’ Plantronics, Inc. v. Aliph, Inc., 724 F.3d 1343, 1354 (Fed. Cir. 2013), quoting KSR, 550 U.S. at 418–21, 127 S. Ct. at 1741–42. Furthermore, the Federal Circuit has explained that a reason to optimize prior art parameters may be found in a PHOSITA’s desire to improve on the prior art. In re Ethicon, Inc., 844 F.3d 1344, 1351 (Fed. Cir. 2017)
…
When formulating an obviousness rejection, Office personnel may use any clearly articulated line of reasoning that would have allowed a PHOSITA to draw the conclusion that a claimed invention would have been obvious in view of the facts. MPEP 2143, subsection I, and MPEP 2144. Acknowledging that, in view of KSR, there are ‘‘many potential rationales that could make a modification or combination of prior art references obvious to a skilled artisan,’’ the Federal Circuit has also pointed to MPEP 2143, which provides several examples of rationales gleaned from KSR. Unwired Planet, 841 F.3d at 1003." [emphasis added]
In this instant case, the herein maintained rejections properly identify several rationales to modify the specific illustrated embodiment in Figure 2 of Wang1, supported by factual findings from the interrelated teachings within the same reference, and the general knowledge in the art. The rejections are consistent with the guidance in MPEP and USPTO's Updated Guidance and therefore establishes a proper ground of rejection under 35 U.S.C. 103.
The rationales stated in the rejection set forth in this Office Action are highlighted here for clarity of record:
Rationale 1: “Combining prior art elements according to known methods to yield predictable results,” see rationale (A) in MPEP 2143 I
“the use of affinity selectable reagents such as biotin-streptavidin magnetic beads to separate an untagged single-stranded DNA of interest from a biotin-tagged complementary strand, is well established in the art of single-stranded DNA synthesis.
Eijk (col 33-35, Example VI), Pound (Figure 1), Yuce (Scheme 1, Abstract), Kao (Fig. 1), and Wilson (FIG. 2) each teach the use of streptavidin beads as solid support to remove biotin-tagged antisense strand from solution, thereby isolating the target sense strand. For example, Example VI of Eijk teaches generating single-stranded oligonucleotides for further analysis by biotin-streptavidin magnetic bead separation (Eijk, col 34, lines 60-63), wherein a precursor comprising a biotin tag on the complementary strand is double restriction digested (Eijk, col 34, lines 50-67) and denatured by alkali or heat treatment to separate the untagged strand from the tagged complementary strand. Because the biotin-tagged strand is resistance to nuclease, only the untagged strand are released from the beads for downstream analysis (Eijk, col 35, lines 1-20).
Therefore, as evidenced by above, both approaches ꟷ amplicons in which both strands are tagged and amplicons in which only the complementary strand is tagged ꟷ were known in the art for the same purpose of isolating synthesized single-stranded DNA products, and both approaches are taught and suggested by Wang1.
Accordingly, it would have been apparent to a skilled artisan that modifying the FIG.2 embodiment such that only one amplification primer is tagged, thereby producing an amplicon having one tagged strand and one untagged strand, represents an obvious variation of the embodiments disclosed in Wang1 in view of established single-stranded DNA synthesis methods in the art. Wang1 already teaches amplification using one tagged primer and another primer lacking the same tag, as shown in FIG. 8. Given the known function of each element as explained by Wang1 and the general knowledge in the field, the combination of such elements represents an assemblage of known elements according to known methods that yields predictable results. “
Rationale 2: Optimization among obvious variations through routine experimentation with reasonably expectation of success, see rationale (F) in MPEP 2143 I
"The difference between FIG. 2 and 8 in relation to the claimed invention, lies in whether a single primer or both primers binding to the first and second strands of a nucleic acid molecule include the same affinity tag. It is well within the capability of a skilled artisan to use primers with or without affinity tags. Wang1 also teaches in paragraph [0137] that variations of the examples provided are included within the scope of its disclosure, thus implying that alternatives and substitutions are obvious to a skilled artisan. "
Rationale 3: Need or problem known in the field of endeavor, PHOSITA’s desire to improve on the prior art, suggestion in reference's teachings, See USPTO's updated guidance
“A person of ordinary skill in the art, motivated by the general desire to enhance the process of oligonucleotide probes synthesis, which will further enable multiplexed target-specific nucleic acid detection at larger scale, as suggested by Wang1([0003]), would have found it obvious to perform routine optimization and make such modification, because affinity-based separation of differently tagged strands in ssDNA synthesis, were well established.
Given that each element performs a known function as per its prior art teaching, their combination to achieve a predictable result would have been obvious, as per MPEP 2143.”
Regarding Applicant's assertion of unexpected results (Remarks, page 10), Applicant appears to rely on Table 2 of the specification, which shows a net fold increase in probe yield of 550 using RPA amplification and chemical denaturation using NaOH at an end concentration of 0.9 M (specification, Table 2; p. 43, lines 3-5). Applicant asserts that, by contrast, PCR amplification achieves only 5-fold amplification.
This assertion has been previously presented and found unpersuasive. Upon reconsideration, the assertion remains unpersuasive for reasons below.
Specifically, the working example disclosing the "net fold increase in probe yield of 550," relied upon by Applicant is not commensurate in scope with the claims, and is insufficient to support the asserted "increased probe yield" as an unexpected property compared to the closest prior art.
According to Applicant's own disclosure in example 2, which includes Table 2 as result. The use of both RPA amplification and chemical denaturation using NaOH at an end concentration of 0.9 M (specification, Table 2; p. 43, lines 3-5) leads to high probe yield. In contrast, the base claim 1 broadly recites "isothermal amplification method," and does not specifically require RPA. Claim 4 only recites using "chemical denaturing." Claim 5 recites using "alkali hydroxide at a concentration of about 0.5 - 1.5 M," but does not require specifically NaOH at an end concentration of 0.9 M.
Therefore, the claimed subject matter is much broader than what the disclosure supports for the "increased prob yield" property. In other words, the working example disclosed in Example 2 with results shown in Table 2, does not commensurate in scope with the breath of the claims.
Whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the “objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support.” In other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range. See In re Grasselli, 713 F.2d 731, 741, 218 USPQ 769, 777 (Fed. Cir. 1983) (Claims were directed to certain catalysts containing an alkali metal. Evidence presented to rebut an obviousness rejection compared catalysts containing sodium with the prior art. The court held this evidence insufficient to rebut the prima facie case because experiments limited to sodium were not commensurate in scope with the claims.).
Furthermore, even assuming that the claim has been amended with a narrower scope, with RPA and using NaOH at an end concentration of 0.9 M specifically for chemical denaturation; the result of example 2 still fails to demonstrate unexpected properties. The disclosure does not provide evidence to substantiate the assertion that the results are unexpected.
The question of unexpected result is whether the claimed invention possesses unexpected properties compared to the closest prior art. The evidence relied upon should establish “that the differences in results are in fact unexpected and unobvious and of both statistical and practical significance.” Ex parte Gelles, 22 USPQ2d 1318, 1319 (Bd. Pat. App. & Inter. 1992) (Mere conclusions in appellants’ brief that the claimed polymer had an unexpectedly increased impact strength “are not entitled to the weight of conclusions accompanying the evidence, either in the specification or in a declaration.”)
As discussed in the rejections, Wang1 already teaches methods for synthesizing pools of probes by isothermal amplification, comprising chemically denature amplified DNA products via increasing of pH ([0135] lines 7-10). While Wang1 does not teach the exact molar concentration of the specific alkaline solution used to increase the pH. Wang2 fills this gap by teaching chemically denaturing DNA by increasing the pH by the addition of sodium hydroxide(NaOH) at a concentration of 1M (Page 6, 1M NAOH shown 100% denaturation capability), in order to ensure complete denaturation.
Therefore, the prior art already teaches probes synthesis using isothermal amplification (not PCR) and NaOH denaturation at 1M, as required by the claims. While the specification concludes that its method using RPA and chemical denaturation with NaOH at an end concentration of 0.9 M results in higher probe yield compared to using PCR instead of RPA ([0330])); and that chemical denaturation is more efficient than heat denaturation, these comparisons are not the proper basis for determining unexpected results as they do not compare the disclosed method to the closest prior art.
Third, the asserted outcome ꟷ greater amplification yield from isothermal amplification methods compared to PCR ꟷ is itself expected in view of the prior art.
For instance, Chang (Chang et al. Diagnostic Devices for Isothermal Nucleic Acid Amplification. Sensors 2012, 12, 8319-8337. doi.org/10.3390/s120608319) explicitly teaches: "Overall, isothermal nucleic acid amplifications have greater amplification efficiency and produce higher DNA yields than PCR owing to their undisrupted and sustained enzyme activity." (page 8320, lines 13-14). Therefore, a higher amplification yield by isothermal amplification compared to PCR would have been reasonably expected by a person of ordinary skill in the art.
For the reasons stated above, Applicant's assertion regarding unexpected results is not persuasive.
Claim Rejections - 35 USC § 112(b) -- New
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.
Claims 5 and 30 are rejected under 35 U.S.C. 112(b), 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.
A) Regarding claim 5, it recites "the addition of an alkali hydroxide," which lacks antecedent basis as the claim does not previously recite any step of adding alkali hydroxide.
B) Regarding claim 30, it recites "wherein the amplified precursors consist of double-stranded deoxyribonucleic acid (DNA)," whereas claim 1, from which claim 30 depends, recites "amplified double-stranded deoxyribonucleic acid (DNA) precursors comprising an untagged first strand and a tagged complementary second strand."
It is therefore unclear whether the closed phrase "consist of double-stranded DNA" in claim 30, excludes the tag required by claim 1 for the amplified DNA precursors, or whether the tagged strand nevertheless falls within the scope of the amplified precursors consist of double-stranded DNA.
Accordingly, the metes and bounds of the claim cannot be determined with reasonable certainty.
For the purpose of compact prosecution and applying prior art under 35 USC§ 102 and 103, "the amplified precursors consist of double-stranded deoxyribonucleic acid (DNA)" is interpreted as excluding additional DNA strands other than the recited double-stranded DNA, such as triple-stranded DNA, or DNA origami structures that comprises more than two strands. However, the amplified precursors consist of double-stranded DNA may include tagged strands.
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.
Claims 1-4, 6, 8-10, 13-16, 18-19, 26 and 29-30 are rejected under 35 U.S.C. 103 as being unpatentable over Wang1 (US20130072390 A1-Methods for Synthesizing Pools of Probes; published March 21, 2013; cited as US Patent Documents #A15, on IDS filed 12/10/2020), as evidenced by
Eijk (US7166429B2- Method for generating oligonucleotides, in particular for the detection of amplified restriction fragments obtained using AFLP®; published 2007-01-23);
Pound (Pound et al., Polymerase chain reaction based scaffold preparation for the production of thin, branched DNA origami nanostructures of arbitrary sizes. Nano Lett. 2009 Dec;9(12):4302-5. doi: 10.1021/nl902535q. PMID: 19995086);
Yuce (Yuce et al., Characterization of a dual biotin tag for improved single stranded DNA production. Anal Methods. 2014;6:548–557);
Kao (Kao et al., An Efficient Bead-captured Denaturation Method for Preparing Long Single-stranded DNA. J Chin Chem Soc. 2017;64:1065–1070);
Wilson (Wilson R. Preparation of single-stranded DNA from PCR products with streptavidin magnetic beads. Nucleic Acid Ther. 2011 Dec;21(6):437-40. doi: 10.1089/nat.2011.0322. Epub 2011 Nov 2. Erratum in: Nucleic Acid Ther. 2012 Apr;22(2):137. PMID: 22047177).
The rejection of claims 1-4, 6, 8-10, 13-16, 18-19, 26 and 29 is maintained and updated in view of the amendments. Claim 30 is newly presented and is rejected over the same prior art.
A) Wang1 teaches a method for synthesizing pools of DNA probes by amplification for multiplex amplification and analysis of nucleic acid targets (entire document).
Regarding claim 1, Wang 1 teaches a method for producing a pool of multiple single-stranded oligonucleotides having a distinct sequence of interest (FIG. 2; FIG. 8, [0118], lines 11-14; [0133] lines 1-8), wherein the method comprises:
(a) providing a pool of multiple single- or double-stranded nucleic acid precursors each comprising a first strand (FIG. 2; FIG. 8, [0118], lines 11-14),
wherein the first strand comprises the following elements in a 5' to 3' direction:
(i) a first primer binding site (FIG. 2, region 102a; [0098]);
(ii) a first endonuclease recognition site (FIG. 2, region 107a; [0098]);
(iii) the sequence of interest (FIG. 2 ; FIG. 8, 103a,105a, 109, 105b,103b;[0090]);
(iv) a second endonuclease recognition site (FIG.2, 107b; [0098]); and
(v) a second primer binding site (FIG. 2, region 102b);
wherein the first endonuclease recognition site is designed such that, after duplexing, a first endonuclease cleaves the sugar-phosphate backbone of the first strand immediately upstream of the sequence of interest (FIG. 2, 151, 107a; [0098], lines 9-11); and,
wherein the second endonuclease recognition site is designed such that, after duplexing, a second endonuclease cleaves the sugar-phosphate backbone of the first strand immediately downstream of the sequence of interest (FIG. 2, 151, 107b; [0098], lines 9-11);
(b) amplifying the pool of multiple precursors of (a) by an isothermal amplification method (FIG 2, 150; [0098]; ([0041], line 11, isothermal amplification method, for instance, SDA), using a first primer (FIG 2, 111a) capable of hybridizing to the first primer binding site and a second primer (FIG 2, 111b) capable of hybridizing to the second primer binding site,
wherein the second primer comprises an affinity-tag (FIG 8, 111a comprises an affinity tag, 111b does not; [0118], lines 7-8) that is not present on the first primer, to produce amplified double-stranded deoxyribonucleic acid (DNA) precursors (FIG 8, 850) comprising an untagged first strand and a tagged complementary second strand ([0118]lines8-9,24-25; Figure 8, 113; [0086]);
(c) digesting the amplified double-stranded DNA precursor obtained in (b) with the first and the second endonuclease (FIG 2, 152 ; [0098], lines 13-21) to produce amplified double-stranded nucleic acid precursors with cleavages of the sugar-phosphate backbone immediately up- and downstream of the sequence of interest and with an intact sugar-phosphate backbone between the tag up to and including the sequence complementary to the sequence of interest (FIG 2, 121 ; [0098], lines 13-21);
(d) immobilizing the amplified double-stranded nucleic acid precursor on a solid support by affinity capture of the tagged complementary second strand ([0135] lines1-5);
(e) denaturing the amplified double-stranded precursor, thereby releasing the single-stranded oligonucleotide having the sequence of interest ([0135] lines 12-15); and
(f) discarding the solid support thereby removing only the second strand, or only a part thereof comprising the reverse complement of the sequence of interest to obtain the pool of multiple single-stranded oligonucleotide having the sequence of interest (Fig. 2; [0135] the MIPs (which are not biotinylated) are released from the streptavidin beads and recovered").
Claim 1 has been amended to recite in step (f): "discarding the solid support," which is interpreted as encompassing separating or removing the solid support from a reaction mixture, including by magnetic separation. This is taught by Wang1.
Wang1 teaches magnetic separation of tagged strand and untagged strands in FIG. 2 ([0135]).
Specifically, Wang1 teaches separating the solid support from single-stranded oligonucleotides, thereby obtaining a pool of multiple single-stranded oligonucleotide having the sequence of interest ([0135] the MIPs (which are not biotinylated) are released from the streptavidin beads and recovered").
Regarding the limitation "amplified double-stranded deoxyribonucleic acid (DNA) precursors " in part (b), it is taught by Wang1 as it is clear that the amplification products disclosed in both FIGs 2 and 8 are DNA products. Wang1 is directed to the preparation of DNA probes via amplification, as stated in the abstract. Accordingly, the amplification products from which the DNA probes are derived are DNA. Therefore, a person of ordinary skill in the art would understand that Wang 1 discloses DNA amplification products, which meets the recited limitation.
Although Wang1 does not explicitly disclose all claimed limitations in a single embodiment, a person of ordinary skill in the art, in view of the teachings of Wang1 and the knowledge in the art, would have found the claimed method to be an obvious variation of known methods.
FIG. 2 of Wang1 teaches most of the claimed limitations. FIG. 2 teaches amplification using two primers (FIG. 2, 111a and 111b; [0091]), to produce an amplified double-stranded nucleic acid precursor (114).
The primary difference is that FIG. 2 teaches amplification using two tagged primers, thereby producing an amplicon in which both strands are tagged, whereas the claim requires that one primer comprises a tag and the other primer does not comprise the same tag, thereby producing a DNA product comprising one tagged strand and one untagged strand. However, this feature is taught In FIG. 8 of Wang1.
Specifically, in Wang1's FIG. 8 and para. [0118] teach a first strand nucleic acid (FIG. 8, 800) amplified by using a first primer (111b) and a tagged second primer (111a), to produce an amplified double-stranded nucleic acid precursor (FIG. 8, 814) comprising an untagged first strand (FIG. 8, bottom sequence is the same sequence as the starter first strand) and a tagged complementary second strand (FIG. 8, top sequence comprising tag).
Further, the use of affinity selectable reagents such as biotin-streptavidin magnetic beads to separate an untagged single-stranded DNA of interest from a biotin-tagged complementary strand, is well established in the art of single-stranded DNA synthesis.
Eijk (col 33-35, Example VI), Pound (Figure 1), Yuce (Scheme 1, Abstract), Kao (Fig. 1), and Wilson (FIG. 2) each teach the use of streptavidin beads as solid support to remove biotin-tagged antisense strand from solution, thereby isolating the target sense strand. For example, Example VI of Eijk teaches generating single-stranded oligonucleotides for further analysis by biotin-streptavidin magnetic bead separation (Eijk, col 34, lines 60-63), wherein a precursor comprising a biotin tag on the complementary strand is double restriction digested (Eijk, col 34, lines 50-67) and denatured by alkali or heat treatment to separate the untagged strand from the tagged complementary strand. Because the biotin-tagged strand is resistance to nuclease, only the untagged strand are released from the beads for downstream analysis (Eijk, col 35, lines 1-20).
Therefore, as evidenced by above, both approaches ꟷ amplicons in which both strands are tagged and amplicons in which only the complementary strand is tagged ꟷ were known in the art for the same purpose of isolating synthesized single-stranded DNA products, and both approaches are taught and suggested by Wang1.
Accordingly, it would have been apparent to a skilled artisan that modifying the FIG.2 embodiment such that only one amplification primer is tagged, thereby producing an amplicon having one tagged strand and one untagged strand, represents an obvious variation of the embodiments disclosed in Wang1 in view of established single-stranded DNA synthesis methods in the art. Wang1 already teaches amplification using one tagged primer and another primer lacking the same tag, as shown in FIG. 8. Given the known function of each element as explained by Wang1 and the general knowledge in the field, the combination of such elements represents an assemblage of known elements according to known methods that yields predictable results. The combination of these elements in the manner claimed does not impart any new or unexpected results beyond the teaching of Wang1.
The person having ordinary skill in the art would have had a reasonable expectation of success in making such modification because the detailed teaching in Wang1 provide a strong technical foundation for their successful integration. The difference between FIG. 2 and 8 in relation to the claimed invention, lies in whether a single primer or both primers binding to the first and second strands of a nucleic acid molecule include the same affinity tag. It is well within the capability of a skilled artisan to use primers with or without affinity tags. Wang1 also teaches in paragraph [0137] that variations of the examples provided are included within the scope of its disclosure, thus implying that alternatives and substitutions are obvious to a skilled artisan.
A person of ordinary skill in the art, motivated by the general desire to enhance the process of oligonucleotide probes synthesis, which will further enable multiplexed target-specific nucleic acid detection at larger scale, as suggested by Wang1 ([0003]), would have found it obvious to perform routine optimization and make such modification, because affinity-based separation of differently tagged strands in ssDNA synthesis, were well established.
Given that each element performs a known function as per its prior art teaching, their combination to achieve a predictable result would have been obvious, as per MPEP 2143.
The "thereby removing only the second strand, or only a part thereof comprising the reverse complement of the sequence of interest to obtain the pool of multiple single-stranded oligonucleotides having the sequence of interest" clause in the step (f) is obvious in view of Wang1 because it merely states the intended result of the process taught and suggested by Wang1 and the knowledge in the art as discussed above. Specifically, as shown in each of the cited references, in affinity separation, the tagged strand will be affinity-bound to the solid support and removed from the untagged strand that remain in solution.
B) Regarding claim 2, Wang1 teaches steps (d) and (e) are reversed, meaning the denaturation takes place prior to immobilizing the nucleic acid amplification products on a solid support (page 11, lines3-5).
Regarding claim 3, Wang1 teaches purifying the single-stranded oligonucleotide (page 16, right col, lines 1-2).
Regarding claim 4, Wang1 teaches denaturing comprises chemical denaturing ([0135] lines 7-10).
Regarding claim 6, Wang 1 teaches the nucleic acid precursor consists of 20 - 200 nucleotides ([0133], lines 2-3, "The length of the oligos was between 110 and 121 bases in length').
Regarding claim 8, Wang 1 teaches the sequence of interest is at least partly complementary to a predetermined genomic sequence ([0133] lines 1-8. "where the genomic homology regions are represented by N20 and vary between MIPs depending on the target."; [0101]).
Regarding claim 9, Wang 1 teaches the produced oligonucleotide is suitable for use in a multiplex oligonucleotide based amplification assay ([0005] lines 1-3).
Regarding claim 10, Wang 1 teaches the nucleic acid precursors are single-stranded (FIG 2; FIG 8).
Regarding claim 13, Wang1 teaches the first and the second endonuclease are two different enzymes (FIG 2. 152).
Regarding claim 14, Wang1 teaches the first endonuclease in (c) cleaves the first DNA strand (FIG 2. 152; [0118], lines 11-14).
Regarding claim 15, Wang1 teaches the amplified double-stranded precursor is purified prior to binding the solid support ([0133] lines 19-21).
Regarding claim 16, Wang1 teaches the tag is biotin and the solid support comprises streptavidin ([0135] lines 1-4).
Regarding claim 18, Wang1 teaches the first primer can selectively anneal only to the first primer binding site and a second primer can selectively anneal to only the second primer binding site ([0133] lines 11-15, the PCR primers have different sequences for binding selectively to distinct sites).
Regarding claim 19, Wang1 teaches the sequence of interest does not comprise the first and the second endonuclease recognition sites or reverse complement thereof (FIG 2. 151, 152; [0098] line 18. "…This cuts the top strand into three pieces…" indicates the sequence of interest does not comprise any endonuclease recognition sites or reverse complement thereof).
Regarding claim 26, Wang1 teaches performing providing step (a), amplifying step (b), digesting step (c), immobilizing step (d), denaturing step (e), and removing step (f) sequentially (FIG. 2; [0098]; [0135]).
Regarding claim 29, Wang1 teaches wherein in (a) the pool of multiple single- or double-stranded nucleic acid precursors comprises at least 3000 unique sequences ([0120]).
Regarding claim 30, Wang1 teaches wherein the amplified precursors consist of double-stranded deoxyribonucleic acid (DNA) (FIG.2, see double-stranded DNA amplicon).
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Wang1, as applied to claims 1 and 4 above and further in view of Wang2 (Wang et al. Characterization of denaturation and renaturation of DNA for DNA hybridization. Environ Health Toxicol. (2014) doi: 10.5620/eht.2014.29.e2014007. PMID: 25234413; PMCID: PMC4168728.)
The teachings of the Wang1 are recited above, applied as for base claims 1 and 4 and incorporated here.
Wang1 teaches a method for synthesizing pools of probes by amplification for multiplex amplification and analysis of nucleic acid targets (entire document).
Regarding claim 5, Wang1 teaches chemically denature the amplified DNA products via increasing of pH ([0135] lines 7-10). Although Wang1 does not teach the exact molar concentration of the specific alkaline solution used to increase the pH, this feature is obvious in view of Wang2.
Wang2 teaches the characterization of the denaturation of DNA using alkaline solutions of varied concentrations (introduction). Wang2 teaches chemically denaturing DNA by increasing the pH by the addition of sodium hydroxide(NaOH) at a concentration of 1M (Page 6, 1M NAOH shown 100% denaturation capability). Wang2 also suggests (page 6, right-hand col, para 2.): "To ensure and leverage the complete denaturation in the follow-up study for gDNA, the 1 mol/L NaOH can be selected as an effective chemical denaturation method."
Accordingly, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method for synthesizing pools of probes by amplification with chemical denaturation via increasing pH in Wang1 with the teachings of the use of 1M NaOH for DNA denaturation disclosed by Wang2 because both references are in the overlapping field of molecular biology and DNA denaturation techniques. A person skilled in the art would commonly encounter and consider teachings of denaturation in Wang2, when working on nucleic acid synthesis and analysis of Wang1, as effective denaturation is integral to the method taught by Wang1. The person of ordinary skill would have had a reasonable expectation of success in apply the specific NaOH concentration taught by Wang2 in the method taught by Wang1 because of Wang2's clear teaching that the use of 1M NaOH shows high denaturation capability, and doing so would yield the predictable result of effective denaturation of double stranded DNA. The skilled artisan would have been motivated to do so because using 1M NaOH as an effective chemical denaturation method would ensure complete DNA denaturation, as suggested by Wang2.
Claims 12 and 27 are rejected under 35 U.S.C. 103 as being unpatentable over Wang1, as applied to claim 1 above and further in view of Lobato (Lobato et al. Recombinase polymerase amplification: Basics, applications and recent advances. Trends Analyt Chem. (Epub 2017 Oct 26) PMID: 32287544; PMCID: PMC7112910.)
The teachings of the Wang1 are recited above, applied as for base claim 1 and incorporated here.
Regarding claims 12 and 27, Wang1 teaches the use of isothermal amplification method, for instance, SDA ([0041], line 11). Although Wang1 does not specifically teach other isothermal amplification methods such as Recombinase Polymerase Amplification (RPA), this feature would have been obvious in view of Lobato.
Lobato provides an overview for Recombinase polymerase amplification and its applications (entire document).
Regarding claims 12 and 27, Lobato teaches the isothermal amplification methods including RPA and SDA (entire document). Lobato specifically teaches the use of both RPA and SDA in DNA amplification applications (page 19, introduction), and when comparing RPA and SDA, RPA does not require initial heating and takes less time to amplify (page 20, Table 1).
Lobato further suggests specific advantages of RPA (page 19, introduction):
"RPA is remarkable due to its simplicity, high sensitivity, selectivity, compatibility with multiplexing, extremely rapid amplification, as well as its operation at a low and constant temperature, without the need for an initial denaturation step or the use of multiple primers. Overall, RPA positions itself very favourably for widespread exploitation in kits and assays for use at the point of-care or point-of-need, as well as in affordable, sensitive, specific, user friendly, rapid, robust, equipment-free and delivered (ASSURED) devices, in low-resource settings."
Accordingly, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the isothermal amplification method taught by Wang1 with the teachings of the RPA isothermal amplification disclosed by Lobato because both references are in the same field of nucleic acid amplification and analysis. The references are related in their use of isothermal amplification methods, a common technique known in the art per the applicant's specification (page 27,line 26-27), thus a skilled artisan would have likely encountered and considered both references in the context of nucleic acid amplification. The person of ordinary skill would have had a reasonable expectation of success in applying RPA in the method taught in Wang1 because Lobato provides detailed information of the application of RPA and insights into the advantages of RPA over SDA. Doing so would have yielded the predictable result of increased operational simplicity and time efficiency. The skilled artisan would have been motivated to use RPA as suggested by Lobato because of several advantages such as not requiring initial heating and faster amplification, which are beneficial in the context of nucleic acid assays such as the probe synthesis methods taught by Wang1.
Prior Art
Other prior art also teach single stranded DNA generation from biotin-tagged amplification products using affinity separation followed by alkali denaturation:
Marimuthu et al., Single-stranded DNA (ssDNA) production in DNA aptamer generation. Analyst. 2012 Mar 21;137(6):1307-15. doi: 10.1039/c2an15905h. Epub 2012 Feb 7. PMID: 22314701. (see Fig. 4; page 1310-1311)
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Wakimoto et al. (2014), Isolation of Single-Stranded DNA. Current Protocols in Molecular Biology, 107: 2.15.1-2.15.9. doi.org/10.1002/0471142727.mb0215s107 (see Figure 1)
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“Asymmetric PCR, enzyme digestion, and magnetic separation with streptavidin beads are the most commonly used methods to isolate ssDNA from dsDNA.” (p. 2.15.7, Background Information., para 2)
“ Streptavidin has an extraordinary binding affinity (K d of 10−15 M) for biotin (Green, 1975). This high-affinity interaction between streptavidin and biotin is resistant to extremes of temperature, pH, organic solvents, and other denaturing agents. Immobilization of biotinylated samples using streptavidin-coated magnetic beads as a solid support is a well-established procedure (Hultman et al., 1989, 1991; Pourmand et al., 2002). Immobilized biotinylated dsDNA bound to the beads is denatured to release the desired non-biotinylated ssDNA strand while the biotinylated strand remains bound to the beads (Fig. 1B).
Alkaline and thermal treatments of dsDNA are the two most common methods of denaturation. Both methods have been reported to release small amounts of streptavidin and biotinylated DNA from the magnetic beads (Avci-Adali et al., 2009; Maricic and Pääbo, 2009; Paul et al., 2009). However, a direct comparison between alkaline and thermal methods confirmed that the alkaline denaturation generates higher yields of ssDNA with the least amount of contamination (Wilson, 2011).” (p. 2.15.7- p. 2.15.8, Background Information, para 3-4)
Conclusion
Claims 7, 16 and 30 are objected to; claims 1-6, 8-10, 12-16, 18-19, 26-27 and 29-30 are rejected. No claims are allowed.
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/TIAN NMN YU/Examiner , Art Unit 1681
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