DETAILED ACTION
Election/Restrictions
Applicant’s election without traverse of Group V, claims 20-29, in the reply filed on April 7 2026 is acknowledged. Claims 1, 3, 5-7, 11, 13-16 and 20-29 are pending in the application. Claims 1, 3, 5-7, 11 and 13-16 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on April 7 2026. Accordingly, claims 20-29 are being examined on the merits herein.
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 is a CON of PCT/US2020/054318 (10/05/2020) which claims benefit of 62/985,401 (03/05/2020) and claims benefit of 62/910,643 (10/04/2019) as reflected in the filing receipt issued on July 25 2023.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on April 7 2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Objections
Claim 20 is objected to because of the following informalities: for depending upon a withdrawn claim. Appropriate correction is required.
Claim Rejections - 35 USC § 103
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 of this title, 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 set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied 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 20-29 are rejected under 35 U.S.C. 103 as being unpatentable over Canard et al. (Gene, 1994) in view of Winz et al. (Nucleic Acids Research, 2015) and Jatsch et al. (Organic Letters, 2008) as evidenced by Jena BioScience (2019).
Applicant Claims
The instant application claims a method for determining the sequence of an immobilized target polynucleotide, comprising: (a) monitoring the sequential incorporation of nucleotides complementary to the immobilized target polynucleotide, wherein each of the nucleotides independently is a nucleoside 5'- triphosphate analog of claim 1, and wherein the identity of each nucleotide incorporated is determined by detection of a detectable label linked to 3' oxygen of the nucleotide incorporated; and(b) removing the detectable label from the 3' oxygen by cleavage of a covalent linker between the 3' oxygen and the detectable label, wherein the cleavage breaks a disulfide bond or an ester bond; wherein non-incorporated nucleotides are removed prior to detection and the detectable label is removed subsequent to detection.
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Determination of the Scope and Content of the Prior Art
(MPEP §2141.01)
Canard et al. is directed to DNA polymerase fluorescent substrates with reversible 3’-tags. Taught is protection of the 3’-end of the extending DNA molecule in which a 3’-free hydroxy group could be eventually regenerated. Taught are 3’-modified 2’-deoxynucleotides 5-triphosphte substrates for DNA polymerases such that the 3’-moeity would be different for each base G, A, T or C can be easily identified (e.g. fluoresce) and then be removed under conditions compatible with DNA stability to restore an unprotected 3’-hydroxy end (paragraph bridging pages 1-2). Taught is esterifying the 3’-hydroxy group with distinct anthranilate or fluorescein based fluorescent residue (page 2, (a) and Fig. 1). Since these nt analogs did not contain a 3’-hydroxy group their incorporation into an elongating DNA resulted in chain termination. This point was assessed using a solid-phase assay (page 2, left column (b)) Taught is the use of modified T7 DNA polymerase which shows rapid extension (page 3, left column and Figure 3). Ethers or esters are both expected to restore a hydroxyl group upon deprotection. Ether bonds may be hard to cleave under mild conditions compatible with DNA chemical stability, whereas chemical deprotection using alkali has the present disadvantage of melting the primer-template duplex. The ester bond is a very attractive candidate to fulfill appropriate conditions for attachment of the tag as it is amendable to reaction with hydroxy groups through several activated forms. Esterases of broad specificity are ubiquitous in nature (page 5, left column). Solid-phase sequencing reactions of immobilized template are taught (page 5, last paragraph). Removal of unbound oligo before detection is taught (Fig. 3).
Ascertainment of the Difference Between Scope the Prior Art and the Claims
(MPEP §2141.02)
While Canard et al. suggest a method for determining the sequence of an immobilized target by using a 5’-triphosphate in which a detectable label is linked to the 3’ oxygen of the nucleotide incorporated, removing the detectable label via an ester bond and removal of non-incorporated nucleotides before detection, Canard et al. does not expressly teach a 5’-triphosphate as claimed or the use of click chemistry to connect the detectable linker to the 3’-position of the sugar. However, these deficiencies are cured by Winz et al. and Jantsch et al.
Winz et al. is directed to nucleotidyl transferase assisted DNA labeling with different click chemistries. Click chemistry is a class of highly efficient, biorthogonal chemical reactions and has become very popular for labeling nucleic acids and other biomolecules. DNA label is required to generate fluorescent for example for DNA detection and purification. For click labeling of DNA, the reactive moieties are generally introduced co-synthetically either chemically or by template-dependent polymerase (page 1). Shown in Figure 1: is the strategy for terminal or internal DNA labeling using click chemistry. Specifically shown is reaction of an alkyne with an azide which forms a 1,2,3-triazole moiety. Shown also is modification at the 3’ position with an azide.
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Jatsch et al. is directed to self-organizing oligothiophene-nucleoside conjugates: versatile synthesis via “click”-chemistry. Taught are thymidine functionalized deoxyadenosine using Click reaction which is the 1,3-dipolar cycloaddition of azides and terminal alkynes. A flexible alkyl spacer bearing a terminal azide group was attached to nucleosides which were subsequentially clicked (page 962). Synthesis of the azide is shown in Scheme 1 which includes an ester.
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As evidenced by Jena Bioscience, there are a variety of different alkyne-containing fluorescent dyes which are used for the fluorescent labeling of azide-tagged molecules with have different color emissions.
Finding of Prima Facie Obviousness Rationale and Motivation
(MPEP §2142-2143)
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Canard et al., Winz et al. and Jatsch et al. and utilize click chemistry to connect the 3’-OH to the fluorophore. One skilled in the art would have been motivated to utilize click chemistry as Winz et al. teaches that this is a simple and efficient strategy to add a 3’-terminal labeling group. One skilled in the art would have been motivated to utilize a 5’triphosphate sugar with the ester coupled azide such as that taught in Jatsch et al. as Canard et al. teaches the importance of the ester group to cleave the attached fluorophore and Jatsch et al. teaches the use of azide modified nucleosides with the 3’-OH converted to an azide to be used in click chemistry. Therefore, one skilled in the art would have a reasonable expectation of success as Jatsch et al. teaches how to attach the azide with an ester linking group and Winz et al. teaches that the azide functionality at the 3’ can be utilized in click chemistry, specifically CuAAC with an alkyne to form a 1,2,3-triazole. Since as evidenced by Jena Bioscience there are a variety of commercially available alkyne containing fluorescent dyes there is a reasonable expectation of success.
Regarding the claimed method steps in claim 20 and 22, it would have been obvious to one of ordinary skill in the art to first form the fluorophore linked nucleotide by reacting an alkyne containing fluorescent dye with an azide functional group. One skilled in the art would have used click chemistry to attach the fluorophore due to the simple chemical method of attachment as taught by Winz et al. One skilled in the art would have been motivated to have the label attached to the 3’ oxygen as taught by Canard et al. Once the label modified nucleotide is formed, it would have been obvious to use the nucleotide(s) (i.e. use different nucleotides with different fluorophores) to monitor incorporation of the nucleotides complementary to the immobilized target polynucleotide followed by washing to remove the non-incorporated nucleotides and then followed by removal of the detectable label via cleavage of an ester bond. One skilled in the art would have been motivated to utilize these method steps as this is taught by Canard et al.
Regarding the claimed compound of formula II, Canard et al. teaches a 5’ triphosphate. The use of the ester azide of Jatsch et al. corresponds to the instantly claimed compound when n is 3 which means that the alkyl chain taught in Jatsch et al. is a homolog of the instant claims (i.e. the compounds differ by one carbon atom). An obviousness rejection based on similarity in chemical structure and function entails the motivation of one skilled in the art to make a claimed compound, in the expectation that compounds similar in structure will have similar properties. In re Payne, 606 F.2d 303, 313, 203 USPQ 245, 254 (CCPA 1979). MPEP 2144.09. Here Canard et al. makes it clear that in order to achieve cleavage of the fluorophore the ester bond must be used. Since this linker is being used in Jatsch et al. to attach an azide to the 3’ position of the nucleotide which would be the same as the instant claims and the difference is merely the length of the alkyl chain between the ester and the azide, absent a demonstration of the criticality it is the examiners position one skilled in the art would recognize that different length of the alkyl chain can be utilized.
Regarding claim 21, Canard et al. teaches the use of different fluorescent residues which can be easily identified. Since the desire is DNA sequencing, it would have been obvious to repeat the steps with different bases in order to identify the total DNA sequence. Therefore depending on the length, the number of repeats would be routinely varied.
Regarding claims 23-24, Canard et al. teaches the use of a polymerase and specifically teaches a modified T7 DNA polymerase reading on engineered polymerase.
Regarding claim 25, Canard et al. teaches an anthranilate or fluorescein based fluorescent residue.
Regarding claims 26-29, as taught by Winz et al. the click reaction occurs between the azide (aka azido group) and the alkyne (aka ethynyl group) and forms a 1,23-triazole.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ABIGAIL VANHORN whose telephone number is (571)270-3502. The examiner can normally be reached M-Th 6 am-4 pm EST.
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/ABIGAIL VANHORN/Primary Examiner, Art Unit 1636
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