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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 December 10, 2025 has been entered.
Status of Claims / Response to Amendment
This office action is in response to an amendment filed on December 10, 2025.
Claims 1-10, 12-16, 18-19 and 26-28 were previously pending. Applicant amended claim 1; claim 29 is newly added.
Claims 1-10, 12-16, 18-19 and 26-29 are currently pending, with claim 28 withdrawn.
Claims 1-10, 12-16, 18-19, 26-27 and 29 are under consideration.
All of the previously presented rejections have been withdrawn as being addressed or obviated by the amendment of the claims.
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.
Response to Arguments
Applicant's arguments filed on December 10, 2025 have been fully considered.
Rejections under 35 U.S.C. §112(b)
In the prior Office Action (Final Office Action - 09/11/2025):
Claims 1-10, 12-16, 18-19 and 26-27 were rejected in independent claim 1, under 35 U.S.C. 112(b), on the basis that "it is unclear whether each individual precursor in the group is required to comprise both an untagged first strand and a tagged second strand, or whether the limitation is satisfied so long as one tagged and one untagged strand are present in the group collectively" (Final Office Action - 09/11/2025, page 15-16).
This rejection has been withdrawn in view of Applicant's amendments and arguments.
Specifically, Applicant argues that this rejection under 35 U.S.C. 112(b) should be withdrawn in view of amendments to independent claim 1, and provided clarification on indefiniteness issue raised by the office (Remarks, page 6):
"For the sole purpose of expediting prosecution, Applicant has amended claim 1 to recite" providing a pool of multiple single- or double-stranded nucleic acid precursors, each precursor comprising a first strand .... " Since the first strand is amplified in step (b) of claim 1 by using "a first primer capable of hybridizing to the first primer binding site and a second primer capable of hybridizing to the second primer binding site, wherein the second primer comprises an affinity-tag that is not present on the first primer," it follows that after amplification, each precursor in the pool thus comprises an untagged first strand and a tagged second strand, thereby resolving the issue." [emphasis added]
Thus, Applicant's remarks provides clarification on the record, stating that each of "amplified double-stranded deoxynucleic acid (DNA) precursors" in part (b) of claim 1 is required to comprise an untagged first strand and a tagged complementary second strand. This is found sufficient to resolve the indefiniteness issue. Accordingly, the rejection has been withdrawn.
35 U.S.C. § 103 rejections
In the prior Office Action (Final Office Action - 09/11/2025):
Claims 1-4, 6, 8-10, 13-16, 18-19 and 26 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);
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.);
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 above 103 rejections have been withdrawn as being obviated by the recent claim amendment filed on December 10, 2025, which added new limitations to the claims (namely, further specifying the amplified double-stranded nucleic acid precursors being DNA in claim 1, part b), that were not considered in the previous rejections.
This Office Action contains new grounds of rejections necessitated by Applicant's amendments. Although the claims were previously rejected as being unpatentable over the same reference(s), Applicant's amendments have necessitated the inclusion of new grounds of rejections in this Office action. It is noted that, to the extent that they apply to the present rejection; Applicant's arguments are addressed below.
With regard to obviousness, Applicant's arguments have been fully considered but are not found persuasive.
First, Applicant argues that Wang1 does not teach "produce amplified double-stranded deoxynucleic acid (DNA) precursors" because Fig 8 in [0118] teaches primers including U bases (Remarks, pages 7-8).
This argument is not persuasive. Applicant's argument reflects a misreading of Wang1's teachings by equating the "U bases" disclosed in para. [0118] with ribonucleotides. On the contrary, in para. [0118] the "U bases" actually refer to DNA bases, specifically deoxyuridine bases that serve as substrates for Uracil-DNA Glycosylase (UDG).
Para. [0118] in Wang1 discloses the following:
"The product of the amplification step 850 is a double stranded precursor 814 that has cleavage site 801 comprising 1 or more U bases in the top strand and cleavage site 803 comprising a type IIs recognition site. …
In step 853 the product 818 is cleaved at cleavage site 801 using UDG treatment followed by cleavage so that the 5′ end of the MIP is generated at location 820. The product of step 853 is the full length MIP 100."
Therefore, Wang1 teaches UDG is active at cleavage site 801 comprising U bases.
UDG is active on both single stranded and double-stranded DNA and catalyzes the hydrolysis of deoxyuridine in DNA 1. Thus, in view of the teachings in Wang1 and the well-established understanding of UDG activity and substrate specificity, a person of ordinary skill in the art would recognize that the "U bases" disclosed in Wang1 are deoxyuridine incorporated into DNA.
Additionally, it is clear that the amplification products shown in both FIGs 2 and 8 of Wang1 are DNA products, as the cleaving enzymes employed in these embodiments, such as UDG, BspQI, and BtsI require DNA substrates (see New England Biolabs, www. neb.com).
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Second, Applicant insists that the rejection lacks motivation to combine (Remarks, page 8-10). However, as set forth in the prior office action (Final Office Action - 09/11/2025, pages 9-11; 17-23) , multiple rationales were expressly provided to support a prima facie case of obviousness. The rejection therefore does not lack motivation or rationale to combine. Rather, Applicant disagrees with the examiner's stated reasons for combining the teachings of the references.
Applicant's disagreement has been considered but is not persuasive. Combining two embodiments disclosed in the same prior art patent is "predictable variation” that does not “require a leap of inventiveness. See Boston Sci. Scimed (Fed. Cir. 01/15/09).
The prior art does not teach away. Obviousness does not require that the motivation be the best option, only that it be a suitable option from which the prior art did not teach away. The examiner is not persuaded of error in the rejection, and maintains that the rationales for combining and modifying the prior art teachings are properly made.
Third, Applicant argues unexpected results, asserting the following:
"In this regard, claims 4-5 should be considered independently allowable because the present Application teaches that chemical denaturation improved the probe yield as explained at paragraph [0330] of the published specification.
The cited references cannot establish the required motivation and guidance for a person of ordinary skill in the art to arrive that the present claims with a reasonable expectation of success in view of such unexpected results." (Remarks, page 10).
This argument has been fully considered but is not persuasive.
MPEP provides the following guidance regarding unexpected results:
The question of unexpected result is whether the claimed invention possesses unexpected properties compare to the closest prior art. Where the unexpected properties of a claimed invention are not shown to have a significance equal to or greater than the expected properties, the evidence of unexpected properties may not be sufficient to rebut the evidence of obviousness. MPEP 716.02 (b)
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. MPEP 716.02 (d)
Applicant's specification as filed does not include paragraph labels; however, paragraph [0330] of the pre-grant publication # US20210164021A1 of the present application states:
“[0330] Initial experiments on probe amplification of a multiplex of 9 probe precursors using a method comprising PCR amplification, amplicon nicking, purification of the nicked amplicons by acrylamide-gel separation, and subsequent heat-denaturation to release of the probes, did not result in a satisfying probe yield. This problem was overcome using biotin-bead purification instead of acrylamide-gel separation, in combination with chemical denaturation instead of heat denaturation. However, increasing the multiplex level to 3912 probes again resulted in low yield and hetero-duplex formation (see Example 1). These problems were overcome by using an isothermal amplification method instead of PCR, together with using biotin-bead for amplicon purification and chemical denaturation for probe release. This amplification method resulting in high yield without hetero-duplex formation is described in detail in Examples 2 and 3.”
Here. The application's disclosure in para.[0330] of the pre-grant publication, and in the referenced examples 2 and 3 have been thoroughly reviewed in accordance with the MPEP's guidance. However, these disclosures do not commensurate with the scope of the claims, and are 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 para.[0330] and example 2. The use of both RPA amplification and chemical denaturation using NaOH at an end concentration of 0.9 M ([0343]) 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 examples disclosed in Examples 2-3, as referenced in para.[0330], do 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 results of examples 2 and 3 still fail 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 compare 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 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.
Priority
The priority date of the instant claims 1-10, 12-16, 18-29 and 26-27 and 29 is June 12, 2018, filling date of the European Patent Application Number 18177178.3, to which the present application claims priority.
Claim Objections
Claim 1 is objected to because of the following informalities:
· In claim 1, lines 24-25,
"(c) digesting the amplified double-stranded precursor obtained in (b) the first and the second endonuclease" should read "(c) digesting the amplified double-stranded DNA precursors obtained in (b), using the first and the second endonuclease."
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 29 is 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.
Regarding claim 29, it recites "wherein the pool of multiple single-stranded
oligonucleotides comprises at least 3000 unique sequences."
This claim language is indefinite, because it is unclear whether this wherein clause is intended to modify any of the steps in the claimed method, and if so, which steps and how.
The wherein clause describes "the pool of multiple single-stranded oligonucleotides," which is an end product of the process recited in base claim 1. However, this end product is not required to be used in any of the steps, and none of the method steps require or reference the feature "at least 3000 unique sequences." None of the materials involved in the claimed method steps are described as comprising unique sequences.
Thus, it is unclear where the "at least 3000 unique sequences" in "the pool of multiple single-stranded Oligonucleotides" comes from, whether they are required to be added and during which step, or whether they are inherent results of performing the claimed method.
Per MPEP 2111.04, a wherein clause can limit a method claim if it contributes meaning and purpose to the manipulative steps. In the instant claim, however, the clause merely describes the intended outcome of the overall process, but it is unclear whether it makes any manipulative difference.
For the purpose of compact prosecution and applying prior art under 35 USC§ 102 and 103, this wherein clause interpreted as descriptive statements without any associated active steps and do not further limit the scope.
Claim Rejections - 35 USC § 103 -- New
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 following are new grounds of rejections necessitated by Applicant's amendments. Although the claims were previously rejected as being unpatentable over the same reference(s), Applicant's amendments have necessitated the inclusion of new grounds of rejections in this Office action. It is noted that, to the extent that they apply to the present rejection; Applicant's arguments are addressed in the "Response to Arguments" section above.
Claims 1-4, 6, 8-10, 13-16, 18-19, 26 and 29 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).
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 deoxynucleic 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 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) removing the solid support to obtain the pool of multiple single-stranded oligonucleotide having the sequence of interest ([0135] lines 15-16).
Claim 1 has been amended to recite "amplified double-stranded deoxynucleic acid (DNA) precursors," which is narrower in scope than the previously recited "amplified double-stranded nucleic acid precursors."
Nevertheless, this limitation is still taught by Wang1. In view of the teachings of Wang1, 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 teach all the limitations and elements of claim 1, Wang1 do not explicitly teach they are arranged or combined in the same way as the claim.
Specifically, in Wang1's FIG. 8 (reproduced below) 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).
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Similarly, FIG. 2 (reproduced below) of Wang1 also teach a first strand nucleic acid (FIG. 2, 101), amplified by two primers (FIG. 2, 111a and 111b; [0091]), to produce an amplified double-stranded nucleic acid precursor (114).
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While FIG. 2 of Wang1 does not explicitly illustrate a first primer does not comprise an affinity tag comprised by the second primer, Wang1 makes it clear that biotin is merely an example of hapten, and numerous variations, changes, and substitutions, presented as alternatives to the embodiments are within the scope of its teaching:
"Biotin is an exemplary hapten but other haptens are available and may be used." (para.[0098])
"While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. All cited references, including patent and non-patent literature, are incorporated herein by reference in their entireties for all purposes and particularly to disclose and describe the methods or materials in connection with which the publications are cited." (para. [0137])
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 combine the elements of claim 1 as taught in separate sections by Wang1, because 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.
While FIG. 2 of Wang1 does not explicitly illustrate a first primer does not comprise an affinity tag comprised by the second primer, and instead shows both primers with a biotin tag, Wang1 makes it clear that biotin is merely an example of hapten, and other haptens could be used. In the teaching of FIG. 2 with both primers having the same biotin tag, it would have been obvious to a skilled artisan that one can apply the same concept with a primer comprising a biotin tag and another comprising a different hepten, thereby producing a biotin-tagged strand and a un-tagged strand in the amplification product. 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 combining the elements taught by Wang1 into a single method 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. Therefore, it would have been obvious to a skilled artisan, based on Wang1's teaching, that FIG. 2 could also include one primer with a biotin tag and another primer without, resulting in an amplified double-stranded nucleic acid precursor with one tagged and one untagged strand. This is further supported by the explicit teaching of the same limitation in FIG. 8, which provides another example of the same invention in Wang1. Since FIG. 8 and FIG. 2 are both embodiments of the same invention, they represent different variations of the same overall technique. Therefore, the teaching in FIG. 8 reinforces the objective fact that Wang1 encompasses different combinations and variations of tagged and untagged primers, a skilled artisan, while performing routine optimization, would have found it obvious to apply these variations to achieve the claimed limitation, with reasonable expectation of success.
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 combine these separate teachings of Wang1 in the manner claimed.
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.
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, it is obvious in view of the teachings in Wang1 because it does not further limit the claimed method under the current claim interpretation. See "Claim Rejections - 35 USC § 112(b)" for detailed claim interpretation.
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.)
A) 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). However, Wang1 does not teach the exact molar concentration of the specific alkaline solution used to increase the pH.
B) 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."
C) All limitations in claim 5 are taught by the combination of Wang1 and Wang2.
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 combine the method for synthesizing pools of probes by amplification with chemical denaturation via increasing pH of 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.)
A) 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). However, Wang1 does not specifically teach other isothermal amplification methods such as Recombinase Polymerase Amplification (RPA) or Helicase Dependent Amplification (HDA).
B) 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."
C) All limitations in claims 12 and 27 are taught by the combination of Wang1 and Lobato.
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 combine the isothermal amplification method taught by Wang1 with the teachings of the isothermal amplification method RPA 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 generating single stranded oligonucleotides using amplification primer comprising tags, solid support immobilization, cleaving enzymes, and alkali denaturation:
US7166429B2 - Method for generating oligonucleotides, in particular for the detection of amplified restriction fragments obtained using AFLP®
Subject Matter Not Taught/Suggested in Prior Art
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.
Conclusion
Claims 1 and 7 are objected to; claims 1-6, 8-10, 12-16, 18-19, 26-27 and 29 are rejected. No claims are allowed.
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/TIAN NMN YU/Examiner , Art Unit 1681 /AARON A PRIEST/Primary Examiner, Art Unit 1681
1 see New England Biolabs, www. neb.com/en-us/products/m0280-uracil-dna-glycosylase-udg