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 .
Election/Restrictions
Applicant’s election without traverse of Species A (claim 7) and Species D (claims 19-20) in the reply filed on 9/10/2026 is acknowledged.
Claims 8 and 15-18 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim.
Claims 1-7, 9-14, and 19-20 are pending and are examined on the merits herein.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 4/9/2025 and 9/10/2026 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 20 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 20 is rejected because in (a) of the claim, the use of “a first sequencing primer” is recited, where this primer binds to a first portion of the concatemer template molecule. This is part of the “one or more cycles of sequencing reactions” first described in claim 1 (and also stated in claim 19, from which claim 20 depends). Separate from the one or more cycles of sequencing reactions in claim 1, the use of a first sequencing primer is already recited, where this primer attaches to a first region of the nucleic acid template molecule. Thus, this hybridization of the first sequencing primer with the target is already described outside of the “one or more cycles of sequencing reactions.” Firstly, it is unclear if the “a first sequencing primer” as recited in both claims is intended to be the same sequencing primer, and if so, it is unclear if the hybridization of the first sequencing primer is to be repeated. Secondly, if such repetition is required, it is unclear how the repetition of this limitation would function in the sequencing reactions. These issues render the scope of the claim indefinite.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-4, 6-7, and 9-14 are rejected under 35 U.S.C. 103 as being unpatentable over Zhou et al. (US 2021/0180123 A1; cited in Applicant’s IDS) as evidenced by The American Heritage Dictionary (“Room Temperature,” 2022).
Zhou teaches methods for sequencing a large number of long nucleic acid fragments (Abstract). The method involves hybridizing an extension primer with a target nucleic acid, extending said primer, performing sequencing, and then removing the extension primer/sequencing product and repeating the same process with a second primer (para. 32). The extension product can specifically be a sequencing primer (para. 131). The removing can involve enzymatic degradation of the sequencing product, and this can specifically be a 5’-3’ exonuclease (paras. 41 and 50). The target nucleic acid can be attached to a substrate via a capture probe (para. 20), where said probe can be complementary to the target and thus would be capable of hybridization with said target (para. 197). This hybridization protects the template from exonuclease digestion as the exonuclease needs a 5’ end to begin digestion, and the template would not have an available 5’ end (though protecting bases may also be used; para. 97). This thus meets the requirements of the enzyme of step (c) of instant claim 1 (and similar steps throughout the instant claims). Washing steps can be used and combined with enzyme degradation (paras. 134-136). Para. 136 notes that the washing can be used to remove residual nucleotides (from the extension reaction) or by products. Though Zhou focuses on the washing step to remove excess nucleotides (e.g. paras. 50 and 60), it would be prima facie obvious that the washing step would also be used generally to remove any excess reagents in the method that are not needed for the next step (e.g. the exonucleases). Here, after removal, a second primer is applied in the method of Zhou, and so exonuclease presence would likely interfere with the second primer binding to the template and being successfully extended. Therefore, the ordinary artisan would be motivated to wash away the exonuclease enzymes once enzyme degradation was complete.
Regarding the temperature of the washing reaction, Zhou notes in Examples 1 and 2 that extension reactions were carried out at room temperature, where said reactions included the exonuclease enzymes (paras. 272 and 274, and see paras. 15 and 50 which describe apyrase and pyrophosphatase as degrading enzymes). Para. 143 also notes that extension reactions can be performed at room temperature, and this can include enzyme degradation reactions. While Zhou does not specify the particular temperature(s) that encompass “room temperature,” as evidenced by The American Heritage Dictionary, this term is defined as “an indoor temperature around 20 or 22°C.”
MPEP 2144.05 I states, “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990)… Similarly, a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 783, 227 USPQ 773, 779 (Fed. Cir. 1985),” and Section II (A) of this portion of the MPEP notes that, “Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical.” In the instant specification, the washing temperature range of 22-27°C does not appear to be critical or produce unexpected results. Particularly, para. 135 notes temperature ranges for removing the exonuclease of 22-25°C and 25-30°C, as do many other paragraphs in the instant specification (e.g. paras. 148-149 and 151), with no indication that the claimed range is specifically desired compared to these other ranges for any particular purpose. Thus, the washing temperature of Zhou as evidenced by The American Heritage Dictionary renders the temperature of the claimed washing step prima facie obvious.
Therefore, claim 1 is prima facie obvious over Zhou as evidenced by The American Heritage Dictionary.
Regarding claims 2-4, Zhou teaches that their method may be repeated to obtain one or more additional sequence reads by removing the primer extension product on the target nucleic acid and performing the hybridization, extension, and sequencing (para. 18). This paragraph states that, “The sequence of the target nucleic acid can be determined by assembling the first, second, and optional, one or more additional sequence reads,” and thus the targeting regions for each of the extension primers need not be the same so that the entirety of a target nucleic acid can be sequenced. Para. 88 also notes that this process for sequencing can be repeated “many times,” as does para. 151. No upper limit is placed on the number of repetitions, and so 2-10 repetitions, as noted in the instant claims, would be prima facie obvious.
Regarding claim 6, Zhou teaches that nucleic acid sequencing libraries may be created by being amplified in a clonal fashion using bridge amplification (para. 106). Para. 101 states that a target nucleic acid can be genomic DNA derived from a particular organism, and can be clones from a clone library.
Regarding claim 7, Zhou teaches that the target nucleic acid can be DNA, RNA, or a hybrid, and can contain any combination of nucleotides, including those with modified bases and modified backbones (paras. 107-110). Thus, this would encompass the use of uridine nucleotides.
Regarding claims 9-10, Zhou teaches that target nucleic acids can contain target DNA or RNA and may also be outfitted with amplification or sequencing adaptors (para. 106). The adaptor sequences can be used to hybridize to the sequencing primers (paras. 128-129) and extension primer sequences (para. 130). Thus, the adaptor sequences would be the same for each of the target sequences to ensure the primers hybridize to the correct position, and this is supported by para. 104, which notes that universal primer sequences can be included in target nucleic acids. Thus, these adaptors would be considered universal adaptor sequences under the definition provided by Applicant (para. 92 of the instant specification).
Regarding claims 11-13, Zhou teaches that their sequencing comprises extension reactions (para. 32), and these extension reactions can include extension with nucleotides and a polymerase (para. 131). As noted in Figures 3-6, the extended sequence is extended based on the template nucleic acid sequence, and so the newly synthesized portion would be complementary to the target sequence and joined to the initial extension/sequencing primer. It would be prima facie obvious that each sequencing cycle with each new extension/sequencing primer in Zhou would occur by the same method, as this would ensure consistent result among all of the sequence reads. Thus, as Zhou renders prima facie obvious claims 1-3 as described above, the reference would also render claims 11-13 prima facie obvious.
Regarding claim 14, as noted above in the rejection of claims 9-10, Zhou teaches adaptors with universal primer sequences. As these sequences would be the same for all adaptors/targets used, in a singleplex reaction, this universal sequence would act as the claimed sample index sequence, as they would label each target from a particular sample. It is noted that “index” and “sample index sequence” do not have a particular definition in the instant specification. As the adaptors of Zhou may be ligated to the target nucleic acid sequences (paras. 106 and 113), and the reference does not specify which end of the target nucleic acid may be ligated with the adaptor, ligation to either end would be prima facie obvious. However, in either instance, the ordinary artisan would want to sequence the index sequence, as this would label the sequence read, which would be of importance for sequence read analysis. As the extension primer would hybridize to the 3’ end of the target sequence and sequencing would then proceed, the index sequence would either be sequenced before the sequence of interest (if the adaptor was ligated to the 3’ end of the target sequence) or after the sequence of interest (if the adaptor was ligated to the 5’ end of the target sequence). Thus, Zhou renders prima facie obvious both options i) and v) of instant claim 14.
Claims 5 and 19-20 are rejected under 35 U.S.C. 103 as being unpatentable over Zhou et al. (US 2021/0180123 A1; cited in Applicant’s IDS) as evidenced by The American Heritage Dictionary (“Room Temperature,” 2022), and further in view of Arslan et al. (US 11,220,707 B1; cited in Applicant’s IDS)
Zhou as evidenced by The American Heritage Dictionary teaches the methods of claims 1-4, 6-7, and 9-14
Regarding claim 5, Zhou does not teach the use of a T7 exonuclease.
Arslan teaches methods of sequencing concatemers (Abstract). These sequencing methods involve immobilizing surface primers to a concatemer template, sequencing the concatemers with forward sequencing primers by extending said sequencing primers, and then removing the extended forward sequencing primers while retaining the concatemer template strands (column 1, para. 6). The extended forward sequencing primers can be removed using enzyme degradation, and specifically, using the 5’-3’ double-stranded DNA exonuclease T7 (column 76, para. 1).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to substitute the T7 Exonuclease described by Arslan for the enzyme degrading exonuclease described by Zhou. MPEP 2143 I (B) states, “The rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.” The exonucleases of both Arslan and Zhou have the same result – namely degrading one strand of a double-stranded complex, and both work in the same 5’-3’ direction. Thus, substituting the T7 Exonuclease of Arslan in Zhou would have predictable results.
Therefore, claim 5 is prima facie obvious over Zhou in view of Arslan.
Regarding claims 19-20, Zhou does not teach information about multivalent molecules or concatemers.
As noted above, Arslan teaches sequencing of concatemers. In the specific sequencing of Arslan, the sequencing of the forward primer comprises the use of (i) a plurality of a first sequencing polymerase and a plurality of multivalent molecules and (ii) a plurality of a second sequencing polymerase and a plurality of nucleotide analogs, and where the multivalent molecules comprises the following: (1) a core; and (2) a plurality of nucleotide arms which comprise (i) a core attachment moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms via their core attachment moiety, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit (columns 1-2 joining para.). The multivalent molecules may also comprise fluorophores (column 3, para. 2). Column 29, para. 3 and column 230, para. 3 note that the spacer can specifically include a PEG moiety. The nucleotide analogs used can be dATP, dGTP, dCTP, dTTP, and dUTP (column 3, para. 4), and though they can be fluorescently labeled, this is not required (column 3, para. 5), and so can be left unlabeled. Column 257, paras. 3-4 show the actual practice of this sequencing method, where it is done in two-stages, the first stage involving the first sequencing primers/polymerases/multivalent molecules and the second stage involving second polymerases and nucleotide analogs.
These teachings thus meet each limitation of instant claim 19.
Columns 24-25, joining para. teaches that sequencing with the forward primers can comprise the following: “a) contacting a plurality of first sequencing polymerases and a plurality of second sequencing primers with different portions of a nucleic acid template molecule to form at least first and second complexed polymerases on the same nucleic acid template molecule;
b) contacting a plurality of multivalent molecules to the at least first and second complexed polymerases on the same nucleic acid template molecule, under conditions suitable to bind a single multivalent molecule from the plurality to the first and second complexed polymerases, wherein at least a first nucleotide unit of the single multivalent molecule is bound to the first complexed polymerase which includes a first sequencing primer hybridized to a first portion of the nucleic acid template molecule thereby forming a first binding complex, and wherein at least a second nucleotide unit of the single multivalent molecule is bound to the second complexed polymerase which includes a second sequencing primer hybridized to a second portion of the same nucleic acid template molecule thereby forming a second binding complex, wherein the contacting is conducted under a condition suitable to inhibit polymerase-catalyzed incorporation of the bound first and second nucleotide units in the first and second binding complexes, and wherein the first and second binding complexes which are bound to the same multivalent molecule forms an avidity complex;
c) detecting the first and second binding complexes on the same nucleic acid template molecule, and
d) identifying the first nucleotide unit in the first binding complex thereby determining the sequence of the first portion of the nucleic acid template molecule, and identifying the second nucleotide unit in the second binding complex thereby determining the sequence of the second portion of the same nucleic acid template molecule.” As noted above, the nucleic acid template can be a concatemer (see columns 1-2).
This language equates to the method described in instant claim 20.
Arslan generally teaches that their sequencing methods improve the quality of sequencing data by increasing signal intensity and base call accuracy, and save time compared to other sequencing methods (column 67, para. 2). As noted in at least Figures 1-2, these methods work with target sequences attached to solid supports, and multiple sequencing reactions may be used to sequence the entirety of the target (see also column 36, para. 7).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the sequencing methods involving multivalent molecules described by Arslan as the sequencing in Zhou, particularly when target sequences involving repeats are used. Zhou notes that their methods can involve analyzing target sequences that have variable numbers of repeats (para. 253), and as the general focus of the method of Zhou is on long nucleic acid fragments (e.g. paras. 11, 13, 64-65, and 84), concatemers are encompassed as nucleic acid targets. Arslan teaches a particular sequencing method that improves the quality of and efficiency benefits for sequencing these types of long, repetitive nucleic acid sequences, and where said method is compatible with sequential rounds of sequencing where primers attach to different spots on a particular target, and thus the ordinary artisan would be motivated to utilize this method in Zhou. This would only add to the sequencing step of each sequencing cycle of Zhou, without affecting other portions of the method, particularly in view of the fact that Arslan teaches removal of the extended forward sequencing primers and further downstream analysis after said removal (e.g. column 2, para. 1 and column 16, para. 1), thus providing a reasonable expectation of success.
Therefore, claims 19-20 are prima facie obvious over Zhou in view of Arslan.
Conclusion
No claims are currently allowable.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to FRANCESCA F GIAMMONA whose telephone number is (571)270-0595. The examiner can normally be reached M-Th, 7-5pm.
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/FRANCESCA FILIPPA GIAMMONA/Examiner, Art Unit 1681
