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 .
This action is in response to the papers filed December 19, 2025.
Currently, claims 1, 2, 7, 9-11, 13, 15, 17, 19, 21-23, 25, 27-28, 30, and 34-41 are pending. Claims 28, 30 and 34-35 were withdrawn as directed to a non-elected invention. Claims 3-6 and 29 were canceled by applicant. Claims 37-41 were added in the amendment filed on August 11, 2025. Therefore, claims 1, 2, 7, 9-11, 13, 15, 17, 19, 21-23, 25, 27, and 36-41 are under examination.
Any objections and rejections not reiterated below are hereby withdrawn.
It is acknowledged that no amendments have been made to the claims with the submission of the response filed December 19, 2025.
The arguments presented in the response filed December 19, 2025 have been thoroughly reviewed and are not persuasive for the reasons which follow.
Election/Restrictions
Applicant’s election without traverse of Group I (claims 1-7, 9-11, 13, 15, 17, 19, 21-23, 25, 27, and 36) in the reply filed on October 25, 2024 is acknowledged.
Priority
This application claims the benefit of 63/127,043 filed December 17, 2020.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-2, 7, 9-10, 13, 15, 17, 19, 21-23, and 36-41 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Romanov, WO 2016/189287, published December 1, 2016.
Regarding claims 1-2, Romanov teaches a method of determining the sequence of a target polynucleotide on an automated sequencing instrument… containing two lasers operating at different wavelengths…” and “a single emission channel, which may therefore reduce or avoid the need for multiple emission filters.” (Romanov, page 25, lines 13-30) using fluorescently labelled reversible terminator nucleotides with long stokes shifts. (Romanov, page 22-page 24) Said labeled nucleotides comprise a first nucleotide conjugate comprising a first label, a second nucleotide conjugate comprising a second label, and a third nucleotide comprising a mixture of the first and second labels, (Romanov, page 24, lines 16-25) (i.e. the three labels are spectrally distinct) wherein two or more of the labels may be excited using a different excitation source at different wavelengths, which may be lasers. (Romanov, page 24, line 17-20) Romanov teaches that four separate nucleotides can be identified using only two labels. A first nucleotide can be labeled with a first label, a second nucleotide can be labeled with a second label, a third nucleotide can be labeled with a mixture of the first and second labels, and a fourth nucleotide can be unlabeled. (Romanov, page 24, line 5-10) (i.e. contacting a primer with a mixture comprising nucleotide conjugates, incorporating a nucleotide conjugate into the primer, performing a first imaging event with a first laser (a first light source), performing a second imaging event with a second laser, and collecting the two emission signals in a single emission channel.)
Quoting MPEP 2123:
"The use of patents as references is not limited to what the patentees describe as their own inventions or to the problems with which they are concerned. They are part of the literature of the art, relevant for all they contain." In re Heck, 699 F.2d 1331, 1332-33, 216 USPQ 1038, 1039 (Fed. Cir. 1983) (quoting In re Lemelson, 397 F.2d 1006, 1009, 158 USPQ 275, 277 (CCPA 1968)).
A reference may be relied upon for all that it would have reasonably suggested to one having ordinary skill in the art, including nonpreferred embodiments. Merck & Co. v. Biocraft Labs., Inc. 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir. 1989), cert. denied, 493 U.S. 975 (1989). See also Upsher-Smith Labs. v. Pamlab, LLC, 412 F.3d 1319, 1323, 75 USPQ2d 1213, 1215 (Fed. Cir. 2005) (reference disclosing optional inclusion of a particular component teaches compositions that both do and do not contain that component); Celeritas Technologies Ltd. v. Rockwell International Corp., 150 F.3d 1354, 1361, 47 USPQ2d 1516, 1522-23 (Fed. Cir. 1998) (The court held that the prior art anticipated the claims even though it taught away from the claimed invention. "The fact that a modem with a single carrier data signal is shown to be less than optimal does not vitiate the fact that it is disclosed.")”
In addition to the specific preferred embodiment taught by Romanov previously described above, Romanov teaches that in other embodiments, “each of the different nucleotide triphosphates (A, T, C, and G) may be labelled with a unique fluorophore and also comprises a blocking group at the 3’ position… Alternatively one of the four nucleotides may be unlabeled (dark)” (Romanov, page 41 line 32- page 42 line 5).
Therefore, the full scope of the disclosure of Romanov teaches that three nucleotide conjugates can be labelled with three “unique” fluorophores and the fourth nucleotide conjugate is unlabeled).
Romanov further teaches that sets of spectrally distinguishable fluorescent dyes that can be excited at different wavelengths but emit at the same wavelength (i.e. are excited using different light sources and their emission is collected in a single emission channel) wherein at least one of the dyes exhibits a large Stokes shift are known in the art and that such dye sets can be selected from the prior art (Romanov, page 49, line 12- page 51, line 26).
Romanov further teaches “Each [label] is only excited by one of the excitation wavelengths, and thus the dyes are separately detectable due to their distinct excitation profiles. In particular embodiments, the excitation from the two or more labels will occur in different regions of the spectrum such that presence of at least one of the labels can be determined by optically distinguishing the excitation.” (i.e. the first nucleotide is detected only in the first imaging event, the second nucleotide is detected only in the second imaging event.)
Regarding claim 7, Romanov teaches labeled nucleotides comprise dATP, dGTP, dCTP, dTTP, dUTP (Romanov, page 32, line 26-page 33, line 5), as well as non-natural nucleotide analogs (Romanov, page 33, line 15-page 34, line 5). Finally, Romanov teaches that the nucleotides comprise a 3’ hydroxyl blocking group (Romanov, page 35, line 15-21) Regarding claims 9-10, Romanov teaches a method wherein: “…the fluorescent signal from each incorporated nucleotide can be "read" optically by suitable means, such as a charge-coupled device using laser excitation and suitable emission filters. The 3’ blocking group and fluorescent dye compounds can then be removed… to expose the nascent chain for further nucleotide incorporation.” (Romanov, page 42, line 12-15) (i.e. repeating steps (a)-(e) for multiple cycles and determining the sequence…)
Regarding claims 13, 15, 17, and 19, Romanov teaches a pair of fluorescent nucleotide conjugates may be excited at different wavelengths (one labeled with a dye with a long Stokes shift taught by Romanov, “NR5201S” excited at 450 nm and a second labeled with a dye with a short Stokes shift, excited at 532 nm) (Romanov, figures 1-2 and page 53, line 24-page 54, line 7)
Regarding claim 21, Romanov teaches the two labels may be detected in a single emission channel between 550-570 (Romanov, page 4, line 12) and the two dyes have emission maxima around 580 nm when excited by the corresponding light source (Romanov, figure 1 and 2) (i.e. the single emission channel has a detection spectrum range above 560)
Regarding claim 22, the methods taught by Romanov do not comprise a chemical modification of the nucleotide conjugates between the imaging steps. In fact, Romanov teaches distinguishing incorporation of any of the four nucleotides by distinguishing their excitation wavelengths. (Romanov, page 23, line 16-31)
Regarding claims 23 and 36, Romanov teaches that the nucleic acid template (i.e. the target polynucleotide) may be attached to a solid support (Romanov, page 44, line 14-18) and that said templates may form an array and “the method is applicable to all types of high -density arrays, including single-molecule arrays, clustered arrays, and bead arrays” (Romanov, page 45, line 30- page 46, line 6) (i.e. multiple target polynucleotides are sequenced in parallel).
Regarding new claims 37 and 41, Romanov teaches a pair of fluorescent nucleotide conjugates may be excited at different wavelengths (one labeled with a dye with a long Stokes shift taught by Romanov, “NR5201S” excited at 450 nm and a second labeled with a dye with a short Stokes shift, excited at 532 nm) (Romanov, figures 1-2 and page 53, line 24-page 54, line 7) Romanov further teaches the two labels may be detected in a single emission channel between 550-570 (Romanov, page 4, line 12) and the two dyes have emission maxima around 580 nm when excited by the corresponding light source (Romanov, figure 1 and 2) (i.e. the single emission channel has a detection spectrum range above 560) Furthermore, Romanov teaches kits for sequencing using fewer than four lasers and detection channels (Romanov, page 61, line 15-19) can comprise a further nucleotide that is labelled with a dye that absorbs in the region of 520 to 560 nm (Romanov, page 52 lines 10-25).
Regarding new claims 38-40, Romanov teaches that any one of the four nucleotides comprising a 3’ blocking group (dATP, dTTP, dGTP, and dCTP) can be labelled with any one of the unique fluorophores, and that any of the nucleotides can be unlabeled (Romanov, page 41, line 32- page 42, line 5).
Response to arguments:
The response traverses the rejection of claims 1-7, 9-10, 13, 15, 17, 19, 21-23, and 36 under 35 U.S.C. 102(a)(1) as being anticipated by Romanov, WO 2016/189287. The response argues that “Romanov fails to disclose using three different fluorophores to label three types of nucleotides.” However, as described in the 102(a)(1) rejection above, Romanov specifically teaches that “each of the different nucleotide triphosphates (A, T, C, and G) may be labelled with a unique fluorophore and also comprises a blocking group at the 3’ position… Alternatively one of the four nucleotides may be unlabeled (dark)” (Romanov, page 41 line 32- page 42 line 5). (i.e. Romanov teaches that three nucleotide conjugates can be labelled with three “unique” fluorophores and the fourth nucleotide conjugate is unlabeled).
The response filed December 19, 2025 traverses the 102 rejection of record over Romanov. The response alleges that “Romanov fails to disclose… how the four types of nucleotides are decoded” and discusses a particular exemplary embodiment of Romanov wherein two types of nucleotides are each labeled with distinct fluorophores, a mixture of a third type of nucleotide having either of the two distinct fluorophores, and a fourth nucleotide that is unlabeled.
This argument has been thoroughly reviewed and is not persuasive.
As was previously described in the 102 rejection of record, Romanov explicitly describes embodiments wherein three types of nucleotides are each labeled with spectrally distinct labels, which are each different fluorophores, and a fourth type of nucleotide that is unlabeled, wherein a first label absorbs at a first excitation wavelength and emits at a first emission wavelength, a second label absorbs at a second excitation wavelength and emits at the first emission wavelength, and a third label absorbs at both excitation wavelengths and emits at the first emission wavelength. As was previously described, Romanov explicitly describes decoding schemes wherein particular labels are distinguished by their excitation profiles (i.e. the first label is excited by the first wavelength, the second label is excited by the second wavelength, the third label is excited by both wavelengths, and the fourth type of nucleotide is unlabeled (i.e. is excited by neither of the wavelengths).
Quoting MPEP 2123:
"The use of patents as references is not limited to what the patentees describe as their own inventions or to the problems with which they are concerned. They are part of the literature of the art, relevant for all they contain." In re Heck, 699 F.2d 1331, 1332-33, 216 USPQ 1038, 1039 (Fed. Cir. 1983) (quoting In re Lemelson, 397 F.2d 1006, 1009, 158 USPQ 275, 277 (CCPA 1968)).
A reference may be relied upon for all that it would have reasonably suggested to one having ordinary skill in the art, including nonpreferred embodiments. Merck & Co. v. Biocraft Labs., Inc. 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir. 1989), cert. denied, 493 U.S. 975 (1989). See also Upsher-Smith Labs. v. Pamlab, LLC, 412 F.3d 1319, 1323, 75 USPQ2d 1213, 1215 (Fed. Cir. 2005) (reference disclosing optional inclusion of a particular component teaches compositions that both do and do not contain that component); Celeritas Technologies Ltd. v. Rockwell International Corp., 150 F.3d 1354, 1361, 47 USPQ2d 1516, 1522-23 (Fed. Cir. 1998) (The court held that the prior art anticipated the claims even though it taught away from the claimed invention. "The fact that a modem with a single carrier data signal is shown to be less than optimal does not vitiate the fact that it is disclosed.")”
In addition to the specific preferred embodiment taught by Romanov previously described above, Romanov teaches that in other embodiments, “each of the different nucleotide triphosphates (A, T, C, and G) may be labelled with a unique fluorophore and also comprises a blocking group at the 3’ position… Alternatively one of the four nucleotides may be unlabeled (dark)” (Romanov, page 41 line 32- page 42 line 5).
Therefore, the full scope of the disclosure of Romanov teaches that three nucleotide conjugates can be labelled with three “unique” fluorophores and the fourth nucleotide conjugate is unlabeled).
For these reasons and the reasons already of record, the rejection is maintained.
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, 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.
Claims 1, 2, 7, 9-10, 13, 15, 17, 19, 21-23, and 36-41 are rejected under 35 U.S.C. 103 as being unpatentable over Romanov, WO 2016/189287, published December 1, 2016 in view of Graham et al., US 10,738,072 B1, granted August 11, 2020 as evidenced by "Quest Graph™ Fluorescence Spectrum Viewer." AAT Bioquest, Inc., 29 May. 2025, https://www.aatbio.com/fluorescence-excitation-emission-spectrum-graph-viewer/.
This rejection overlapping with the 102(a)(1) rejection over Romanov is directed at the preferred embodiment applicant argues is not anticipated by the preferred embodiment of Romanov.
Regarding claims 1-2, Romanov teaches a method of determining the sequence of a target polynucleotide on an automated sequencing instrument… containing two lasers operating at different wavelengths…” and “a single emission channel, which may therefore reduce or avoid the need for multiple emission filters.” (Romanov, page 25, lines 13-30) using fluorescently labelled reversible terminator nucleotides with long stokes shifts. (Romanov, page 22-page 24) Said labeled nucleotides comprise a first nucleotide conjugate comprising a first label, a second nucleotide conjugate comprising a second label, and a third nucleotide comprising a mixture of the first and second labels, (Romanov, page 24, lines 16-25) (i.e. the three labels are spectrally distinct) wherein two or more of the labels may be excited using a different excitation source at different wavelengths, which may be lasers. (Romanov, page 24, line 17-20) Finally, Romanov teaches that four separate nucleotides can be identified using only two labels. A first nucleotide can be labeled with a first label, a second nucleotide can be labeled with a second label, a third nucleotide can be labeled with a mixture of the first and second labels, and a fourth nucleotide can be unlabeled. (Romanov, page 24, line 5-10) (i.e. contacting a primer with a mixture comprising nucleotide conjugates, incorporating a nucleotide conjugate into the primer, performing a first imaging event with a first laser (a first light source), performing a second imaging event with a second laser, and collecting the two emission signals in a single emission channel.)
Quoting MPEP 2123:
"The use of patents as references is not limited to what the patentees describe as their own inventions or to the problems with which they are concerned. They are part of the literature of the art, relevant for all they contain." In re Heck, 699 F.2d 1331, 1332-33, 216 USPQ 1038, 1039 (Fed. Cir. 1983) (quoting In re Lemelson, 397 F.2d 1006, 1009, 158 USPQ 275, 277 (CCPA 1968)).
A reference may be relied upon for all that it would have reasonably suggested to one having ordinary skill in the art, including nonpreferred embodiments. Merck & Co. v. Biocraft Labs., Inc. 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir. 1989), cert. denied, 493 U.S. 975 (1989). See also Upsher-Smith Labs. v. Pamlab, LLC, 412 F.3d 1319, 1323, 75 USPQ2d 1213, 1215 (Fed. Cir. 2005) (reference disclosing optional inclusion of a particular component teaches compositions that both do and do not contain that component); Celeritas Technologies Ltd. v. Rockwell International Corp., 150 F.3d 1354, 1361, 47 USPQ2d 1516, 1522-23 (Fed. Cir. 1998) (The court held that the prior art anticipated the claims even though it taught away from the claimed invention. "The fact that a modem with a single carrier data signal is shown to be less than optimal does not vitiate the fact that it is disclosed.")”
In addition to the specific preferred embodiment taught by Romanov previously described above, Romanov teaches that in other embodiments, “each of the different nucleotide triphosphates (A, T, C, and G) may be labelled with a unique fluorophore and also comprises a blocking group at the 3’ position… Alternatively one of the four nucleotides may be unlabeled (dark)” (Romanov, page 41 line 32- page 42 line 5).
Therefore, the full scope of the disclosure of Romanov teaches that three nucleotide conjugates can be labelled with three “unique” fluorophores and the fourth nucleotide conjugate is unlabeled).
In yet another embodiment, Romanov teaches that a single nucleotide can be labeled with two dyes (i.e. one nucleotide molecule can be labeled with two different dye molecules simultaneously) (Romanov, page 52, line 10-25).
Even more, Romanov teaches that dyes with long Stokes shifts, such as NR5201s are particularly advantageous for sequencing platforms because they allow for improved signal to noise ratios, and allow sequencing using “fewer than the conventional four detection channels”.
Romanov teaches that sets of spectrally distinguishable fluorescent dyes that can be excited at different wavelengths but emit at the same wavelength (i.e. are excited using two different light sources and their emission is collected in a single emission channel) wherein at least one of the dyes exhibits a large Stokes shift are known in the art and that such dye sets can be selected from the prior art (Romanov, page 49, line 12- page 51, line 26).
Romanov does not teach a particular second prior art dye with a long stokes shift that would be suitable for the embodiment wherein each of three nucleotides are labelled with a unique fluorophore excited at ~460 and/or 540 nm and one nucleotide is unlabeled and the three nucleotides are detected in a single emission channel above 560nm.
However, Graham et al. teach a variety of nucleotide analogues (i.e. conjugates) that are suitable for methods of sequencing nucleic acids (Graham et al., Abstract) including nucleotide conjugates that are labeled with “fluorescent dyes” such as PromoFluor-500LSS (Graham et al., column 26, line 46) OR ATTO 490LS (Graham et al., column 23, line 24). As evidenced by AAT Bioquest (see graph below), PromoFluor-500LSS is a fluorophore that is similarly excited at 450 nm and at 532 nm and emits above 560 nm (the shaded area). As further evidenced by AAT Bioquest (see second graph below), ATTO 490LS is another nucleotide sequencing-compatible dye taught by Graham et al. with a similarly suitable (i.e. interchangeable) excitation/emission profile to PromoFluor-500LSS. Graham apparently teaches a plurality of suitable fluorescent labels for nucleic acid sequencing with long stokes shifts and similar excitation/emission profiles to those required by the method taught by Romanov et al.
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Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to select PromoFluor-500LSS (or ATTO 490LS) as a unique label for a third nucleotide conjugate that is not a combination of the first and second labels. The ordinary artisan would have been motivated to select a single fluorophore that satisfies the criteria required by the method taught by Romanov (i.e. is excited at both excitation wavelengths and emits within the single emission detection range (above 600 nm) rather than the dual label or mixture of labels comprising both the first and second fluorophores taught by Romanov, because the ordinary artisan would expect nucleotide conjugates with more chemical differences from their natural counterpart (i.e. more fluorophores) to be more likely to exhibit reduced incorporation into polynucleotides by enzymes (i.e. DNA polymerases). Alternatively, the ordinary artisan would have been motivated to select a second known nucleotide conjugate with a long stokes shift and an absorbance profile that is excited at both wavelengths because Romanov teaches that long stokes shift dyes allow for more efficient data analysis by improving signal to noise, and allowing for sequencing platforms to operate using fewer than the conventional four detection channels. The ordinary artisan would have been reasonably confident that either the PromoFluor-500LSS-labeled nucleotide conjugate or the ATTO 490LS-labeled nucleotide conjugate taught by Graham et al. would have successfully replaced the dual-fluorophore labeled nucleotide conjugate in the sequencing method taught by Romanov because Graham et al. teaches that the nucleotide conjugates of their invention are useful as unique detectable nucleotide conjugates in sequencing by synthesis applications (Graham et al., column 1, lines 15-26). Regarding the selection of either of the dyes taught by Graham as a suitable fluorescent label from the list of labels taught by Graham, it is well within the ability of the ordinary artisan to compare between published excitation and emission spectra using publicly available online tools as the "Quest Graph™ Fluorescence Spectrum Viewer".
Regarding claim 7, Romanov teaches labeled nucleotides comprise dATP, dGTP, dCTP, dTTP, dUTP (Romanov, page 32, line 26-page 33, line 5), as well as non-natural nucleotide analogs (Romanov, page 33, line 15-page 34, line 5). Finally, Romanov teaches that the nucleotides comprise a 3’ hydroxyl blocking group (Romanov, page 35, line 15-21) Regarding claims 9-10, Romanov teaches a method wherein: “…the fluorescent signal from each incorporated nucleotide can be "read" optically by suitable means, such as a charge-coupled device using laser excitation and suitable emission filters. The 3’ blocking group and fluorescent dye compounds can then be removed… to expose the nascent chain for further nucleotide incorporation.” (Romanov, page 42, line 12-15) (i.e. repeating steps (a)-(e) for multiple cycles and determining the sequence…)
Regarding claims 13, 15, 17, and 19, Romanov teaches a pair of fluorescent nucleotide conjugates may be excited at different wavelengths (one labeled with a dye with a long Stokes shift taught by Romanov, “NR5201S” excited at 450 nm (i.e. about 450 to about 460 nm) and a second labeled with a dye with a short Stokes shift, excited at 532 nm (i.e. about 510 to about 530 nm)) (Romanov, figures 1-2 and page 53, line 24-page 54, line 7)
Regarding claim 21, Romanov teaches the two labels may be detected in a single emission channel between 550-570 (Romanov, page 4, line 12) and the two dyes have emission maxima around 580 nm when excited by the corresponding light source (Romanov, figure 1 and 2) (i.e. the single emission channel has a detection spectrum range above 560)
Regarding claim 22, the methods taught by Romanov do not comprise a chemical modification of the nucleotide conjugates between the imaging steps. In fact, Romanov teaches distinguishing incorporation of any of the four nucleotides by distinguishing their excitation wavelengths. (Romanov, page 23, line 16-31)
Regarding claims 23 and 36, Romanov teaches that the nucleic acid template (i.e. the target polynucleotide) may be attached to a solid support (Romanov, page 44, line 14-18) and that said templates may form an array and “the method is applicable to all types of high density arrays, including single-molecule arrays, clustered arrays, and bead arrays” (Romanov, page 45, line 30- page 46, line 6) (i.e. multiple target polynucleotides are sequenced in parallel).
Regarding new claims 37 and 41, Romanov teaches a pair of fluorescent nucleotide conjugates may be excited at different wavelengths (one labeled with a dye with a long Stokes shift taught by Romanov, “NR5201S” excited at 450 nm and a second labeled with a dye with a short Stokes shift, excited at 532 nm) (Romanov, figures 1-2 and page 53, line 24-page 54, line 7) Romanov further teaches the two labels may be detected in a single emission channel between 550-570 (Romanov, page 4, line 12) and the two dyes have emission maxima around 580 nm when excited by the corresponding light source (Romanov, figure 1 and 2) (i.e. the single emission channel has a detection spectrum range above 560) Furthermore, Romanov teaches kits for sequencing using fewer than four lasers and detection channels (Romanov, page 61, line 15-19) can comprise a further nucleotide that is labelled with a dye that absorbs in the region of 520 to 560 nm (Romanov, page 52 lines 10-25).
Regarding new claims 38-40, Romanov teaches that any one of the four nucleotides comprising a 3’ blocking group (dATP, dTTP, dGTP, and dCTP) can be labelled with any one of the unique fluorophores, and that any of the nucleotides can be unlabeled (Romanov, page 41, line 32- page 42, line 5).
Claims 11, 23, 25, and 27 are rejected under 35 U.S.C. 103 as being unpatentable over Romanov, WO 2016/189287, published December 1, 2016 in view of Graham et al., US 10,738,072 B1, granted August 11, 2020 as evidenced by "Quest Graph™ Fluorescence Spectrum Viewer." AAT Bioquest, Inc., 29 May. 2025, https://www.aatbio.com/fluorescence-excitation-emission-spectrum-graph-viewer/ as applied to claims 1-7, 9-10, 13, 15, 17, 19, 21-23, and 36 above, and further in view of Illumina Inc., December 31, 2018, Illumina CMOS chip and one-channel SBS chemistry, https://www.illumina.com/content/dam/illumina-marketing/documents/products/techspolights/cmos-tech- note-770-2013-054.pdf, 4 pp. (previously cited on 04/28/2022 IDS and in International search report PCT/EP2021/086349)
Romanov in view of Graham et al. teaches a method of determining the sequence of a target polynucleotide on an automated sequencing instrument… containing two lasers operating at different wavelengths…” and “a single emission channel, which may therefore reduce or avoid the need for multiple emission filters.” (Romanov, page 25, lines 13-30) using fluorescently labelled reversible terminator nucleotides with long stokes shifts. (Romanov, page 22-page 24) Said labeled nucleotides comprise a first nucleotide conjugate comprising a first label, a second nucleotide conjugate comprising a second label, and a third nucleotide comprising a mixture of the first and second labels, (Romanov, page 24, lines 16-25) (or alternatively, a separate nucleotide conjugate labeled with PromoFluor-500LSS) (i.e. the three labels are spectrally distinct) wherein two or more of the labels may be excited using a different excitation source at different wavelengths, which may be lasers. (Romanov, page 24, line 17-20) Finally, Romanov in view of Graham et al. teaches that four separate nucleotides can be identified using only two (or three) spectrally distinguishable labels. A first nucleotide can be labeled with a first label, a second nucleotide can be labeled with a second label, a third nucleotide can be labeled with a mixture of the first and second labels (or PromoFluor-500LSS), and a fourth nucleotide can be unlabeled. (Romanov, page 24, line 5-10) (i.e. contacting a primer with a mixture comprising nucleotide conjugates, incorporating a nucleotide conjugate into the primer, performing a first imaging event with a first laser (a first light source), performing a second imaging event with a second laser, and collecting the two emission signals in a single emission channel.)
Romanov in view of Graham et al. teaches a method wherein: “…the fluorescent signal from each incorporated nucleotide can be "read" optically by suitable means, such as a charge-coupled device using laser excitation and suitable emission filters. The 3’ blocking group and fluorescent dye compounds can then be removed… to expose the nascent chain for further nucleotide incorporation.” (Romanov, page 42, line 12-15) (i.e. repeating steps (a)-(e) for multiple cycles and determining the sequence…)
Romanov does not teach that the method of sequencing comprising repeating the sequencing-by-synthesis steps for at least 50 cycles.
However, Illumina 2018 teaches “Two-channel SBS chemistry” wherein a first nucleotide (thymine) is labeled with a first label (a green fluorophore), a second nucleotide (cytosine) is labeled with a second label (a red fluorophore), a third nucleotide (adenine) is labeled with both fluorophores, and a fourth nucleotide (guanine) is unlabeled (Illumina, page 2, column 1, paragraph 2). Illumina 2018 further demonstrates quality score information from a 2x151 bp sequencing run (i.e. more than 50 cycles) (Illumina 2018, figure 4).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the method taught by Romanov in view of Graham et al. to read lengths (i.e. number of cycles) greater than 50 as taught by Illumina 2018. The ordinary artisan would have been motivated to perform at least 50 sequencing cycles in the method taught by Romanov in view of Graham et al. because of the teaching of Illumina 2018 that the 2-channel chemistry produces high quality data in a 2x151 bp (>300 cycles) sequencing run. (Illumina 2018, figure 4) The ordinary artisan would have reasonably expected the method of Romanov in view of Graham et al. would produce high-quality 150 base reads.
Regarding claims 25 and 27, while Romanov in view of Graham et al. teaches that the nucleic acid template (i.e. the target polynucleotide) may be attached to a solid support (Romanov, page 44, line 14-18) and that said templates may form an array and “the method is applicable to all types of high density arrays, including single-molecule arrays, clustered arrays, and bead arrays” (Romanov, page 45, line 30- page 46, line 6) Romanov in view of Graham et al. does not teach that the solid support comprises a patterned flow cell, comprising a plurality of nanowells in which the plurality of target polynucleotides are immobilized.
However, Illumina 2018 teaches sequencing a plurality of immobilized target polynucleotides in a patterned flow cell comprising a plurality of nanowells fabricated over a CMOS chip (Illumina 2018, Figure 3).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the method of sequencing a plurality of immobilized polynucleotide templates comprising two excitation wavelengths and a single emission channel using the set of labeled and unlabeled nucleotides, distinguishable by their excitation wavelengths, taught by Romanov in view of Graham et al. with the CMOS flow cell taught by Illumina 2018. The ordinary artisan would have been motivated to modify the method taught by Romanov in view of Graham et al. with the CMOS flow cell taught by Illumina 2018 because Illumina teaches that one-channel CMOS sensor detection technology has “simplified chemistry and optics, an entry-level price point, and the convenience of a small footprint.” (Illumina 2018, Page 3, column 2, paragraph 3) Therefore, the ordinary artisan would have reasonably expected the method taught by Romanov in view of Graham et al. to successfully implement the 2-channel chemistry (not requiring the one-channel intermediate chemistry step taught by Illumina 2018) on the cost-effective CMOS Flow Cell taught by Illumina 2018.
Response to Arguments
The response argues that the combination of Romanov, Graham, and AAT Bioquest does not teach a method wherein three nucleotides are labeled with different fluorophores and one nucleotide is unlabeled. However, as noted in the revised 102 and 103 rejections above, Romanov contemplates sequencing with three nucleotide conjugates with “unique” fluorophores, 3’ blocking groups, and a fourth unlabeled nucleotide conjugate (Romanov, page 24, line 5-10) The response further argues that Romanov and Graham do not provide an explicit motivation to select PromoFluor-500LSS as the label for the third type of nucleotide because Graham suggests sequencing with four distinctly labeled nucleotides and one embodiment of Romanov extolls particular benefits of sequencing using only two dyes compared to sequencing with four distinct dyes or three distinct dyes and one unlabeled nucleotide. This argument has been thoroughly reviewed, but is not persuasive. As described in the updated 102 and 103 rejections above, Romanov provides for embodiments wherein three nucleotides are each labeled with a unique fluorophore and the fourth is unlabeled and particularly discusses the benefits of selecting sets of dyes known in the prior art that are distinguishable based on their excitation wavelengths but are all detectable in a single emission channel. As discussed above, Romanov further teaches that dyes with particularly long Stokes shifts are especially well suited to this methodology and beneficially reduce signal to noise and allow for fewer than the traditional four emission filters to be used. Graham provides for a plurality of dyes that are suitable for labelling nucleotide conjugates for sequencing and, as evidenced above, it is well within the ability of a person with ordinary skill in this art to evaluate the publicly available excitation/emission spectra of dyes listed by Graham for dyes that have the excitation and emission maxima (and therefore stokes shift) properties required by the teachings of Romanov.
The response filed December 19, 2025 traverses the 103 rejection of record, on the grounds that: a) a person of ordinary skill would not be motivated to select the particular dye cited in the rejection of record because although the response acknowledges Romanov “includes a single sentence… suggesting that each of the different dNTPs may be labeled with a unique fluorophore… alternatively one of the four dNTPs may be unlabeled”, the response asserts that Romanov does not provide guidance for a person of ordinary skill on a decoding scheme when nucleotide 3 is labeled uniquely because Romanov does not teach an example of this embodiment and b) the particular dye cited is disclosed in a “laundry list” of dyes that can be used for nucleotide labeling.
This argument has been thoroughly reviewed and is not persuasive. As previously discussed, as prior art, Romanov is relevant for all that that it contains.
"The use of patents as references is not limited to what the patentees describe as their own inventions or to the problems with which they are concerned. They are part of the literature of the art, relevant for all they contain." In re Heck, 699 F.2d 1331, 1332-33, 216 USPQ 1038, 1039 (Fed. Cir. 1983) (quoting In re Lemelson, 397 F.2d 1006, 1009, 158 USPQ 275, 277 (CCPA 1968)).
A reference may be relied upon for all that it would have reasonably suggested to one having ordinary skill in the art, including nonpreferred embodiments. Merck & Co. v. Biocraft Labs., Inc. 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir. 1989), cert. denied, 493 U.S. 975 (1989). See also Upsher-Smith Labs. v. Pamlab, LLC, 412 F.3d 1319, 1323, 75 USPQ2d 1213, 1215 (Fed. Cir. 2005) (reference disclosing optional inclusion of a particular component teaches compositions that both do and do not contain that component).
Further, Romanov further teaches/suggests that sets of spectrally distinguishable fluorescent dyes that can be excited at different wavelengths but emit at the same wavelength (i.e. are excited using different light sources and their emission is collected in a single emission channel) wherein at least one of the dyes exhibits a large Stokes shift are known in the art and that such dye sets can be selected from the prior art (Romanov, page 49, line 12- page 51, line 26). As such, the citation of a particular dye, evidenced by an exemplary widely known and publicly available fluorophore selection tool, having the specific properties suggested by Romanov was included by the examiner as an example that the ordinary artisan knows how to identify suitable fluorophores having emission and excitation properties suggested by Romanov.
Finally, the response argues that in the examples provided by Romanov, a decoding scheme is demonstrated that utilizes signal intensity to distinguish labeled nucleotides in the embodiments wherein the third nucleotide comprises a mixture of nucleotides having either the first or second fluorophore label.
This argument has been thoroughly reviewed and is not persuasive.
As described above, the teachings of Romanov are not limited to particular preferred embodiments or examples. Romanov specifically teaches that the three labeled nucleotides may be labeled with spectrally distinct fluorophores that are distinguishable based upon their excitation and/or emission properties.
Conclusion
No claim is allowed.
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/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682