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. Priority This application 17/986,357 filed on 11/24/2022 claims the benefit of European Patent Application No. EP 21210099.4, filed on 11/24/2021. The priority date of claim 1 and its dependent claims is determined to be 11/24/2021 , the filing date of European Patent Application No. EP 21210099.4 Status of Claims Applicant’s amendments to claims filed 12/01/2025 in response to the Non-Final Rejection mailed 09/05/2025 are acknowledged. Claims 1-6 and 8-14 are amended. Claim 7 has been canceled. Claims 1-6 and 8-14 are pending and under examination. Response to Remarks filed 12/01/2025 The amendments and arguments presented in the papers filed 12/01/2025 ("Remarks”) have been thoroughly considered. The issues raised in the Office action dated 09/05/2025 listed below have been reconsidered as indicated. a) The objections to claims 1-6 and 8-14 are withdrawn in view of the amendments to the claims . b) FILLIN "Enter claim or figure number or specification or abstract etc" \* MERGEFORMAT The 35 USC 112(b) indefiniteness rejections of claim 7 has been withdrawn as moot in view of the cancellation of the claim. c ) The 35 USC 112(b) indefiniteness rejections of claims 1 ,3 -5 and 8-14 have been withdrawn in view of the amendments to claims. d ) The rejection s of claims 1-5, 8, 9, and 11-14 under 35 U.S.C. 103 as being unpatentable over Guo et al. (An Integrated System for DNA Sequencing by Synthesis Using Novel Nucleotide Analogues. 2010. Acc Chem Res. 43 (4): 551-563) in view of Turcatti et al. (A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis. 2008. Nucleic Acids Res. 36(4): e25) and the rejection of claims 6 and 10 under 35 U.S.C. 103 as being unpatentable over Guo et al. (An Integrated System for DNA Sequencing by Synthesis Using Novel Nucleotide Analogues. 2010. Acc Chem Res. 43 (4): 551-563) in view of Turcatti et al. (A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis. 2008. Nucleic Acids Res. 36(4): e25), further in view of Mok et al. (US20200010875) has been withdrawn in view of the amendments to claims . e) The rejection of claim 7 under 35 U.S.C. 103 as being unpatentable over Guo et al. (An Integrated System for DNA Sequencing by Synthesis Using Novel Nucleotide Analogues. 2010. Acc Chem Res. 43 (4): 551-563) in view of Turcatti et al. (A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis. 2008. Nucleic Acids Res. 36(4): e25), further in view of Xie et al. (US20130053252) has been withdrawn as being moot in view of the cancellation of the claim. New and modified grounds of rejection necessitated by amendment are detailed below and this action is made FINAL. Claim Rejections - 35 USC § 112(b) - maintained and modified 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 FILLIN "Enter claim indentification information" \* MERGEFORMAT s 2 and 6 remain/ are 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 6 recites the limitation " the adapter sequence " in line 3 . There is insufficient antecedent basis for this limitation in the claim. No “adapter sequence” is cited in claim 1, which claim 6 depends from. The following are modified rejections necessitated by claim amendments. The term “ at least a part of the DNA or RNA molecule” in claim 2 is a relative term which renders the claim indefinite. The term “ at least a part of ” in the context of “RNA or DNA molecule to be detected is completed” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The limitation "repeated until all the nucleotides required for the sequencing" is rendered indefinite by use of the term "at least a part of the DNA or RNA molecule" . Response to Arguments against Claim Rejection - 35 U.S. C § 112(b) The response asserts that amendments to the claims render ed these rejections moot. claim 2 Applicant's arguments have been fully considered but are not persuasive. The amendment of claim 2 did not modify the phrase “at least a part of”. claim 6 Applicant's arguments have been fully considered but are not persuasive. Claim 6 was not amended to correct the phrase “the adapter sequence”. Claim Rejections - 35 USC § 103 In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status. The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action: A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made. Claims 1-5 and 8-14 are rejected under 35 U.S.C. 103 as being unpatentable over Huang et al. (US20120046177) in view of Eshoo et al. ( US20150111762 ). These are new rejections necessitated by claim amendment s. Regarding claim 1 , Huang teaches a method for sequencing by synthesis, the method comprising: contacting a template comprising a DNA sequence with DNA polymerase and a first single nucleotide solution and washing to remove unincorporated nucleotides before determining the presence or absence of an incorporated labeled nucleotide (para 11). The method further comprises repeating steps with a second, third, and fourth single nucleotide (para 12) and further cleaving the label from the incorporated labeled nucleotide (para 12) . The addition of the nucleotides may be sequential, with repeated addition of all four single nucleotide solutions comprising labeled nucleotides (para 58). Huang teaches the nucleotides are non-terminating ( have an unprotected 3'OH position ) fluorescently labeled nucleotides (para 39) and the label is attached to the nucleotide via a cleavable linker (para 14 and exampled in Fig. 5) . Huang further teaches each nucleotide can be labeled with different labels (fluorescent dyes with different emission maxima) (para 103) , pointing to, for instance Alexa dyes that cover the whole spectra (para 93) . Huang teaches the addition of adapter sequences to DNA templates (para 19, Fig. 1) but does not teach the RNA or DNA molecule comprises a sequence of 4 to 50 A, T, C and G nucleotides, each with at least one fluorescent dye as a calibration sequence for fluorescent emission radiation, wherein the fluorescent emission of the incorporated nucleotides is detected with its relative intensity against the fluorescent emission of the calibration sequence. Eshoo teaches a method of sequencing comprising incorporating fluorescently labeled nucleotides (para 6) and using a 4-base calibration sequence at the beginning of a sequencing run contain ing each of the 4 bases in a known order to provide a calibration reference, e.g., to calibrate a sequencing instrument to recognize the appropriate signal intensities for each of the bases (para 8). Neither Huang nor Eshoo teach the detection step c) is performed once the sequential steps a) and b) for the nucleotides A, T, C and G have been performed. However, i t would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Huang and Eshoo to arrive at the instantly claimed invention. The modification would have entailed incorporating a calibration sequence as taught by Eshoo in the DNA molec ule of Huang , using the different fluorescent dyes as taught by Huang. One would have been motivated to do so to improve the accuracy of the fluorescent signal. Huang teaches the addition of adapter sequences to DNA target templates and one of skill in the art would have known how to design a specific calibration sequence. The modification would further have entailed modifying the method of Huang to detect incorporated nucleotides after the sequential addition of A, T, C, and G nucleotides rather than after each individual addition. The changing of order in the protocol is deemed a matter of routine optimization. Reordering the steps of a protocol would have been well-known to one of skill in the art and would have simplified the method by decreasing the number of imaging steps. T here would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art. Regarding claim 2 , Huang teaches repeating the steps of the method to generate a longer sequence (para 12) , which is encompassed by the limitations of the claim. Regarding claim 3 , Huang teaches repeating the steps of the method more than 10 times (para 12) . Regarding claim 4 , Huang teaches the single nucleotide solution comprises labeled and nonlabelled nucleotides (para 11). Regarding claim 5 , Huang teaches the cleavable linker may be cleaved through a chemical reaction (para 14). Regarding claim 8 , Huang teaches nucleotides (Fig. 5 B, final structure shown below ) satisfying the claimed formula. Regarding claim 9 , Huang does not teach a specific temperature for incorporation of nucleotides in the presence of polymerase. However, Huang teaches compatibility of the method with a variety of polymerases (para 88-90), and further states that routine molecular biology techniques are encompassed by the invention (para 84) and selecting an appropriate polymerase based on incorporation efficiency of incorporated nucleotides (para 90). It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Huang and Eshoo to arrive at the temperature range disclosed by the instant invention. Determining an appropriate temperature range is deemed merely a matter of judicious selection and routine optimization which is well within the purview of the skilled artisan. One of ordinary skill in the art would have been motivated to select polymerases active at different temperatures in order optimize conditions and increase efficiency of the incorporation reaction to improve sequencing performance, a stated desire of Huang . There would have been a reasonable expectation of success given the underlying materials and methods of DNA sequencing by synthesis and nucleotide analogs are widely known, successfully demonstrated, and commonly used as evidenced by the prior art. Regarding claim 10 , Huang teaches the target DNA sequence can be amplified by rolling circle amplification before addition of nucleotides (para 18), which satisfies the requirement of DNA rolonies comprising multiple concatemers of the DNA molecules. Regarding claim 11 , Huang teaches the DNA molecules are single-stranded (Figs. 1, 3). Regarding claim 12 , Huang teaches fragmenting the DNA molecules before the incorporation of nucleotides (Fig. 1). Regarding claim 13 , Huang teaches polynucleotides immobilized on a substrate (para 137) . C laim s 6 and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Huang et al. (US20120046177) in view of Eshoo et al. ( US20150111762 ) as applied to claim s 1-5 and 8-13 above, and further in view of Fields et al. ( US20170335369 ) . Regarding claim 6 , Huang teaches the addition of adapter sequences (para 19, Fig. 1) and states that the method includes routine molecular biology techniques such as adapter molecules (para 84). Huang does not teach a sequence of 4 to 50 nucleotides as an unique identifier (UMI) as part of the adapter . Fields teaches methods for tagging nucleic acids for sequencing. Fields teaches the method comprises attaching DNA comprising a specific sequence (para 15) such as a unique molecular barcode (para 57). Fields further teaches barcodes can be at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleotides in length (para 31 2 ). Fields states that the barcode allows some feature of a polynucleotide , such as the sample from which the polynucleotide is derived , with which the barcode is associated to be identified (para 312). It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Huang and Eshoo with Fields to arrive at the instantly claimed invention. The modification would have entailed adding the adapter of Fields as the adapters to DNA templates as taught by Huang. One would have been motivated by the ability to track and identify a feature of the DNA molecule, a well-known goal at the time in the art. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art. Regarding claim 14 , Huang teaches polynucleotides immobilized on a substrate (para 137) but does not teach immobilized by interacting with the surface via electrostatic charges or via NHS ester-activated crosslinkers. Fields teaches immobilizing libraries of DNA binding probes to a solid phase by any suitable method known in the art , including electrostatic charge (para 374). Fields teaches binding DNA molecules to binding probes (paras 9-11), which is encompassed by immobilizing the DNA molecule by interacting with the surface via electrostatic charge. It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Huang and Eshoo with Fields to arrive at the instantly claimed invention. The modification would have entailed using the method of Fields to immobilize probes on a surface and adding the DNA molecule of Huang. One would have been motivated to do so by the desire to use a known method for immobilization as recited in Fields. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art. Conclusion Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL . See MPEP § 706.07(a). 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. Any inquiry concerning this communication or earlier communications from the examiner should be directed to FILLIN "Examiner name" \* MERGEFORMAT JESSICA GRAY whose telephone number is FILLIN "Phone number" \* MERGEFORMAT (571)272-0116 . The examiner can normally be reached FILLIN "Work Schedule?" \* MERGEFORMAT Monday-Friday 8-5 with second Fridays off . Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, FILLIN "SPE Name?" \* MERGEFORMAT WINSTON SHEN can be reached at FILLIN "SPE Phone?" \* MERGEFORMAT (571)272-3157 . The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000. /JESSICA GRAY/ Examiner, Art Unit 1682 /WU CHENG W SHEN/ Supervisory Patent Examiner, Art Unit 1682