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
Applicant's filing dated 10/08/2025 is acknowledged. Claims 8, 16, 19, 23, 27-32, 34, 36, 38-59 have been cancelled previously. Claims 1-7, 9-15, 17, 18, 20-22, 24-26, 33, 35, 37, and 60 are pending in the instant application and the subject of this final office action.
All of the amendments and arguments have been reviewed and considered. Any rejections or objections not reiterated herein have been withdrawn in light of amendments to the claims or as discussed in this office action.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Previous Rejection
Status of Prior Rejections/Objections:
The prior art rejection(s) under 35 USC 103 directed to the following claims are maintained:
Claims 1-4, 15, 17-18, 20-22, 24-26, 33, and 35 over Kain as evidenced by Sigma and Lumiprobe
Claim 5 over Kain as evidenced by Sigma, Lumiprobe, and FluoroFinder, and in view of Romanov
Claims 6-7, 9, and 11-13 over Kain as evidenced by Sigma and Lumiprobe, in view of Drmanac 2022 and Drmanac 2017
Claim 10 over Kain as evidenced by Sigma and Lumiprobe, in view of Drmanac 2022 and Drmanac 2017 and further in view of Romanov
Claim 14 over Kain as evidenced by Sigma and Lumiprobe, in view of Drmanac 2022 and Drmanac 2017 and further in view of Yazaki
Claim 37 over Kain as evidenced by Sigma and Lumiprobe, in view of Illumina
Claim 60 over Kain as evidenced by Sigma and Lumiprobe, in view of Fedurco
Claim Rejections - 35 USC § 103
Claim(s) 1-4, 15, 17-18, 20-22, 24-26, 33, and 35 is/are rejected under 35 U.S.C. 103 as being unpatentable over Kain (US 2013/0079232 A1; as cited in the IDS dated 02/23/2022), as evidenced by Sigma (Atto Dyes for Superior Fluorescent Imaging [online]. Millipore Sigma; 2025 [retrieved 2025 Jul 11]. Retrieved from the Internet: https://web.archive.org/web/20250322155819/https://www.sigmaaldrich.com/US/en/technical-documents/protocol/research-and-disease-areas/cancer-research/atto-dyes-for-superior-fluorescent-imaging) and Lumiprobe (AMCA NHS Ester [online]. Lumiprobe; 2023 [retrieved 2025 Jul 11]. Retrieved from the Internet: https://www.lumiprobe.com/p/amca-nhs-ester).
Regarding claims 1 and 21, Kain teaches a method for determining the sequence of a target polynucleotide (claim 1):
contacting a primer polynucleotide with a mixture comprising one or more of a first type of nucleotide, a second type of nucleotide, a third type of nucleotide, and a fourth type of nucleotide, wherein the primer polynucleotide is complementary to at least a portion of the target polynucleotide (claim 1: “detecting in a sequencing reaction the incorporation of three different types of detectable nucleotide conjugates into a polynucleotide and determining the incorporation of a fourth type of nucleotide”; claims 2-3), wherein the polynucleotide may be attached to a substrate [i.e., solid support] (para [0017]; [0140])
incorporating one type of nucleotide from the mixture to the primer polynucleotide to produce an extended primer polynucleotide (claim 1: “the incorporation of three different types of detectable nucleotide conjugates is detected from a signal state and wherein the incorporation of the fourth type of nucleotide is determined from a dark state”; instant claim 21)
performing a first imaging event using a first excitation light source and collecting a first emission signal from the extended primer polynucleotide with a first emission filter (claim 5; claim 8; para [0011]); and
performing a second imaging event using a second excitation light source and collecting a second emission signal from the extended primer polynucleotide with a second emission filter (claim 5: “the one or more different fluorescent moieties are detected by one or more emission filters”; claim 8: “the fluorescence detection pattern is determined by a first and a second imaging event”; para [0011]: “A first image obtained using the first excitation wavelength and emission in the first channel can detect and show features that incorporate the first and/or third nucleotide type (e.g. A and/or T). A second image obtained using the second excitation wavelength and emission in the second channel can detect and show features that incorporate the second and/or third nucleotide type (e.g., C and/or T).”)
Kain teaches the use of two or more dyes of different fluorescence spectra (para [0008]). Kain further teaches labeled dNTPs and non-natural analogues thereof comprising fluorescent moieties (claim 4) and/or a nucleotide conjugate comprising a nucleotide linked to a hapten, which may comprise biotin, wherein the nucleotide conjugate may further comprise a streptavidin-fluorescent moiety conjugate (para [0018]).
Kain teaches exposing fluorescent moieties to the appropriate wavelength of light and recording during an imaging event (para [0044]), wherein said wavelength is the light of the excitation wavelength for the fluorescent moiety (para [0050]). Kain teaches that fluorescently labelled nucleotides are typically excited and measured by one optical filter for each distinct dye (para [0024]).
Kain teaches a dye set comprising Atto 465, wherein the dye set comprises an emission offset of at least 60 nm (para [0112]). Kain teaches a dye with an excitation maximum of about 453 nm and an emission maximum of about 508 nm by teaching Atto 465, as evidenced by Sigma. While Kain does not explicitly teach the excitation/emission maxima of Atto 465, it is inherently taught because these are inherent properties of the fluorophore, as taught by Sigma (Reactive Atto Dyes and Conjugates).
Kain also teaches the exemplary fluorescent moiety AMCA-NHS (para [0117]). In teaching AMCA-NHS, Kain teaches a dye with an excitation maximum of about 348 nm and emission maximum of about 435 nm, as evidenced by Lumiprobe. While Kain does not explicitly teach the excitation/emission maxima of AMCA-NHS, it is inherently taught because these are inherent properties of the fluorophore, as taught by Lumiprobe (Spectral properties).
Kain teaches that embodiments of the present disclosure provide [methods that would decrease size and/or cost associated with fluorescence detection with more time efficiency, lower reagent usage, and smaller less expensive instrumentation; see para 0006] by providing investigators with methods for determination of a polymer nucleic acid sequence comprising using a minimal dye set, minimal light sources, and minimal excitation/emission filters, while still allowing for the differentiation of different nucleotides incorporated in a sequencing reaction (para [0007]).
Thus, in teaching the use of appropriate sources and filters and dyes with spectra in the claimed ranges, Kain teaches an excitation light source with a wavelength of about 350nm to about 410 nm and an emission filter with detection of about 415 nm to about 450 nm and another excitation light source with a wavelength of about 450 nm to about 460 nm and another emission filter with detection of about 480 nm to about 525 nm.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to simply substitute these dyes of Kain in the methods of Kain and further to use the appropriate sources and filter, as taught by Kain, motivated by the desire to appropriately record the dyes and to reduce the size, cost, time, and/or use of reagents associated with sequencing, as taught by Kain. There would have been a strong expectation for success and a further motivation to select this pair of dyes (i.e., substitute for the dyes taught in other examples) as Kain teaches that emission spectra off-set should be at least 60 nm (AMCA-NHS and Atto465 have an offset of about 73 nm) and this amounts to applying a known technique to a known method to predictable results.
Further, it is noted that the courts have found that “where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). See MPEP 2144.05 II. Thus, the claimed ranges of light sources and excitation filters merely represent routine optimization of the values of the cited prior art.
Applicant is advised that MPEP 716.01(c) makes clear that “[t]he arguments of counsel cannot take the place of evidence in the record” (In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965)). Thus, Applicant should not merely rely upon counsel's arguments in place of evidence in the record.
Regarding claims 2-4, in the method of Kain evidenced by Sigma and Lumiprobe, Kain teaches a method of utilizing four types of nucleotide analogues wherein a first type is conjugated to a first detectable label [i.e., Atto 465] the (instant claim 2), a second type is conjugated to a second detectable label [i.e., AMCA-NHS] (instant claim 3), wherein the first label and second label are spectrally distinguishable, and a third type comprising a mixture of the nucleotide conjugated to the first detectable label or to the second detectable label (instant claim 4), wherein the first and second detectable labels utilize different excitation and emission spectra (para [0087- 0095]).
As above, it follows that that the chosen dye pairs would be utilized with appropriate sources and filters under the same motivation and expectation for success as above.
Regarding claims 15 and 17, in the method of Kain evidenced by Sigma and Lumiprobe, Kain teaches a labelling method using two detectable labels excitable with different light sources and detectable with different detection filters wherein a first type of nucleotide is labeled with a first detectable label, a second type of nucleotide is labeled with a second type of detectable label, and a third type of nucleotide is labeled with a mix of the two detectable labels (para [0087-0095]). Kain further teaches a method wherein a first type of nucleotide is labeled with a first detectable label, a second type of nucleotide is attached to biotin, a third type of nucleotide is a mixture of the nucleotide that is unlabeled and that is labeled with the first type of detectable label, wherein the biotin is conjugated to a fluorescently labeled streptavidin prior to a second imaging event (para [0055-0062]).
Kain further teaches a nucleotide conjugate comprising a nucleotide linked to a hapten, which may comprise biotin, wherein the nucleotide conjugate may further comprise a streptavidin-fluorescent moiety conjugate (para [0018]; also para [0116-117])).
Kain teaches that AMCA-NHS is a fluorescent moiety (para [0117]), wherein fluorescent moieties may be conjugated to a binding partner as part of a hapten, wherein a hapten comprises biotin (para [0116]]).
Kain does not explicitly teach contacting the extended primary polynucleotide within an affinity reagent prior to the first imaging event or wherein the affinity reagent carries a second detectable label excitable by a second excitation light source and detectable by a second emission filter.
However, first, it is held to be prima facie obvious variant to change the sequence of adding ingredients such that the streptavidin with the fluorescent label may be added prior to the first imaging event rather than the second imaging event. With respect to the order of steps, it is noted that the courts have held that any order of performing process steps is prima facie obvious in the absence of new or unexpected results (In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930); Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959)). See MPEP §2144.04 IV C.
Second, given that in method of Kain evidenced by Sigma and Lumiprobe, Kain teaches a method of attaching detectable labels via an affinity label conjugated to a hapten such as biotin and (wherein the affinity reagent would correspondingly be streptavidin; see para [0164] and [0173], for example) and a two-color method of sequencing by synthesis as cited above, it would have been obvious to one of ordinary skill before the effective filing date of the claimed invention to combine these methods to conjugate one of the dyes in a two-color SBS system so as to utilize the biotin and streptavidin as all of the elements are taught, motivated by the desire to reduce the size, cost, time, and/or use of reagents associated with sequencing, as taught by Kain (see claim 1). There would be a strong expectation for success as AMCA-NHS is taught as a fluorescent moiety, wherein fluorescent moieties are taught as compatible with hapten conjugation.
Regarding claims 18 and 20, in the method of Kain evidenced by Sigma and Lumiprobe, it follows therefore, given the same reasons as in claims 15 and 17, that this is also an obvious variant as the identities of the “first type” and “second type” of nucleotide are not further specified and thus are interchangeable so long as there two distinct types, given that claim 18 also only depends from claim 1. Likewise, the “first” and “second” detectable labels of each are also interpreted to be interchangeable so long as there are two distinct types, as the first and second light sources and emission filters may utilize either of the sets of relevant wavelength ranges so long as both sets are utilized.
Regarding claim 22, in the method of Kain evidenced by Sigma and Lumiprobe, Kain teaches the nucleotide conjugate comprises nucleotide types selected from the group consisting of dATP, dTTP, dUTP, dCTP, dGTP or non-natural nucleotide analogs thereof (claim 2). Kain further teaches the non-natural nucleotide analog comprises a reversible terminator moiety and is selected from the group consisting of rbATP, rbTTP rbUTP, rbCTP and rbGTP (claim 3). Kain additionally teaches the modified base is reversibly blocked wherein the NTP comprises a reversible terminator 3' blocking group which, once removed, allows for continued extension in a sequence by synthesis sequencing reaction; the 3' blocking group may comprise an azido and/or alkoxy group and is removable by cleavage with a phosphine reagent; and that nucleotides are termed “reversibly blocked' or “rb’, a type of which is a “fully functional” or “ff NTP, which is commercially available at Illumina, Inc. (para [0128]).
Regarding claim 24, in the method of Kain evidenced by Sigma and Lumiprobe, Kain teaches that blockers present on the incorporated nucleotides are removed and washed away along with other reagents present after the second imaging event in preparation for the next sequencing cycle (para [0040]), wherein the blockers may be a 3’ terminator that comprises, on the sample 3' ribose position, both alkoxy and azido functionalities which is removable by cleavage with a phosphine reagent, thereby creating a nucleotide that is reversibly blocked and once again functional for further elongation (i.e., fully functional or ff) (para [0043]).
Regarding claim 25-26, in the method of Kain evidenced by Sigma and Lumiprobe, Kain teaches that disclosed methods for nucleic acid detection may comprise sequential addition of one or more fluorescently labeled nucleotides to a growing polynucleotide chain in the 5' to 3' direction using a polymerase and determining the identity of an incorporated nucleotide in the extended chain after two imaging steps thereby providing real time incorporation sequence data (para [0129]; also para [0017]: “determining a plurality of nucleic acid sequences”). Kain teaches a method utilizing two dyes, one of which is conjugated using streptavidin, comprising:
a standard incorporation step
a step wherein clusters were laser excited and a fluorescent image was acquired
a subsequent second imaging
a standard de-block and incorporation step
wherein rbNTPs are used and wherein the sequencing was repeated for at least 100 cycles (Example 4); Example 5 of Kain teaches 150 cycles (instant claim 26).
Thus, Kain teaches repeating the steps of sequencing for least 50 cycles.
Therefore, while Kain does not explicitly teach the steps (a)-(e) repeated at least 50 time, in teaching the combination of (a)-(e) and repeating cycles at least 50 times, the combination would have been obvious to one of ordinary skill before the effective filing date of the claimed invention, motivated by the same rationale as previous claims with a strong expectation of success as this is applying a known technique to known method.
Regarding claim 33 and 35, in the method of Kain evidenced by Sigma and Lumiprobe, Kain teaches that the method is performed on a substrate, wherein multiple nucleic acid targets are detected and sequenced in parallel, for example in a flow cell or array type format (para [0035]). Kain teaches that clonally amplified target sequences are typically covalently immobilized on the substrate, wherein the immobilization may be in channels of a flow cell or specific locations on an array (para [0035]). Kain teaches performing patterned photolithography and dry etching so as to create discrete locations [i.e., patterned nanowells; see also para 0037] for immobilization of nucleic acids for use in sequencing methodologies (para [0038]).
Thus, Kain teaches performing the method using immobilized target polynucleotides, in parallel, in an array format, on a patterned flow cell, and with patterned nanowells.
Kain teaches that many methods exist for creating slides/chips with discrete locations for immobilization of nucleic acids for use in sequencing methodologies and the present methods are not limited by the method in which a substrate is prepared for sequencing (para [0038]).
While Kain doesn’t explicitly teach the combination of the array format and flow cell, such would be obvious at least under MPEP 2144.04(IV)(V) Changes in shape and because the all the elements were taught, the artisan would find it obvious to combine them motivated by the same rationale as above with a strong expectation for success as Kain teaches that each of the types of substrates are compatible and the methods are not limited by the method of the substrate preparation.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Kain (US 2013/0079232 A1; as cited in the IDS dated 02/23/2022), as evidenced by Sigma (Atto Dyes for Superior Fluorescent Imaging [online]. Millipore Sigma; 2025 [retrieved 2025 Jul 11]. Retrieved from the Internet: https://web.archive.org/web/20250322155819/https://www.sigmaaldrich.com/US/en/technical-documents/protocol/research-and-disease-areas/cancer-research/atto-dyes-for-superior-fluorescent-imaging) and Lumiprobe (AMCA NHS Ester [online]. Lumiprobe; 2023 [retrieved 2025 Jul 11]. Retrieved from the Internet: https://www.lumiprobe.com/p/amca-nhs-ester), as applied to claim 1 above and as further evidenced by FluoroFinder (Cascade Blue Dye [online]. FluoroFinder; 2025 [retrieved 2025 Jul 11]. Retrieved from the Internet: https://app.fluorofinder.com/dyes/19-cascade-blue-ex-max-396-nm-em-max-410-nm) and in further view of Romanov (WO 2019/0077331 A1; published 04/25/2019).
Regarding claim 5, in the method of Kain evidenced by Sigma and Lumiprobe, Kain teaches a method of using set of four dyes detectable with two imaging events to perform sequencing by synthesis, i.e., utilizing one type of nucleotide comprising a mixture of a third type of nucleotide labeled with a third label and a third type of nucleotide with a fourth label, wherein the third label is excitable by a first excitation light source and detectable by a first emission filter and wherein the fourth label is excitable by the second excitation light source and detectable by the second emission filter (para [0101-0109]).
Kain also teaches the exemplary fluorescent moiety Cascade Blue (para [0117]). In teaching Cascade Blue, Kain teaches a dye with an excitation maximum of about 396 nm and emission maximum of about 410 nm, as evidenced by FluoroFinder. While Kain does not explicitly teach the excitation/emission maxima of Cascade Blue, it is inherently taught because these are inherent properties of the fluorophore, as taught by FluoroFinder (Profile).
Thus, Kain teaches a fourth label excitable by the second light source and detectable by the second emission filter.
As cited above, Kain teaches in the example with set of two dye sets [that can be detected together], that the example is not limited to any particular two dye sets or conjugate combinations and any two dye sets of different emission spectra could be used, and further that, in any combination of rbNTP-dye conjugate combination while following the strategy for conjugation as disclosed herein [e.g. different intensity dyes are used] (para [0097]; see also [0099]).
Kain teaches that the fluorescent moieties of the invention may include coumarin and coumarin derivatives (para [0117]).
Kain fails to explicitly teach a second dye (i.e., third label) detectable by the first excitation source and detectable by the first emission filter.
Romanov rectifies this by teaching coumarin compounds useful for fluorescent labels for nucleic acids in nucleic acid sequencing applications (Abstract). Romanov teaches at least four exemplary dye compounds (I-1-1, I-1-3, I-1-4, I-2A) that have absorbance maxima between about 450 nm and about 460 nm and have emission maxima between about 480 nm and about 525 nm (Table 2). Romanov teaches a comparison of intensity to Atto465 wherein the intensity is >2X (Table 2).
Romanov teaches that the dyes of the invention absorb light optimally at a wavelength of 450-460 nm, wherein blue wavelength excitation allows detection and resolution of a higher density of features per unit area due to the shorter wavelength of fluorescence emission, and wherein when used in conjugates with nucleotide, improvements are seen in the length, intensity, accuracy, and quality of sequencing reads (pg. 3, Summary, para 1).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further substitute the Cascade Blue of Kain for the fourth label of Kain and one of the dyes of Romanov with absorbance between 450-460 nm. The artisan would have been motivated first by the desire to reduce the size, cost, time, and/or use of reagents associated with sequencing, as taught by Kain above, and further to utilize blue wavelength excitation to improve the method of sequencing by allowing to an improved density of detection and features, as taught by Romanov. There would be a high expectation of success as the dyes were known and taught for sequencing.
Further, it remains obvious that selecting appropriate sources and filters is obvious under Kain as above and that it is noted that the courts have found that “where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). See MPEP 2144.05 II. Thus, the claimed ranges of light sources and excitation filters merely represent routine optimization of the values of the cited prior art.
Applicant is advised that MPEP 716.01(c) makes clear that “[t]he arguments of counsel cannot take the place of evidence in the record” (In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965)). Thus, Applicant should not merely rely upon counsel's arguments in place of evidence in the record.
Claims 6-7, 9, and 11-13 are rejected under 35 U.S.C. 103 as being unpatentable over Kain (US 2013/0079232 A1; as cited in the IDS dated 02/23/2022), as evidenced by Sigma (Atto Dyes for Superior Fluorescent Imaging [online]. Millipore Sigma; 2025 [retrieved 2025 Jul 11]. Retrieved from the Internet: https://web.archive.org/web/20250322155819/https://www.sigmaaldrich.com/US/en/technical-documents/protocol/research-and-disease-areas/cancer-research/atto-dyes-for-superior-fluorescent-imaging) and Lumiprobe (AMCA NHS Ester [online]. Lumiprobe; 2023 [retrieved 2025 Jul 11]. Retrieved from the Internet: https://www.lumiprobe.com/p/amca-nhs-ester), as applied to claim 1 above and in further view of Drmanac 2022 (US 2022/0162693 A1; effectively filed Nov. 9, 2018) and Drmanac 2017 (US 2017/0240961 A1; published 08/24/2017).
Regarding claim 6-7, 9, 11, and 13, in the method of Kain evidenced by Sigma and Lumiprobe, Kain teaches a method wherein at least two of the nucleotides are unlabeled and the extended primary polynucleotide is contacted with an affinity reagent prior to a second imaging event, wherein the affinity reagent bind specifically to the incorporated nucleotide (para [0055-0061]).
Kain further teaches a first type of nucleotide is conjugated to a first detectable label, a second type is conjugated to a second detectable label, wherein the first label and second label are spectrally distinguishable, a third type of nucleotide comprising a mixture of the nucleotide conjugated to a first detectable label or to a second detectable label, wherein the first and second detectable labels utilized different excitation and emission spectra (para [0087- 0095]).
Kain teaches multiple haptens—including biotin, digoxigenin and dinitrophenol—and affinity reagents—including anti-hapten antibodies, such as anti-digoxigenin and anti-dinitrophenol, or streptavidin (para [0016]), i.e., Kain teaches a set of affinity reagents.
However, Kain fails to explicitly teach the use of a set of affinity reagents for labeling multiple unlabeled nucleotides.
Drmanac 2022 rectifies this by teaching a method for sequencing that employs all non-labeled reversible terminator nucleotides (para [0009]) that is identified by binding of an affinity reagent (e.g., antibody, aptamer, aptamer, knottin, etc.) in the last incorporated nucleotide (para [0008]). Drmanac 2022 also incorporates by reference Drmanac 2017 and teaches the method therein of utilizing an affinity tag [hapten] attached via a linker to the nucleotide (para [0042]). Drmanac 2022 further teaches that the affinity reagent (e.g., antibody) may be directly labeled (e.g., a fluorescent labeled antibody), i.e., by conjugation to a fluorophore, or may be detected indirectly (e.g., by binding of a labeled anti affinity reagent secondary affinity reagent) (para [0043]). Drmanac 2022 further teaches a method wherein a DNA molecule being sequenced is contacted with a primer to form a hybrid and wherein the hybrid is contacted with a set of four different nucleotide analogs to form extended primers, wherein a first labelling reaction is performed by contacting the extended primers with an affinity reagent to form a first reaction product, wherein the affinity reagents comprise:
a first affinity reagent specific for one of the four nucleotide analogs, bearing a label that fluoresces at a first wavelength, and
a second affinity reagent specific for another of the analogs, bearing a label that fluoresces at a second wavelength
determining the nucleotide analogue that has been added in each of products of the first reaction by measuring fluorescence at the first and second wavelengths (claim 59).
Drmanac 2022 further teaches that the label may comprise fluorogenic dyes including fluorescein, a rhodamine, a phenoxazine, an acridine, a coumarin, or a derivative (para [0065]). Drmanac 2022 further teaches that the hapten may be selected from the hapten is selected from biotin, digoxigenin and dinitrophenol and the nucleotide conjugate may comprise a streptavidin-fluorescent moiety conjugate or an anti-hapten antibody-fluorescent moiety conjugate selected from the group consisting of anti-digoxigenin and anti-dinitrophenol (para [0015]).
Drmanac 2022 additionally teaches that bases without linked labels may offer cost savings and improved incorporation rates (para [0007] and [0051]), and that current [i.e., other] SBS methods require a cleavable linker that results in a chemical scar, less efficient incorporation, quenching, excited induced termination of extension, and reduced signal in each sequencing cycle (para [0007]). Drmanac 2022 further teaches that this method enables an artisan to vary the intensity by attaching different numbers of dye molecules by antibody, for example, cheaply to create different four distinct intensities (para [0283]).
Therefore, it would have been obvious to one of ordinary skill before the effective filing date of the claimed invention to combine the method of Kain evidenced by Sigma and Lumiprobe with the methods of Drmanac 2022 and Drmanac 2017 of using multiple unlabeled nucleotides with a set of affinity reagents specific to each nucleotide in order reduce costs and/or increase incorporation rates of nucleotides into the extension polynucleotide and/or to enable the addition of more information for sequencing systems through varying image intensity by more cheaply adding additional dye molecules and/or to enable the use of commercial available affinity reagents bound to dyes for faster testing of dye combinations, as taught by Drmanac 2022. There would have been a strong expectation for success as Drmanac 2022 and Drmanac 2017 teach overlapping affinity reagents and haptens with Kain, as well as compatible/overlapping dye classes, and because these are known techniques being combined with known methods.
It likewise would have been obvious to one of ordinary skill before the effective filing date of the claimed invention to utilize a hapten on two or more nucleotides, such as the first and second nucleotides, in conjunction with an appropriate first affinity reagent that comprises a first hapten-binding partner that specifically binds to the first hapten and a second hapten-binding partner that specifically binds to the second hapten—given that such a method is taught by Kain as well as Drmanac 2022 and Drmanac 2017—motivated by the same motivations as above with the same expectation for success.
Additionally, it would have to one of ordinary skill before the effective filing date of the claimed invention to utilize the method of sequencing utilizing a mixture of the first and second labels on the third nucleotide taught by Kain in the method of Kain evidenced by Sigma and Lumiprobe with the method of attaching dyes through affinity reagents taught by Drmanac 2022 and Drmanac 2017 to arrive at a third nucleotide that was a mixture of a third nucleotide with any of the combinations of two, for example, haptens-linked to the type of nucleotide for the same reasons articulated above the same expectations of success.
Therefore, it follows that artisans of ordinary skill would likewise choose appropriate excitation light sources and detection filters in the method as discussed above.
Regarding claim 12, in the method of Kain evidenced by Sigma and Lumiprobe, Kain teaches the use of a biotin moiety and a hapten-binding partner comprising streptavidin in numerous exemplary methods (e.g., para [0054-0061]).
Therefore, it would have been obvious to one of ordinary skill before the effective filing date of the claimed invention use streptavidin as one of the affinity reagents of the set, motivated by the same rationales and expectations for success as above.
It is noted that as the first type of nucleotide may still have any identity (i.e., base), the detectable label that complies with the wavelength requirements of either the first set of spectra or the second, etc., the choice of biotin and streptavidin for it specifically as opposed to say, the third, appears to be primarily a matter of nomenclature (i.e., it was picked arbitrarily above for convince in this Office Action). For argument’s sake, it additionally would be an obvious variant of the art based on a rearrangement of parts under MPEP 2144.04(VI)(C) or as a rearrangement of steps as the courts have held that any order of performing process steps is prima facie obvious in the absence of new or unexpected results (In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930); Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959)). See MPEP §2144.04 IV C, absent any showing that the order is essential.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Kain (US 2013/0079232 A1; as cited in the IDS dated 02/23/2022), as evidenced by Sigma (Atto Dyes for Superior Fluorescent Imaging [online]. Millipore Sigma; 2025 [retrieved 2025 Jul 11]. Retrieved from the Internet: https://web.archive.org/web/20250322155819/https://www.sigmaaldrich.com/US/en/technical-documents/protocol/research-and-disease-areas/cancer-research/atto-dyes-for-superior-fluorescent-imaging) and Lumiprobe (AMCA NHS Ester [online]. Lumiprobe; 2023 [retrieved 2025 Jul 11]. Retrieved from the Internet: https://www.lumiprobe.com/p/amca-nhs-ester) in view of Drmanac 2022 (US 2022/0162693 A1; effectively filed Nov. 9, 2018), Drmanac 2017 (US 2017/0240961 A1; published 08/24/2017), as applied to claim 7 above and as further evidenced by FluoroFinder (Cascade Blue Dye [online]. FluoroFinder; 2025 [retrieved 2025 Jul 11]. Retrieved from the Internet: https://app.fluorofinder.com/dyes/19-cascade-blue-ex-max-396-nm-em-max-410-nm) and in further view of Romanov (WO 2019/0077331 A1; published 04/25/2019).
Regarding claim 10, in the method of Kain evidenced by Sigma and Lumiprobe in view of Dramanac 2022 and Dramanac 2017, as cited above Kain teaches at least three types of affinity reagents.
As cited in claim 5 above, Kain teaches Cascade Blue [i.e., a fourth detectable label excitable with the second source and detectable with the second filter], which has an excitation maximum of about 396 nm and emission maximum of about 410 nm, as evidenced by FluoroFinder.
Kain further teaches a method wherein four different detectable labels are used two of which are detectable by one emission filter and two of which are detectable by another, wherein two sets of fluorescent dyes can be excited in a step by step manner, such as first exciting one set of fluorophores followed by a first imaging event and then exciting the second set of fluorophores followed by a second imaging event (para [0100-08]). However, Kain fails to teach a method where a fourth nucleotide is labeled with one or more detectable label and wherein multiple affinity reagents are used to conjugate detectable labels.
Romanov partially rectifies this by teaching a four suitable labels with excitation peaks of about 450 nm to about 460 nm as discussed in claim 5 above.
Drmanac 2022 further rectifies this by teaching a method of sequencing wherein:
a third affinity reagent specific for one of the two remaining analogs, bearing a label that fluoresces or generates a product that fluoresces at the first wavelength, and
a fourth affinity reagent specific for the fourth analog, bearing a label that fluoresces or generates a product that fluoresces at the second wavelength (claim 59)
Therefore, it would have been to one of ordinary skill to further substitute the dyes in the method of Kain evidenced by Sigma and Lumiprobe in view of Drmanac 2022 and Drmanac 2017 with the Cascade Blue of Kain and suitable dye of Romanov, as discussed in claim 5 with the same motivation and expectation for success. Further, it would have been obvious to the artisan to further substitute the nucleotides in the method of further steps (c) and (d) above with the two dyes, motivated by the same motivations as claim 7 above with the same expectation of success.
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Kain (US 2013/0079232 A1; as cited in the IDS dated 02/23/2022), as evidenced by Sigma (Atto Dyes for Superior Fluorescent Imaging [online]. Millipore Sigma; 2025 [retrieved 2025 Jul 11]. Retrieved from the Internet: https://web.archive.org/web/20250322155819/https://www.sigmaaldrich.com/US/en/technical-documents/protocol/research-and-disease-areas/cancer-research/atto-dyes-for-superior-fluorescent-imaging) and Lumiprobe (AMCA NHS Ester [online]. Lumiprobe; 2023 [retrieved 2025 Jul 11]. Retrieved from the Internet: https://www.lumiprobe.com/p/amca-nhs-ester) in view of Drmanac 2022 (US 2022/0162693 A1; effectively filed Nov. 9, 2018), Drmanac 2017 (US 2017/0240961 A1; published 08/24/2017), as applied to claim 13 above and in further view of Yazaki (Yazaki J, et al. HaloTag-based conjugation of proteins to barcoding-oligonucleotides. Nucleic Acids Res. 2020 Jan 24;48(2):e8.).
Regarding claim 14, in the method of Kain evidenced by Sigma and Lumiprobe in view of Drmanac 2022 and Drmanac 2017, Kain teaches the use of haptens and corresponding hapten binding partners but fails to teach chloroalkyl groups and HaloTags.
Yazaki rectifies this by teaching the use of a chloroalkyl group on a DNA oligonucleotide to covalently bind a HaloTag (Fig. 1) and report increased sensitivity compared to conventional protein binding assays involving luciferase (Abstract). Yazaki further teaches the click chemistry involved in the HaloTag binding had advantages including pH insensitivity and reactivity in water (Introduction). Yazaki further utilizes the method of attaching the chloroalkyl group on a DNA oligonucleotide that is a sequencing barcode (Fig. 1).
Yazaki further teaches that size of the HaloTag (33 kDa) is relatively small compared with that of avidin (∼70 kDa) or streptavidin analogs (∼50 kDa) and HaloTag also binds in a 1:1 ratio instead of 1:4, as for of biotin–avidin [or streptavidin] binding (pg. e8, col 2).
Therefore, 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 method of Kain evidenced by Sigma and Lumiprobe in view of Drmanac 2022 and Drmanac 2017 by substituting one of the haptens for the chloroalkyl group of Yazaki, motivated by the desire the decrease the size relative to biotin/streptavidin and/or to decrease the pH sensitivity and reactivity in aqueous environments, as taught by Yazaki. There would be a strong expectation for success as this applying known technique to a known method.
Claim 37 is rejected under 35 U.S.C. 103 as being unpatentable over Kain (US 2013/0079232 A1; as cited in the IDS dated 02/23/2022), as evidenced by Sigma (Atto Dyes for Superior Fluorescent Imaging [online]. Millipore Sigma; 2025 [retrieved 2025 Jul 11]. Retrieved from the Internet: https://web.archive.org/web/20250322155819/https://www.sigmaaldrich.com/US/en/technical-documents/protocol/research-and-disease-areas/cancer-research/atto-dyes-for-superior-fluorescent-imaging) and Lumiprobe (AMCA NHS Ester [online]. Lumiprobe; 2023 [retrieved 2025 Jul 11]. Retrieved from the Internet: https://www.lumiprobe.com/p/amca-nhs-ester), as applied to claim 33 above and in further in view of Illumina (Optimizing Cluster Density on Illumina Sequencing Systems [Internet]. Illumina; 2016 [accessed 2025 Jan 3]. Available from: https://www.illumina.com/content/dam/illumina-marketing/documents/products/other/miseq-overclustering-primer-770-2014-038.pdf).
Regarding claim 37, in the method of Kain evidenced by Sigma and Lumiprobe, Kain incorporates by reference US 2008/0009420 A1, which teaches that a preferred density of single stranded polynucleotide molecules for use in the invention is typically 10,000/mm2 to 100,000/mm2 but higher densities of 100,000/mm2 to 1,000,000/mm2 may be achieved (US 2008/0009420 A1 para. [0079]). Kain teaches that its inventions can be used with a system provided by Illumina such as a MiSeq (para [0145]). Therefore, Kain teaches 100k/mm2.
However, Kain fails to teach a range specifically bounded at about 300 k/mm2.
Illumina rectifies this by teaching two commercially available machines, including the MiSeq, with optimal densities of 170-220 k/mm2 given current nucleotide chemistries (pg. 8, c. Flow Cell Loading) (wherein nucleotide chemistries are known to mean fluorescent labelling methods in the art) and means for optimizing the density of flow cells (pg. 7, IV. Common Causes of Under- and Overclustering and Strategies for Prevention, spanning pg. 8).
Therefore, 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 method of Kain with the optical density of Illumina, motivated to optimize for the MiSeq machine taught for both, wherein there would be a strong expectation of success as both utilize the same machine.
Further, is noted that the courts have found that “where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). See MPEP 2144.05 II.
Claim 60 is rejected under 35 U.S.C. 103 as being unpatentable over Kain (US 2013/0079232 A1; as cited in the IDS dated 02/23/2022), as evidenced by Sigma (Atto Dyes for Superior Fluorescent Imaging [online]. Millipore Sigma; 2025 [retrieved 2025 Jul 11]. Retrieved from the Internet: https://web.archive.org/web/20250322155819/https://www.sigmaaldrich.com/US/en/technical-documents/protocol/research-and-disease-areas/cancer-research/atto-dyes-for-superior-fluorescent-imaging) and Lumiprobe (AMCA NHS Ester [online]. Lumiprobe; 2023 [retrieved 2025 Jul 11]. Retrieved from the Internet: https://www.lumiprobe.com/p/amca-nhs-ester), as applied to claim 1 above and in further in view of Fedurco (US 9,217,178 B2; granted 12/22/2015).
Regarding claim 60, in the method of Kain evidenced by Sigma and Lumiprobe, Kain teaches the use of a “Scan Mix (SMX)” buffer (para [0162]) but fails to disclose the composition of the buffer solution. Therefore, it fails to explicitly teach a buffer in use during the imaging events that contains one or more antioxidants.
Fedurco rectifies this by teaching a method of inhibiting light-induced degradation of nucleic acids during a detection step of nucleic acid sequencing comprising irradiating a template nucleic acid in the presence of a detection buffer comprising ascorbic acid, or a salt thereof, and determining the identify of one or more of the incorporated nucleotides (claim 1) and wherein the buffer further comprises additional antioxidants (claim 7). Fedurco further teaches that light-induced damage may interfere with the ability to read incorporated fluorophores (col. 1, para 6) and that it is well known in the art to add ascorbic acid to fluorescent imaging buffers (col. 2, para 1). Fedurco likewise teaches that the buffer may have a pH of 5.5 to 8.6 (claim 8) and have an ascorbic acid concentration of at least 10 mM to at least 100 mM ascorbic acid (claim 3-5).
Therefore, it would have been prima facie obvious to one of ordinary skill before the effective filing date of the claimed invention to combine the method of Kain evidenced by Sigma and Lumiprobe with the buffer solution that includes one or more antioxidants of Fedurco in order to reduce the light-induced damage and improve the ability to read incorporated fluorophore in the imaging buffer solution. There would have been a strong expectation of success as Fedurco provides a wide range of effective pH and antioxidant concentrations that would enable one of ordinary skill to include the ascorbic acid and/or additional antioxidants in the Scan Mix of Kain according to its requirements within routine experimentation.
Response to Arguments
Applicant's arguments filed 10/08/2025 have been fully considered but they are not persuasive.
Regarding the 103 rejection over Kain evidenced by Sigma and Lumiprobe, Applicant argues that there is no reason for the artisan to use the dye combination of Atto 465 and AMC-NHS in the method of Kain as suggested.
Applicant argues that Atto 465 is disclosed in in the context of a strategy that utilizes two dye sets of similar emission spectra wherein one of the two dyes emits at a detectably higher intensity than the other dye in the set, and wherein the two dye sets differ in fluorescence emission spectra. Therefore, it is alleged that without a showing that there is a higher intensity of Atto 465 or AMCA-NHS, the Examiner has failed to establish a case of obviousness.
Applicant further argues that AMCA-NHS is disclosed as a detection moiety for secondary labelling, citing paragraph [0116]; thus, the artisan would not have been motivated to use Atto465 in combination with AMCA-NHS because Atto 465 is used in a sequencing method that distinguishes the incorporated nucleotide based on both spectra and intensity of the labels.
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, appropriately record the dyes and to reduce the size, cost, time, and/or use of reagents associated with sequencing, as taught by Kain (para [0006-0007]). There would have been a strong expectation for success and a further motivation to select this pair of dyes (i.e., substitute for the dyes taught in other examples) as Kain teaches that emission spectra off-set should be at least 60 nm (AMCA-NHS and Atto465 have an emission offset of about 73 nm).
It is also noted that substituting equivalents known for the same purpose is prima facie obvious, wherein each is taught as a dye and/or fluorescent moiety (which the artisan would understand to be usable for the same purpose herein given the generic recitation and use for fluorescent imaging in the examples) for labelling nucleotides and the bounds for requisite emission spectra distances are taught by Kain. See MPEP 2144.06(II).
Regarding the arguments of about selection of dyes, Kain teaches multiple embodiments, and indeed Kain teaches that the configurations are not mutually exclusive and can be used in various combinations (para [0010]). Reflecting this, the cited claims of Kain recite broad methods the encompass the intensity-based methods and the hapten-based method discussed in the Remarks (pgs. 10-11). Indeed, the claims generically recite “fluorescent moieties” (e.g., claim 8), which is how is AMCA-NHS described in para [0117]. Thus, the argument that the artisan would not consider using Atto 465 with AMCA-NHS is not considered persuasive.
Likewise, it is noted that para [0009] recites that a pair of nucleotide types can distinguished based on a difference in intensity for one member of the pair compared to the other or based on a change to one member of the pair (e.g., via chemical modification or physical modification), which is interpreted to refer to the hapten binding and/or the cleavage events, which are encompassed in the cited claims (see, e.g., claim 17). As such, the argument that a lack of showing of respective intensities negates a showing of obviousness is not found to be persuasive.
However, for the sake of compact prosecution, it is also noted that Lumiprobe teaches that AMCA-NHS has an ε [i.e., molar absorptivity] of 17400 L⋅mol^-1⋅cm^-1 (pg. 4). Sigma teaches that Atto 465 has an ε of 45,000 M^-1*cm^-1 and a ηem [quantum yield] of 90%. Accordingly, even should AMCA-NHS have a 100% ηem, the intensity of emitted photons for Atto 465 would be about 2.3X.
For these reasons, the arguments are not considered persuasive.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Emma R Hoppe whose telephone number is (703)756-5550. The examiner can normally be reached Mon - Fri 11:00 am - 7:00 pm.
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/EMMA R HOPPE/Examiner, Art Unit 1683
/NANCY J LEITH/Primary Examiner, Art Unit 1636
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