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 .
Status of Claims / Response to Amendment
This office action is in response to an amendment filed on June 22, 2026.
Claims 98 and 100-116 were previously pending. Applicant amended claims 98, 102, 108, 111 and 114-115; cancelled claims 100-101.
Claims 98 and 102-116 are currently pending, with claims 103-107 and 113-114 withdrawn. Claims 98, 102, 108-112 and 115-116 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.
Priority
The priority date of the instant claims 98, 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
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 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.
D) 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.
E) 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.
F) Regarding claim 98, the claim recites a method comprising performing cycles of polynucleotide synthesis, and first describes the steps inn each cycle using one set of terms (e.g., double-stranded polynucleotide, first strand, second strand). The claim then recites performing a first cycle and a further cycle using a different set of terms (e.g., scaffold polynucleotide, support strand , synthesis strand).
For purpose of applying prior art and clarity of the record, the following terms are interpreted as equivalent terms referring to the same elements in the claimed method.
Claim term (each cycles)
Equivalent term (first and further cycle)
Basis for interpretation
double-stranded polynucleotide
scaffold polynucleotide
The claim uses both terms to refer to a dsDNA extended via blunt-end ligation comprising the same DNA strands; see Fig. 1, 101,106
first strand of the double-stranded polynucleotide
support strand of the double-stranded scaffold polynucleotide
The claim uses both terms to refer to the strand of a dsDNA that is extended by incorporation of a second nucleotide; see Fig. 1, 101, 106, strand a;
the claim also recites "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"
second strand of the double-stranded polynucleotide
synthesis strand of the double-stranded scaffold polynucleotide
The claim uses both terms to refer to the strand of a dsDNA that is extended by incorporation of a second nucleotide; see Fig. 1, 101, 106, strand b;
the claim also recites "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"
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Response to Arguments
Applicant's arguments filed on June 22, 2026 have been fully considered.
Claim Rejections - 35 USC § 103
In the prior Office Action (Non-Final Office Action- 12/22/2025):
Claims 98, 100-102, 108-112, and 115-116 are rejected under 35 U.S.C. 103 as being unpatentable over ARLOW, in view of McKernan, as evidenced by Güixens-Gallardo, Bentley and Chen.
Applicant amended claim 98 to incorporates the limitations of prior claims 100-101, now cancelled. The rejections for claims 98, 102, 108-112, and 115-116 are maintained in this Office Action for reasons below.
Applicant argues that the rejections above should be withdrawn (Remarks, page 21-25). Applicant's arguments have been fully considered but are not found persuasive.
First, Applicant argues that
"one of ordinary skill in the art at the time of filing would not have considered McKernan to be relevant to the synthesis of double-stranded polynucleotides or to be compatible with the subject matter of Arlow" because McKernan does not relate to synthesis of a double-stranded polynucleotide, but rather relates to a method for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template (Remarks, page 22).
This arguments is not persuasive. These references are related, as explained in the rejection, McKernan's sequencing-by-extension/ligation method and ARLOW's double-stranded polynucleotide synthesis method both involve polynucleotide synthesis workflows comprising nucleotide extension and cleavage steps. Specifically, both ARLOW and McKernan teach the use of cleavable nucleotides in cyclic DNA synthesis.
ARLOW teaches cleavable dU bases and explicitly states that cleavable linkers are not limited to dU, that "[d]ifferent modified nucleobases than uracil as a cleavable linker could have also used, combined with any enzyme that cleaves the backbone at the site of the modified nucleobase leaving a 5′ PO4 terminus." ([0135])
The pairing of deoxyinosine and Endo V, disclosed in McKernan, meets this description.
Thus, this substitution represents a predicable, simple substitution of known, functionally equivalent elements, leading to the predictable result of cleaving of polynucleotide. This rationale aligns with the principle of KSR for a simple substitution of one known element for another to obtain predictable results, see MPEP 2141.
Although Applicant asserts that the claimed invention is performed without the use of a pre-existing template strand, the polymerase-mediated extension steps in both ARLOW and the claimed invention still involve template-dependent nucleotide incorporation. Thus, the extension chemistry in both ARLOW and the claimed invention is similar to the template-dependent extension taught in McKernan.
Applicant's argument merely identifies a difference between the references. However, two related references can be different in some aspect while still sharing relevant mechanisms and technical features. McKernan is still analogous art because it is reasonably pertinent to the same technical problem of performing and controlling cyclic nucleotide extension and cleavage during polynucleotide synthesis. The rejection does not rely on the references having identical teachings, and asserted difference does not amount to teaching away.
Applicant then argues that
"McKernan neither teaches nor suggests that deoxyinosine would be a suitable substitution for dU in Arlow' s cleavage reaction. Instead, McKernan teaches that deoxyinosine acts as a "trigger" residue for Endonuclease V cleavage activity at "the second phosphodiester bond 3' to deoxyinosine" (see McKernan at [0101]), which thus produces different termini and cleaves at a different location than what Arlow' s teachings require. One of ordinary skill in the art would therefore recognize that the deoxyinosine of McKernan is not an appropriate substitute for the dU of Arlow." (Remarks, page 23)"
This argument is not persuasive.
The relevant claim limitation recites :
” (A) a first strand of a double-stranded polynucleotide is extended by incorporation of a first nucleotide of the predefined sequence and a universal nucleotide by the action of a ligase enzyme in a blunt-ended ligation reaction,
wherein the universal nucleotide defines a cleavage site,
wherein the universal nucleotide comprises hypoxanthine, 4-nitroindole, 5- nitroindole, 6-nitroindole, 3-nitropyrrole, nitroimidazole, 4-nitropyrazole, 4- nitrobenzimidazole, 5-nitroindazole, 4-aminobenzimidazole, or phenyl”
As expressly discussed in the rejection below, and reiterated here for clarity of the record, ARLOW teaches extending a first strand by blunt-end ligation through incorporation of a first nucleotide and a cleavable nucleotide base, i.e., a dU base (Figure10, see C. extension), wherein the dU base defines a cleavage site adjacent to the extended first nucleotide (Figure 10E; [0050] “E. The deprotection reagent is introduced to the reaction chamber, which cleaves the backbone at the dU base immediately adjacent to the delivered payload”).
In other words, ARLOW already discloses the position and function of a cleavable nucleotide base incorporated during strand extension.
McKernan teaches a functionally equivalent cleavable nucleotide (i.e., deoxyinosine cleavable by Endo V ) that can be substituted for the dU base of ARLOW and meets the definition of a universal nucleotide as it comprises hypoxanthine ([0105] lines 9-11). Therefore, the rejection does not rely on McKernan teaching identical cleavage location. Rather, the rejection proposes the substitution of one known cleavable nucleotide base for another known cleavable nucleotide base to perform the same function of being cleaved at the site of the cleavable nucleobase and leaving a 5′ PO4 terminus. McKernan does not criticize, discredit, or otherwise discourage the use of deoxyinosine in the method of ARLOW; thus, it does not teach away from the proposed substitution.
Applicant's argument focuses on differences in cleavage location, but does not address the reason for the combination. The rejection relies on the substitution of one known cleavable base for another known cleavable base to perform the same general function (i.e., being cleaved by its corresponding enzyme) in a cyclic polynucleotide synthesis workflow. A difference in cleavage location or resulting termini does not, by itself, show that deoxyinosine would have been unsuitable for use in ARLOW's method.
Additionally, Applicant's argument that the prior art does not expressly suggest substituting deoxyinosine for dU in Arlow' s cleavage reaction is also not persuasive. The rejection is based on the simple substitution of one known element for another to obtain predictable results. See MPEP §2143 (I)B. An express suggestion to substitute one equivalent for another need not be present to render such substitution obvious.
Applicant Argues:
"[t]here would be no reason for the skilled person to consider that the method of Arlow could be successfully modified to incorporate reversible terminator use as recited in claim 98." (Remarks, 24).
This is not persuasive. As detailed below, the rejection provides several reasons 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, to ensure complete and accurate nucleotide incorporation while preventing over-incorporation due to non-templated addition.
As discussed in detail in the rejection below, ARLOW teach using photo-reversible terminator nucleotide which prevents extension by polymerase enzyme to improve fidelity and correctness of the polynucleotides synthesized. The use of nucleotides having reversible terminator groups in DNA synthesis cycles was a well-established approach in molecular biology. In addition, non-templated nucleotide extension by DNA polymerase was a known issue in the art.
Therefore, a skilled artisan would have found it obvious to apply the proposed modification. There would have been a reasonable expectation of success because the use of nucleotides having reversible terminators were well-known in molecular biology, particularly in cyclic nucleic acid synthesis; and ARLOW already teaches both the Figure 10's polynucleotide synthesis method comprising polymerase extension steps and the use of reversible terminator nucleotides in DNA synthesis to control polymerase extension. Because the proposed modification applies a known nucleotide type to the same polymerase-mediated extension chemistry taught in ARLOW, a skilled artisan would have reasonably expected the reversible terminator nucleotide to function predictably in Figure 10's method.
For the reasons above, Applicant's arguments are unpersuasive. Accordingly, since all the claims remain rejected under the same statutory basis and in view of the same prior art references with the same rationales and teachings, the previously set forth rejections under 35 USC § 103 are properly maintained 1and updated to reflect the recent claims amendment.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
The following are maintained rejections with changes to account for the claim amendments. Since all the claims remain rejected under the same statutory basis and in view of the same prior art references with the same rationales and teachings, the previously set forth rejections under 35 USC § 103 are properly maintained and updated to reflect the recent claims amendment. Applicant's arguments are addressed in "Response to Arguments" section.
Claims 982, 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), in view of
McKernan (McKernan et al. US20090181860A1 - Reagents, methods, and libraries for bead-based sequencing; Published 2009-07-16),
Güixens-Gallardo (Güixens-Gallardo et al. Inhibition of non-templated nucleotide addition by DNA polymerases in primer extension using twisted intercalating nucleic acid modified templates. Bioorg Med Chem Lett. 2016 Jan 15;26(2):288-291. doi: 10.1016/j.bmcl.2015.12.034. Epub 2015 Dec 11. PMID: 26707394),
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) Regarding claim 98, the claimed method is an obvious combination of a known double-stranded DNA synthesis method disclosed in ARLOW with known elements, including reversible terminator nucleotides disclosed in ARLOW, Bentley and Chen, and a known cleavable nucleotide base, such as deoxyinosine, disclosed in McKernan.
ARLOW teaches a method for DNA polynucleotide synthesis with repeated cycles involving DNA ligation, nucleotide extension, and strand-specific DNA cleavage (Figure 10).
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Regarding claim 98, ARLOW teaches an in vitro method of synthesizing a double-stranded polynucleotide having a predefined sequence, the method comprising performing cycles of synthesis (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),
wherein 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);
wherein the first nucleotide and the universal nucleotide are components of a polynucleotide ligation molecule (Figure 10, extension Unit comprising "1nt payload" and dU base; [0069]), wherein the polynucleotide ligation molecule is ligated to the double-stranded polynucleotide during step (A) by the action of the ligase enzyme in a blunt-ended ligation reaction (Figure 10), and wherein upon ligation of the polynucleotide ligation molecule to the double-stranded polynucleotide, the first strand of the double-stranded polynucleotide is extended and the cleavage site is created (Figure 10, step C, extension, the immobilized polynucleotide is extended and comprise a dU base cleavage site after ligation );
wherein the method comprises performing a first cycle of synthesis comprising:
(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]).
ARLOW in Figure 10 discloses a filling step where DNA polymerase extends a strand by 1 nucleotide, using the complementary bottom strand as a template, creating a blunt end that enables subsequence extension through blunt-end ligation.
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).
Although ARLOW does not explicitly disclose utilizing photo-reversible terminator nucleotide in the specific embodiment of Figure 10, this feature would have been obvious in view of the knowledge in the art.
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, as disclosed by Bentley and Chen.
Bentley teaches 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 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.
Although ARLOW's DNA synthesis method differs from DNA sequencing by synthesis methods in application (i.e., one synthesizes DNA while the other determines DNA sequence through DNA synthesis), both methods rely on cyclic, template-directed nucleotide incorporation and require accurate, reliable control of nucleotide addition. Therefore, a skilled artisan would have looked to known elements in sequencing-by-synthesis approaches, including reversible terminator nucleotides, to improve control over nucleotide incorporation in ARLOW's DNA synthesis method.
The potential advantage of using photo-reversible terminator nucleotides in the method disclosed in ARLOW's Figure 10 is apparent, as non-templated nucleotide extension by DNA polymerase is a well-known issue in the art, particularly in DNA polynucleotide synthesis, as evidenced by Güixens-Gallardo.
Güixens-Gallardo teaches DNA polymerases are also capable of non-templated addition (NTA) of nucleotides to the 3′-ends of blunt-ended DNA duplexes, resulting in 3′-overhangs, and NTA is disadvantageous in oligonucleotide (i.e., DNA polynucleotide) synthesis:
"Besides incorporation of nucleotides complementary to the template sequence, DNA polymerases are also capable of non-templated addition (NTA) of nucleotides to the 3′-ends of blunt-ended DNA duplexes, resulting in 3′-overhangs.6, 7 In the enzymatic synthesis of DNA, NTA typically leads to the formation of multiple DNA products—the product of the desired length and/or products having one (or more) extra nucleotides at the 3′-end. Any of natural dNTPs can be used by the polymerase for NTA but dATP is the most preferred one.8 PCR-amplified products with 3′-adenine overhangs can be used in so called TA cloning.9 On the other hand, NTA is disadvantageous when an ON of a given length and sequence is to be prepared, for example, in genotyping10, 11 and synthesis of ON probes.12, 13 Especially in probes for electrochemical detection of DNA, the exact number of nucleotides bearing electrochemical labels is crucial for the application of the ON probe. " (page 288, right-hand col)[emphasis added]
Therefore, in view of the teachings in ARLOW and the general knowledge in the art, evidenced here by Güixens-Gallardo, a skilled artisan would recognize the importance of avoiding any non-templated extension by DNA polymerase, because such extensions would interfere with blunt-end ligation and negatively impact the fidelity and sequence accuracy of the synthesized polynucleotides.
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 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, to ensure complete and accurate nucleotide incorporation while preventing over-incorporation due to non-templated addition.
Given the known function of each element as explained by ARLOW and the general knowledge in the field, as disclosed in Bentley, Chen, and Güixens-Gallardo, 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.
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 ensure complete and accurate nucleotide incorporation while preventing over-incorporation due to non-templated addition, as suggested by Bentley and Güixens-Gallardo.
It would also 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, disclosed in Bentley and Chen. 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.
The skilled artisan would have been motivated to leverage the advantages of photo-reversible terminator nucleotides, to address the known problem of non-templated DNA polymerase extension in the art.
There would have been a reasonable expectation of success because the use of nucleotides having reversible terminators were well-known in molecular biology, particularly in cyclic nucleic acid synthesis; and ARLOW already teaches both the Figure 10's polynucleotide synthesis method comprising polymerase extension steps and the use of reversible terminator nucleotides in DNA synthesis to control polymerase extension. Because the proposed modification applies a known nucleotide type to the same polymerase-mediated extension chemistry taught in ARLOW, a skilled artisan would have reasonably expected the reversible terminator nucleotide to function predictably in Figure 10's method.
Regarding the claim limitation specifying the universal nucleotide as comprising: hypoxanthine, 4-nitroindole, 5-nitroindole, 6-nitroindole, 3-nitropyrrole, nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 5-nitroindazole, 4-aminobenzimidazole, or phenyl;
ARLOW teaches extending a first strand by blunt-end ligation through incorporation of a first nucleotide and a cleavable nucleotide base, i.e., a dU base (Figure10, see C. extension), wherein the dU base defines a cleavage site adjacent to the extended first nucleotide (Figure 10E; [0050] “E. The deprotection reagent is introduced to the reaction chamber, which cleaves the backbone at the dU base immediately adjacent to the delivered payload”).
ARLOW also states that nucleobases other than dU can also serve as cleavable linkers, provided that they can be cleaved by enzyme that cleaves the backbone at the site of the nucleobase leaving a 5′ phosphate terminus ([0135]lines 18-22).
Although ARLOW does not explicitly disclose any nucleotide comprising the bases listed in claim 98, such as hypoxanthine, this feature is obvious in view of McKernan.
McKernan discloses methods of polynucleotide synthesis involving cyclic steps of extension, ligation, and cleavage (entire document, see Abstract for example), and specifically teaches the use of deoxyinosine as a cleavable nucleotide in combination with E.coli Endonuclease V (Endo V), which cleaves a nucleic acid containing deoxyinosine at the second phoshodiester bond 3' to the deoxyinosine residue, leaving a 3' OH and 5' phosphate termini. ([00225]). McKernan teaches deoxyinosine comprises hypoxanthine as its nucleobase ([0105] lines 9-11).
Accordingly, it would have been prima facie obvious to a person of ordinary skill in the art to substitute the dU cleavable linker in ARLOW with deoxyinosine comprising hypoxanthine, as taught by McKernan. Both references pertain to cyclic polynucleotide synthesis workflows comprising nucleotide cleavage steps. ARLOW explicitly states that cleavable linkers are not limited to dU, that "[d]ifferent modified nucleobases than uracil as a cleavable linker could have also used, combined with any enzyme that cleaves the backbone at the site of the modified nucleobase leaving a 5′ PO4 terminus." ([0135])
The pairing of deoxyinosine and Endo V, disclosed in McKernan, meets this description.
Thus, this substitution represents a predicable, simple substitution of known, functionally equivalent elements, leading to the predictable result of cleaving of polynucleotide.
This rationale aligns with the principle of KSR for a simple substitution of one known element for another to obtain predictable results, see MPEP 2141.
B) 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
For the purpose of compact prosecution, the examiner has reviewed the application's entire disclosure, but has not readily identified any subject matter that is not taught or suggested by the prior art, or combined in a non-obvious way. However, the examiner remains open to considering any aspect of the invention that the applicant believes may be allowable. To advance prosecution, Applicant is advised to amend the claims in their response, to focus on what they consider as the inventive concept in the present disclosure.
Below are relevant prior art not used in rejection but pertinent to the claims or disclosure.
Other prior art references also teach de novo DNA synthesis via ligation:
See Fig. 7 in US20130309725A1 - Methods and devices for in situ nucleic acid synthesis; published 2013-11-21;
See Fig. 1 in US20060115850A1 - Method for the synthesis of DNA fragments; published 2006-06-01.
Conclusion
No claims are allowed.
THIS ACTION IS MADE FINAL. 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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/TIAN NMN YU/Examiner , Art Unit 1681 /AARON A PRIEST/Primary Examiner, Art Unit 1681
1 See in MPEP 1207.03(a): “There is no new ground of rejection when the basic thrust of the rejection remains the same such that an appellant has been given a fair opportunity to react to the rejection. Where the statutory basis for the rejection remains the same, and the evidence relied upon in support of the rejection remains the same, a change in the discussion of, or rationale in support of, the rejection does not necessarily constitute a new ground of rejection. See In re Kronig, 539 F.2d 1300, 1302-03, 190 USPQ 425, 426-27 (CCPA 1976). at 1303, 190 USPQ at 427.”
2 Amended claim 98 incorporates the limitations of prior claims 100-101, now cancelled, which were previously rejected over the claim references.
Prosecution Insights