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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 April 07, 2025 has been entered.
Status of Claims / Response to Amendment
This office action is in response to an amendment filed on April 07, 2025.
Claims 98 and 100-116 were previously pending. Applicant amended claim 101.
Claims 98 and 100-116 are currently pending, with claims 103-107 and 113-114 withdrawn.
Claims 98, 100-102, 108-112, and 115-116 are under consideration.
Applicant's claim amendments overcame the following rejections:
Rejections of claims 101-102, 108-109, 111, and 115 under 35 U.S.C. 112(b).
All other previously presented rejections are maintained for reasons given in the "Response to Arguments" below. 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 ACTION IS MADE FINAL even though it is a first action after the filing of a request for continued examination and the submission under 37 CFR 1.114. See MPEP § 706.07(b).
Applicant amended dependent claim 101 with "wherein the primer strand portion comprises a terminal end at a 3' end of the synthesis strand," to provide antecedent basis to the later recited "the terminal end of the primer strand portion" in part (4) of the claim. The scope of the claim remains unchanged.
MPEP 706.07(b) States:
"For an application in which an RCE has been filed, claims may be finally rejected in the first action following the filing of the RCE (with a submission and fee under 37 CFR 1.114 ) when all the claims in the application after the entry of the submission under 37 CFR 1.114 and any entered supplemental amendments (A) are identical to, patentably indistinct from, or have unity of invention with the claims in the application prior to the entry of the submission under 37 CFR 1.114 (in other words, restriction (including lack of unity of invention) would not have been proper if the new or amended claims had been entered prior to the filing of the RCE), and (B) would have been properly finally rejected on the grounds and art of record in the next Office action if they had been entered in the application prior to the filing of the RCE under 37 CFR 1.114. Note that applicants cannot use an RCE to obtain continued examination on the basis of claims that are independent and distinct from, or lack unity of invention with, the claims previously claimed and examined as a matter of right (i.e., applicant cannot switch inventions). See 37 CFR 1.145 and MPEP § 706.07(h), subsection VI. Therefore, condition (A) is always met where the RCE is accompanied by a submission that will be entered as a matter of right." [emphasis added]
In this instant case, the present application includes an RCE filling, and the claims could have been rejected on the existing grounds with the previously presented prior art of record if they had been entered before the RCE was filed. This is evidenced by the maintained art rejections for all the claims under examination. Accordingly, the issuance of this first-action final rejection is proper and complies with the guidelines set forth in MPEP 706.07.
Response to Arguments
Applicant's arguments filed on April 07, 2025 have been fully considered.
Claim Interpretation
The following interpretations regarding terms "predefined sequence" and "universal nucleotide" are made In the prior Office Action (Final Rejection - 01/06/2025, page 3-5):
"B) For the purpose of applying prior art, claim 98 recites "predefined sequence," which is a term not defined nor clearly described in the applicant's disclosure. Thus a "predefined sequence" is interpreted under BRI to mean any sequence.
C) For the purpose of applying prior art, claim 98 recites "universal nucleotide," which is defined by the applicant's disclosure on page 86:
"A universal nucleotide is one wherein the nucleobase will bond, e.g. hydrogen bond, to some degree to the nucleobase of any nucleotide of the predefined sequence. A universal nucleotide is preferably one which will bond, e.g. hydrogen bond, to some degree to nucleotides comprising the nucleosides adenosine (A), thymine (T), uracil (U), guanine (G) and cytosine (C). The universal nucleotide may bond more strongly to some nucleotides than to others."
As any nucleotide can form hydrogen bond to any other nucleotide to some degree. For the purpose of applying prior art, "universal nucleotide" is interpreted under BRI, consistent with the specification, as any nucleotide."
Applicant contends that the above interpretations are improper, in view of the following arguments:
"Regarding (B), Applicant asserts that given the plain meaning of "predefined" as "being defined in advance" (i.e., in advance of the claimed method steps, but not yet synthesized), one of ordinary skill in the art at the time of filing would understand that a "predefined" sequence, as recited in the instant claims, is any sequence that one would desire to synthesize prior to performing the method, and that a polynucleotide molecule having that sequence is created using the claimed method.
This interpretation is further supported by the teachings provided in the instant application.
For example, the instant application describes several issues faced in popular synthesis methods of the time, including a requirement of either a maximum length less than approximately 100 nucleotides1 or, for longer sequences, "a DNA template from which a copy is made." The instant application provides solutions to these problems, describing methods that allow for synthesis of polynucleotide molecules of any desired length "de nova in a stepwise manner without the need to copy a pre-existing template molecule". Thus, a person of ordinary skill in the art would understand that the solutions provided described in the instant application relate to synthesis of desired sequences, e.g., sequences for which conventional methods require a template molecule. In contrast, the interpretation of "any sequence" proposed by the Examiner would encompass undesired sequences, such as random sequences, which a person of ordinary skill in the art would understand could be synthesized by other means (e.g., methods not having the same problems to be solved). Accordingly, under broadest reasonable interpretation, the term "pre-defined sequence" does not refer to "any sequence" per se, but rather, "any sequence defined in advance."
Regarding (C), Applicant asserts that the Examiner's interpretation of "universal nucleotide" as "any nucleotide" is inconsistent with the definition provided in the instant application (e.g., at) and the plain meaning of the term. Based on the description in the specification and the examples provided therein, one of ordinary skill in the art would understand that a "universal nucleotide" refers to a nucleotide that bonds to each of the natural DNA/RNA bases (i.e., nucleotides comprising the nucleosides adenosine (A), thymine (T), uracil (U), guanine (G) and cytosine (C)) with little discrimination between them, such as inosine. Indeed, at the time of filing of the instant application, the term "universal base" was already understood by those of ordinary skill in the art to refer to "a class of compounds which pair with all natural bases without discrimination.," e.g., as described by Liang, F., et al. "Universal base analogues and their applications in DNA sequencing technology." RSC advances 3.5 (2013): 14910-14928.
Accordingly, under broadest reasonable interpretation, the term "universal nucleotide", a term of art, refers to "a nucleobase that will bond to some degree to the nucleobase of any nucleotide", as defined in the instant application and as understood by those of ordinary skill in the art." [emphasis added](Remarks, page 23-25)
These arguments have been fully considered but are not found persuasive.
i) Regarding the term "predefined sequence," applicant argues that this term should be interpreted as a sequence "being defined in advance," and further states that a "predefined" sequence is "any sequence that one would desire to synthesize." Applicant further asserts that "undesired sequences," as understood by a person of ordinary skill in the art are not encompassed by "predefined sequence."
This argument is not persuasive and raises additional issues.
In evaluating the patentability of the claims, claim terms are 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. MPEP§ 2111.04 states: "Claim scope is not limited by claim language that suggests or makes optional but does not require steps to be performed, or by claim language that does not limit a claim to a particular structure."
Here, the term "predefined sequence" is not defined in the applicant's disclosure. And the specification does not identify any structural characteristics of "predefined sequence, " that could distinguish a "predefined sequence" from any other sequence in the prior art. Therefore, this term is properly interpreted under BRI as any sequence.
While Applicant's effort to elaborate their position is appreciated, the argument still fails to identify any structural characteristics that could distinguish the claimed "predefined sequence" from any sequence taught in the prior art. Additionally, Applicant's interpretation raises further issues of indefiniteness.
First, the proposed definitions ꟷ "being defined in advance," and "any sequence that one would desire to synthesize" ꟷ are subjective and lack clear metes and bounds. It is unclear who defines the sequence or based on what criteria a sequence is considered "desired." This ambiguity adds confusion to applicant's interpretation.
Second, the definitions provided in the argument appear inconsistent in scope. For example, the phrase "defined in advance" could encompass both sequences that are desired and those that are not, because a person of ordinary skill in the art who identifies a desired sequence would inherently define the other sequences as undesired; whereas "any sequence that one would desire to synthesize" implies a narrower scope. This inconsistency introduces further uncertainty.
ii) Regarding the term "universal nucleotide," Applicant asserts "under broadest reasonable interpretation, the term "universal nucleotide", a term of art, refers to "a nucleobase that will bond to some degree to the nucleobase of any nucleotide."
However, this is consistent with the Examiner's interpretation, which aligns with the definition provided by Applicant in the specification at page 86.
As any nucleotide can form hydrogen bond to any other nucleotide to some degree 1. Accordingly, "universal nucleotide" is interpreted under BRI, consistent with the specification, as any nucleotide. "
To further clarify, the examiner's interpretation is properly made for the following reasons.
MPEP 2111.01 provides guidance on claim interpretation:
"Under a broadest reasonable interpretation (BRI), words of the claim must be given their plain meaning, unless such meaning is inconsistent with the specification. … the best source for determining the meaning of a claim term is the specification - the greatest clarity is obtained when the specification serves as a glossary for the claim terms. Phillips v. AWH Corp., 415 F.3d 1303, 1315, 75 USPQ2d 1321, 1327 (Fed. Cir. 2005) (en banc) ("[T]he specification ‘is always highly relevant to the claim construction analysis. Usually, it is dispositive; it is the single best guide to the meaning of a disputed term.’"
(quoting Vitronics Corp. v. Conceptronic Inc., 90 F.3d 1576, 1582 (Fed. Cir. 1996))."; and
"The only exceptions to giving the words in a claim their ordinary and customary meaning in the art are (1) when the applicant acts as their own lexicographer; and (2) when the applicant disavows or disclaims the full scope of a claim term in the specification. "
In this instant case, Applicant's disclosure sets forth a special definition of the claim term "universal nucleotide" in the specification that differs from the plain and ordinary meaning it would otherwise possess.
Specifically, the specification defines "universal nucleotide" as "a nucleobase [that] will bond, e.g. hydrogen bond, to some degree to the nucleobase of any nucleotide" and that "[t]he universal nucleotide may bond more strongly to some nucleotides than to others"
(specification, page 86).
Applicant has acted as their own lexicographer by providing this broad definition, which deviates from the customary meaning in the art.
In contrast, Feng 2(referenced in Applicant's remarks, page 25, lines 5-6) defines a "universal base" as: "a “universal” base that pairs with each natural nucleobase without discrimination and results in duplexes with similar stability as the corresponding duplexes composed of all natural nucleobases. " (page 14910, right-hand col, lines 7-11)
Thus, Feng teaches a universal base that bonds equally across nucleobases.
Accordingly, the definition of a "universal nucleotide" in the instant application is distinct from the definition of a "universal base" in Feng. The application's specification defines a universal nucleotide as one that binds "to some degree," and explicitly states that the bonding may vary across nucleobases, which introduces discrimination. This contrasts with the nondiscriminatory binding described in the prior art, as evidenced by Feng.
"Some degree" is not equivalent to "same degree," and therefore, the two definitions do not align.
Therefore, the broadest reasonable interpretation of the term "universal nucleotide" is made in accordance with Applicant's own definition as provided in the specification. The claim interpretation in this Office Action is proper and is maintained.
Applicant could further define the term by, for example, reciting in the claim a Markush group consist of specific universal bases (see specification, page 87 for examples) to further distinguish the claimed "universal nucleotide" from the nucleotides in the prior art.
Claim Rejections - 35 USC § 103
In the prior Office Action (Final Office Action- 01/06/2025):
Claims 98, 100-102, 108-112, and 115-116 are rejected under 35 U.S.C. 103 as being unpatentable over ARLOW, as evidenced by Bentley and Chen.
These rejections are maintained in this Office Action for reasons below.
Applicant argues that the rejections above should be withdrawn (Remarks, page 26-29). Applicant's arguments have been fully considered but are not found persuasive.
i) First, Applicant argues that ARLOW does not teach "universal nucleotide" (Remarks, page 26-28), and defines the term "universal nucleotide" by the function of "indiscriminately pair with natural nucleotides" (Remarks, page 27, line 6).
This is not persuasive. As discussed above, the Examiner has applied the proper interpretation of "universal nucleotide" under BRI and in light of the specification. Applicant's own definition in the specification describes a universal nucleotide as a nucleobase that will bond to some degree to the nucleobase of any nucleotide (specification at page 86), which supports the interpretation of "any nucleotide," as any nucleotide may exhibit differential binding by forming hydrogen bond to any other nucleotide to some degree, and is not required to bind indiscriminately.
ARLOW teaches this limitation by teaching a dU base that facilities cleavage (Figure 10; [0050] “… cleaves the backbone at the dU base”), meeting the requirement for universal nucleotide. Therefore, the limitation is met by the cited art.
Applicant's argument is not persuasive because it is based on an interpretation that is narrower than what Applicant has defined in the specification. Applicant's remarks do not disavows or disclaims the full scope of the claim term as defined in the specification, and thus fail to resolve the inconsistency between the broader definition in the application and the narrower interpretation to which the argument relies upon.
ii) Second, Applicant contends that neither Bentley nor Chen teach, suggest, or motivate the Examiner's proposed modifications of ARLOW (i.e. the use of a reversible terminator group in the DNA synthesis method of ARLOW to prevent further extension of the polymerase enzyme and improve fidelity of the synthesized DNA, see Final Rejection - 01/06/2025, page 8-12), based on the rationale that the references differ in their use of a template strand. Specifically, Applicant asserts that Bentley and Chen are only described for use in methods of DNA sequencing-by-synthesis using a template DNA strand, whereas ARLOW describes a DNA synthesis method performed in the absence of a template DNA strand. (Remarks, page 28-29) 3
This above argument is not persuasive for two reasons.
First, the factual basis of Applicant's argument is incorrect. Contrary to Applicant's assertion, all three references ꟷ including ARLOW ꟷ teach templated synthesis. Specifically, Figure 10 of ARLOW shows a filling step ("B. Fill-in" )in which DNA polymerase extends a strand using the complementary bottom strand as a template (See also [0050]; [0009]), confirming that ARLOW also involves templated synthesis.
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Second, even assuming, for the sake of argument, that ARLOW does not disclose templated DNA synthesis, this specific argument is still not relevant to the rationale supporting the legal conclusion of obviousness in the previously set forth rejection (Final Office Action - 01/06/2025, page 7-19). The requirements for a proper response to a rejection may be found in 37 CFR 1.111(b) and MPEP §714.02; see also MPEP §707.07(a). The remarks do not provide any specific reasons as to why either the findings of fact or the legal conclusion of obviousness in the previously set forth rejection is allegedly in error.
Applicant appears to argue that a reference must teach both reversible terminators and template-free synthesis in order to suggest the incorporation of reversible terminators
In the method of ARLOW. However, the rejection focuses on the teaching and suggestion of using reversible terminators in DNA synthesis generally. There is no requirement in the rejection or the cited art that the use of reversible terminators must be limited to template-free systems.
Therefore, Applicant's argument relies on an unsupported assumption and does not address the actual reasoning provided in the rejection (see detailed rejection maintained in section "Claim Rejections - 35 USC § 103" below).
In conclusion, Applicant's arguments have been fully considered but are not sufficient to overcome the previously set forth rejections. Consequently, the rejection of claims 98, 100-102, 108-112, and 115-116 are rejected under 35 U.S.C. 103 as being unpatentable over ARLOW, as evidenced by Bentley and Chen is maintained in this Office Action.
Priority
The priority date of the instant claims 98, 100-102, 108-112, and 115-116 is 07/19/2018, filling date of the UNITED KINGDOM Patent Application Number 1811810.9, to which the present application claims priority.
Claim Interpretation -- Maintained
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.
A) For the purpose of applying prior art, regarding all claims, the term "cycle" is recited in the claims, but not defined in the applicant's disclosure. Under BRI, this term is interpreted to mean "a recurring series of steps."
B) For the purpose of applying prior art, claim 98 recites "predefined sequence," which is a term not defined nor clearly described in the applicant's disclosure. Thus a "predefined sequence" is interpreted under BRI to mean any sequence.
C) For the purpose of applying prior art, claim 98 recites "universal nucleotide," which is defined by the applicant's disclosure on page 86:
"A universal nucleotide is one wherein the nucleobase will bond, e.g. hydrogen bond, to some degree to the nucleobase of any nucleotide of the predefined sequence. A universal nucleotide is preferably one which will bond, e.g. hydrogen bond, to some degree to nucleotides comprising the nucleosides adenosine (A), thymine (T), uracil (U), guanine (G) and cytosine (C). The universal nucleotide may bond more strongly to some nucleotides than to others."
As any nucleotide can form hydrogen bond to any other nucleotide to some degree. For the purpose of applying prior art, "universal nucleotide" is interpreted under BRI, consistent with the specification, as any nucleotide.
D) For the purpose of applying prior art, claim 101 recites "blunt-ended ligation reaction," which is not defined in the applicant's disclosure. Page 134 of the specification provides the following description:
"In method versions 1 and 2 the terminal end of the scaffold polynucleotide comprising the primer strand portion comprises a blunt end, i.e. with no overhanging nucleotides in either strand."
Thus, in light of the specification and under BRI, the term "blunt-ended ligation reaction" is interpreted as a ligation reaction of double stranded DNAs that contain no nucleotide overhang on the ligation end of each strand.
E) For the purpose of applying prior art, claim 101 recites a "primer strand portion," which is not defined nor clearly described in the applicant's disclosure. Thus this term "primer strand portion" is interpreted under BRI to comprise any portion of sequence on the synthesis strand.
F) Claim 100 recites "partner nucleotide" and “nucleotide pair,” which are related terms that are not defined in the applicant's disclosure. Page 20 of the specification provides that a "partner nucleotide" may be one that pairs with the first nucleotide and is potentially complementary:
"In any of the methods described above and herein, in any one, more or all cycles of synthesis a partner nucleotide which pairs with the first nucleotide of the predefined sequence may be a nucleotide which is complementary with the first nucleotide, preferably naturally complementary."
Page 138 of the specification indicates that the nucleotide pairs can consist of nucleotides chosen by the user to be either complementary or non-complementary:
"In method versions 1 and 2 described herein, given that first and second nucleotides of each cycle form a nucleotide pair with respective partner nucleotides, if the first and second nucleotides of each cycle is chosen by the user to be naturally complementary to their respective partner nucleotides of each cycle, then the final synthesised strands will be perfectly complementary. If first and second nucleotides of certain cycles are chosen by the user to be non-complementary to their respective partner nucleotides of those cycles, then the final synthesised strands will not be perfectly complementary. Nevertheless, in either situation the final synthesised strands both comprise sequence which in the context of the synthesised double-stranded polynucleotide as a whole is predefined."
Therefore, in light of the specification and under BRI, the broadest reasonable interpretation of "partner nucleotide" encompass any nucleotide that pairs with another, which does not necessarily need to be complementary. Similarly, a "nucleotide pair" can be any two nucleotides added to the polynucleotide on opposite strands, within the same synthesis cycle, regardless of their complementarity.
Claim Rejections - 35 USC § 103 -- Maintained
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 98, 100-102, 108-112, and 115-116 are rejected under 35 U.S.C. 103 as being unpatentable over ARLOW (US20170218416A1 - Compositions and methods for single-molecule construction of dna; Published August 03 2017; cited as U.S. Patent Document on IDS filed 06/26/24), as evidenced by Bentley (Bentley et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456, 53–59 (2008). doi.org/10.1038/nature07517) and Chen (Chen et al. The history and advances of reversible terminators used in new generations of sequencing technology. Genomics Proteomics Bioinformatics. 2013 Feb;11(1):34-40. doi: 10.1016/j.gpb.2013.01.003. Epub 2013 Jan 23. PMID: 23414612; PMCID: PMC4357665).
A) ARLOW teaches a method for DNA polynucleotide synthesis with repeated cycles involving DNA ligation, nucleotide extension, and strand-specific DNA cleavage (entire document).
Regarding claim 98, ARLOW teaches an in vitro method of synthesising a double-stranded polynucleotide having a predefined sequence, the method comprising performing cycles of synthesis (entire document, Figure 10 for example) wherein in each cycle:
(A) a first strand of a double-stranded polynucleotide (Figure 10, - strand) is extended by incorporation of a first nucleotide of the predefined sequence and a universal nucleotide by the action of a ligase enzyme (Figure 10), wherein the universal nucleotide defines a cleavage site(Figure 10, dU base);
(B) the double-stranded polynucleotide is then cleaved at the cleavage site(Figure 10E); and
(C) a second strand of the double-stranded polynucleotide which is hybridized to the first strand (Figure 10, + strand) is then extended by incorporation of a second nucleotide of the predefined sequence by a nucleotide transferase or polymerase enzyme(Figure 10B; [0050]); and wherein the first and second nucleotides of the predefined sequence of each cycle are retained in the double-stranded polynucleotide following cleavage (Figure 10, the second nucleotide filled in takes place after cleavage and is retained); and wherein in a given cycle of synthesis the second nucleotide of that cycle which is added to the second strand of the double-stranded polynucleotide comprises a reversible terminator group which prevents further extension by the nucleotide transferase or polymerase enzyme ([0144], lines1-9), and wherein the reversible terminator group is removed from the incorporated second nucleotide of that cycle prior to the addition in the next cycle of synthesis of the second nucleotide of the next cycle([0144], lines1-9).
While ARLOW does not explicitly teach all the limitations and elements of claim 98 are arranged or combined in the same way as the claim, it suggests such arrangement as evidenced by Bentley and Chen.
Although the embodiment of Figure 10 does not explicitly disclose using and removing reversible terminator group, para. 142 and 144 of ARLOW teach using photo-reversible terminator nucleotide which prevents extension by polymerase enzyme to improve fidelity and correctness of the polynucleotides synthesized ([0142]; [0144]lines1-9). 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 apply the teaching of reversible terminator group in DNA synthesis to the method of Figure 10 (step B, fill in with single nucleotide), both taught by ARLOW to incorporate the use of a photo-reversible terminator nucleotide as the second nucleotide in its template-dependent extension step, wherein the reversible terminator group is removed from the incorporated second nucleotide of that cycle prior to the addition in the next cycle.
Because given the known function of each element as explained by ARLOW 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 (See MPEP §2143), such as enhanced fidelity and control of polynucleotide synthesis.
In KSR Int'l v. Teleflex Inc., 550 U.S. 398, 415 (2007), the Supreme Court rejected a rigid application of a teaching-suggestion-motivation test in the obviousness determination. The Court emphasized that “[a]s our precedents make clear . . . the [obviousness] analysis need not seek out precise teachings directed to the specific subject matter of the challenged claim, for a court can take account of the inferences and creative steps that a person of ordinary skill in the art would employ.” Id. at 418. Thus, “[t]he combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” Id. at 416. “If a person of ordinary skill can implement a predictable variation, § 103 likely bars its patentability.” Id. at 417. Moreover, it is proper to “take account of the inferences and creative steps that a person of ordinary skill in the art would employ.” Id. at 418; see also id. at 421 (“A person of ordinary skill is also a person of ordinary creativity, not an automaton.”).
“[I]n considering the disclosure of a reference, it is proper to take into account not only specific teachings of the reference but also the inferences which one skilled in the art would reasonably be expected to draw therefrom.” In re Preda, 401 F.2d 825, 826 (CCPA 1968).
According to ARLOW, the use of nucleotides with photo-reversible terminators represents a known approach to enhancing accuracy, and it would have been an obvious, logical step for a skilled artisan with ordinary creativity to apply this technical approach in the single nucleotide extension step in the method of Fig 10 (step B) to achieve similar benefits.
Additionally, it would have been obvious to a skilled artisan to perform removing the reversible terminator group from the incorporated nucleotide prior to the next synthesis cycle to enable operability of subsequence cycle. As could be readily understood by a person of ordinary skill in the art, without removal of the terminator group, the next nucleotide could not have be added, making the removal of the reversible terminator group a logical and necessary step in ARLOW’s teaching of a cyclic DNA synthesis method.
Bentley further supports this specific combination of teachings by explicitly teaching the use of nucleotides comprising reversible terminator group in DNA synthesis cycles (entire document, page 53, right-hand col, para 1 for example), where the terminator group is removed after incorporation to allow the next nucleotide addition (page 53, right-hand col, para 1, “We added tris(2-carboxyethyl)phosphine (TCEP) to remove the fluorescent dye and side arm from a linker attached to the base and simultaneously regenerate a hydroxyl group ready for the next cycle of nucleotide addition”). Bentley explains that the use of nucleotides comprising reversible terminator group in DNA synthesis cycles ensures complete and accurate nucleotide incorporation while preventing over-incorporation (page 53, right-hand col, para 1, lines 6-10). Therefore, Bentley provides additional motivation and evidence that the use of nucleotides comprising reversible terminator group in DNA synthesis cycles and the removal of the terminator group between cycles is a known practice in the art for achieving accuracy and control in DNA synthesis methods.
Chen is a review article that provides a comprehensive overview of reversible terminators in sequencing, such as sequencing-by-synthesis (entire document). Similar to Bentley, Chen discloses the wide use of reversible terminator nucleotides in DNA sequencing-by-synthesis cycles, where the terminator group is removed at the end of each cycle (Figure 3). Thus further demonstrating the use of nucleotides with reversible terminator groups in DNA synthesis cycles is a well-established, routine, and conventional approach within the field of molecular biology.
Therefore, it would have been obvious to modify the method of ARLOW to incorporate the claimed limitations, including the use of a second nucleotide with a reversible terminator group to prevent further extension and the removal of the terminator group prior to the next synthesis cycle. This represents the predictable application of known elements to achieve a predictable result, making such combination obvious. The combination of these elements in the manner claimed does not impart any new or unexpected results beyond the teaching of ARLOW, and would have led to the predictable result of improved fidelity and accuracy in synthesizing the predefined DNA sequence.
The person having ordinary skill in the art would have had a reasonable expectation of success in combining the elements taught by ARLOW into a single method because the detailed teaching in ARLOW provide a strong technical foundation for their successful integration. A person of ordinary skill in the art, motivated by the desire to enhance the process of DNA synthesis, would have found it obvious to combine these separate teachings of ARLOW in the manner claimed.
B) Regarding claim 100, ARLOW teaches in each cycle the first nucleotide is a partner nucleotide for the second nucleotide, and wherein upon incorporation into the double- stranded polynucleotide the first and second nucleotides form a nucleotide pair (Figure 10).
Regarding claim 101, ARLOW teaches :
(1) providing a scaffold polynucleotide (Figure 10 A) comprising a synthesis strand (Figure 10 A, - strand) and a support strand (Figure 10 A, + strand)hybridized thereto, wherein the synthesis strand comprises a primer strand portion, and wherein the support strand is the first strand of the double-stranded polynucleotide and the synthesis strand is the second strand of the double-stranded polynucleotide (Figure 10 A);
(2) ligating a double-stranded polynucleotide ligation molecule to the scaffold polynucleotide by the action of the ligase enzyme in a blunt-ended ligation reaction (Figure 10;[0050] ), the polynucleotide ligation molecule (Figure 10, extension unit) comprising a support strand (Figure 10, extension unit, bottom strand) and a helper strand (Figure 10, extension unit, top strand) hybridised thereto and further comprising a complementary ligation end, the ligation end comprising:
(i) in the support strand a universal nucleotide and a first nucleotide of the predefined sequence (Figure 10, extension unit, bottom strand with 1nt payload and dU); and
(ii) in the helper strand a non-ligatable terminal nucleotide(Figure 10, extension unit, top strand, 5’ OH ); wherein upon ligation the first strand of the double-stranded polynucleotide is extended with the first nucleotide and the cleavage site is created by the incorporation of the universal nucleotide into the first strand(Figure 10);
(3) cleaving the ligated scaffold polynucleotide at the cleavage site (Figure 10, E), wherein cleavage comprises cleaving the support strand and removing the universal nucleotide from the scaffold polynucleotide to provide a cleaved double-stranded scaffold polynucleotide comprising the incorporated first nucleotide (Figure 10);
(4) extending the terminal end of the primer strand portion of the synthesis strand of the double- stranded scaffold polynucleotide by incorporation of a second nucleotide of the predefined sequence by the action of the nucleotide transferase or polymerase enzyme (Figure 10, F-B) , the second nucleotide comprising a reversible terminator group which prevents further extension by the nucleotide transferase or polymerase enzyme ([0144], lines1-9), wherein the second nucleotide is a partner for first nucleotide and wherein upon incorporation the second nucleotide and the first nucleotide form a nucleotide pair (Figure 10) , and
(5) removing the reversible terminator group from the second nucleotide([0144], lines1-9), ;
the method further comprising performing a further cycle of synthesis comprising (Figure 10;[0050] ):
(6) ligating a further double-stranded polynucleotide ligation molecule to the cleaved scaffold polynucleotide by the action of the ligase enzyme in a blunt-ended ligation reaction, the polynucleotide ligation molecule comprising a support strand and a helper strand hybridised thereto and further comprising a complementary ligation end (Figure 10;[0050]), the ligation end comprising:
(i) in the support strand a universal nucleotide and the first nucleotide of the further cycle of synthesis; and
(ii) in the helper strand a non-ligatable terminal nucleotide; wherein upon ligation the first strand of the double-stranded polynucleotide is extended with the first nucleotide of the further cycle of synthesis and the cleavage site is created by the incorporation of the universal nucleotide into the first strand (Figure 10;[0050]) ;
(7) cleaving the ligated scaffold polynucleotide at the cleavage site, wherein cleavage comprises cleaving the support strand and removing the universal nucleotide from the scaffold polynucleotide to provide a cleaved double-stranded scaffold polynucleotide comprising the nucleotide pair(s) from step (4) and the first nucleotide of the further cycle of synthesis (Figure 10, polynucleotide synthesis is cyclic, wherein in each cycle the previously extended bases are retained in cleavage step E ;[0050]);
(8) extending the terminal end of the primer strand portion of the synthesis strand of the double- stranded scaffold polynucleotide by the incorporation of the second nucleotide of the further cycle of synthesis by the action of the nucleotide transferase or polymerase enzyme (Figure 10;[0050]), the second nucleotide comprising a reversible terminator group which prevents further extension by the nucleotide transferase or polymerase enzyme([0144], lines1-9), wherein the second nucleotide of the further cycle of synthesis is a partner for the first nucleotide of the further cycle of synthesis, and wherein upon incorporation the second and first nucleotides of the further cycle of synthesis form a further nucleotide pair(Figure 10;[0050]);
(9) removing the reversible terminator group from the second nucleotide ([0144], lines1-9); and
(10) repeating steps 6 to 9 multiple times to provide the double-stranded polynucleotide having a predefined nucleotide sequence (Figure 10;[0050]).
Regarding claim 102, ARLOW teaches in the ligation step of the first cycle (step 2) and in ligation steps of all further cycles the complementary ligation end of the polynucleotide ligation molecule is structured such that:
i. the first nucleotide of the predefined sequence of that cycle is the terminal nucleotide of the support strand, occupies nucleotide position n in the support strand and is paired with the terminal nucleotide of the helper strand (Figure 10, 1nt payload);
ii. the universal nucleotide is the penultimate nucleotide of the support strand, occupies nucleotide position n+1 in the support strand and is paired with the penultimate nucleotide of the helper strand(Figure 10, dU base);
and iii. the terminal nucleotide of the helper strand is a non-ligatable nucleotide (Figure 10, HO 5’); wherein position n is the nucleotide position which is opposite the second nucleotide of the predefined sequence of that cycle upon its incorporation (Figure 10), and wherein position n+1 is the next nucleotide position in the support strand relative to position n in the direction distal to the complementary ligation end (Figure 10; [0050], next 1bp extension); and wherein upon ligation the terminal nucleotide of the support strand of the polynucleotide ligation molecule is ligated to the terminal nucleotide of the scaffold polynucleotide proximal to the primer strand portion of the synthesis strand and a single-strand break is created between the terminal nucleotides of the helper strand and the primer strand portion of the synthesis strand (Figure 10; [0050]) ;
(b) in the cleavage step of the first cycle (step 3) and in all further cycles the support strand of the ligated scaffold polynucleotide is cleaved between positions n+1 and n, thereby releasing the polynucleotide ligation molecule from the scaffold polynucleotide and retaining the first nucleotide of that cycle attached to the first strand of the cleaved scaffold polynucleotide (Figure 10; [0050]) ; and
(c) in the extension step of the first cycle (step 4) and in all further cycles the second nucleotide of that cycle is incorporated into the second strand opposite the first nucleotide in the first strand and is paired therewith (Figure 10; [0050]); and whereupon the position occupied by the first nucleotide of that cycle in the support strand of the cleaved scaffold polynucleotide is defined as nucleotide position n-1 in the next cycle of synthesis (Figure 10; [0050]).
Regarding claim 108, ARLOW teaches the polynucleotide ligation molecule is provided with a complementary ligation end comprising a first nucleotide of the predefined sequence of the first cycle and further comprising one or more further nucleotides of the predefined sequence of the first cycle (Figure 3, nucleotides added to – strand via ligation; [0068]);
following cleavage the first and one or more further nucleotides of the predefined sequence of the first cycle are retained in the cleaved scaffold polynucleotide(Figure 3; [0068]);
the terminal end of the primer strand portion of the synthesis strand of the double-stranded scaffold polynucleotide is extended by the incorporation of a second nucleotide of the predefined sequence of the first cycle by the action of the nucleotide transferase or polymerase enzyme, and wherein the terminal end of the primer strand portion is further extended by the incorporation of one or more further nucleotides of the predefined sequence of the first cycle by the action of the nucleotide transferase or polymerase enzyme (Figure 3; [0068]), wherein each one of the second and further nucleotides of the first cycle comprises a reversible terminator group which prevents further extension by the enzyme, and wherein following each further extension the reversible terminator group is removed from a nucleotide before the incorporation of the next nucleotide([0144], lines1-9);
the polynucleotide ligation molecule is provided with a complementary ligation end comprising a first nucleotide of the predefined sequence of the further cycle and further comprising one or more further nucleotides of the predefined sequence of the further cycle (Figure 3; [0068]);
following cleavage the first and further nucleotides of the predefined sequence of the further cycle are retained in the cleaved scaffold polynucleotide(Figure 3; [0068]);
the terminal end of the primer strand portion of the synthesis strand of the double-stranded scaffold polynucleotide is extended by the incorporation of a second nucleotide of the predefined sequence of the further cycle by the action of the nucleotide transferase or polymerase enzyme (Figure 3; [0068], blunting polymerase), and wherein the terminal end of the primer strand portion is further extended by the incorporation of one or more further nucleotides of the predefined sequence of the further cycle by the action of the nucleotide transferase or polymerase enzyme(Figure 3; [0068], blunting polymerase), wherein each one of the second and further nucleotides of the further cycle comprises a reversible terminator group which prevents further extension by the enzyme, and wherein following each further extension the reversible terminator group is removed from a nucleotide before the incorporation of the next nucleotide (Figure 3; [0068]).
Regarding claim 109, ARLOW teaches: the complementary ligation end of the polynucleotide ligation molecule is structured such that in steps (3) and (7) prior to cleavage the universal nucleotide occupies a position in the support strand which is the next nucleotide position in the support strand after the nucleotide positions of the first and further nucleotides in the direction distal to the complementary ligation end, and the support strand is cleaved between the position occupied by the last further nucleotide and the position occupied by the universal nucleotide (Figure 3, the cleavage site is distal to ligation junction, the universal nucleotide is at the 6th position to the right of ligation junction).
Regarding claim 110, ARLOW teaches in any one, more or all cycles of synthesis a partner nucleotide which pairs with the first nucleotide of the predefined sequence is a nucleotide which is complementary with the first nucleotide (Figure 10).
Regarding claim 111, ARLOW teaches (a) in any one, more or all cycles of synthesis, prior to step (3) and/or (7) the scaffold polynucleotide is provided comprising a synthesis strand and a support strand hybridized thereto, and wherein the synthesis strand is provided without a helper strand (Figure 3A).
Regarding claim 112, ARLOW teaches each cleavage step comprises a two step cleavage process wherein each cleavage step comprises a first step comprising removing the universal nucleotide thus forming an abasic site, and a second step comprising cleaving the support strand at the abasic site ([0135]) .
Regarding claim 115, ARLOW teaches synthesis strand comprising the primer strand portion and the portion of the support strand hybridized thereto are tethered to a common surface (Figure 3, the DNA strands are attached to a common surface).
Regarding claim 116, ARLOW teaches synthesis cycles are performed in droplets within a microfluidic system ([0123]; figure 4; [0126]).
Prior Art
Other prior art also teach using universal nucleotide (e.g., inosine) in facilitating DNA cleavage: US20090181860A1; US20150292007A1 ; US20140309119A1; US20110281736A1.
Conclusion
No claims are allowed.
All claims are identical to or patentably indistinct from, or have unity of invention with claims in the application prior to the entry of the submission under 37 CFR 1.114 (that is, restriction (including a lack of unity of invention) would not be proper) and all claims could have been finally rejected on the grounds and art of record in the next Office action if they had been entered in the application prior to entry under 37 CFR 1.114. Accordingly, THIS ACTION IS MADE FINAL even though it is a first action after the filing of a request for continued examination and the submission under 37 CFR 1.114. See MPEP § 706.07(b). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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