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 .
DETAILED ACTION
1. A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on May 12, 2026 has been entered.
Status of the Application
2. Claims 1-12 and 14-16 are pending. Claims 13 and 17 are canceled. The allowability of the claims 1-12 and 14-16 has been withdrawn in view of new combination of prior art references.
Claim Rejections - 35 USC § 103
3. 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.
A. Claims 1-12 and 14-16 are rejected under 35 U.S.C. 103 as being unpatentable over Peterson et al. (US 2018/0163259) in view of Crothers et al. (US 2007/0092880).
Peterson et al. teach a method of claim 1, analyzing a sample comprising target nucleic acids, the method comprising the steps of: (a) contacting, in a reaction mixture, any of a first target nucleic acid of the sample with a first primary probe oligonucleotide comprising a sequence complementary thereto, and a FEN-1 endonuclease under conditions such that if the first primary probe oligonucleotide is hybridized to the first target nucleic acid, the first primary probe is cleaved by the FEN-1 endonuclease to generate a first primary cleaved flap, wherein the first primary cleaved flap hybridizes to a flap-hybridizing sequence of a first FRET cassette oligonucleotide contained in the reaction mixture to form an invasive cleavage structure that is cleaved by the FEN-1 endonuclease at a cleavage site between a first fluorophore moiety and a first quencher moiety of the first FRET cassette oligonucleotide to release a first cassette cleaved flap comprising the first fluorophore moiety at a first temperature that is below a first Tm of the primary probe and the first cassette cleaved flap, wherein at a second temperature that is above the first Tm, the first cassette cleaved flap do not form a stable duplex with the target (para 0009, 0013-0014, 0066-0070, 0090-0110);
(b) detecting any fluorescence emitted from the first fluorophore moiety at the second temperature (para 0013-0014, 0066-0070, 0090-0110); and
(c) determining either that the sample comprises the first target nucleic acid if fluorescence emitted from the first fluorophore moiety is detected in step (b), or the sample does not comprise the first target nucleic acid if fluorescence emitted from the first fluorophore moiety is not detected in step (b) (para 0013-0014, 0066-0070, 0090-0110).
With reference to claim 2, Peterson et al. teach that step (a) further comprises contacting, in the reaction mixture, any of a second target nucleic acid of the sample with a second primary probe oligonucleotide comprising a sequence complementary thereto, and the FEN-1 endonuclease under conditions such that if the second primary probe oligonucleotide hybridizes to the second target nucleic acid, the second primary probe is cleaved by the FEN-1 endonuclease to generate a second primary cleaved flap that is different from the first primary cleaved flap, wherein the second primary cleaved flap hybridizes to the cleaved flap-hybridizing sequence of a second FRET cassette oligonucleotide contained in the reaction mixture to form an invasive cleavage structure that is cleaved by the FEN-1 endonuclease at a cleavage site between a second fluorophore moiety and a third quencher moiety of the second FRET cassette oligonucleotide to release a second cassette cleaved flap comprising the second fluorophore moiety; wherein step (b) further comprises detecting any fluorescence emitted from the second fluorophore moiety at the fourth temperature and wherein step (c) further comprises determining either that the sample comprises the second target nucleic acid if fluorescence emitted from the second fluorophore moiety of a 5’ flap cleavage product of the second FRET cassette oligonucleotide is detected in step (b), or the sample does not comprise the first target nucleic acid if fluorescence emitted from the second fluorophore moiety of the 5' flap cleavage product of the second FRET cassette oligonucleotide is not detected in step (b) ((para 0008-0009, 0013-0014, 0066-0070, 0090-0110).
With reference to claim 3, Peterson et al. teach that the step (a) further comprises contacting, in the reaction mixture, any of a second target nucleic acid of the sample with a second primary probe oligonucleotide comprising a sequence complementary thereto, and the FEN-1 endonuclease under conditions such that if the second primary probe oligonucleotide hybridizes to the second target nucleic acid, the second primary probe is cleaved by the FEN-1 endonuclease to generate a second primary cleaved flap, wherein the second primary cleaved flap hybridizes to the cleaved flap-hybridizing
sequence of a second FRET cassette oligonucleotide contained in the reaction mixture to form an invasive cleavage structure that is cleaved by the FEN-1 endonuclease at a cleavage site between a second fluorophore moiety and a third quencher moiety of the second FRET cassette oligonucleotide to release a cleavage product comprising the second fluorophore moiety, wherein step (b) further comprises detecting any fluorescence emitted from the second fluorophore moiety of the cleavage product; and wherein step (c) further comprises determining either that the sample comprises the
second target nucleic acid if fluorescence emitted from the second fluorophore moiety is detected in step (b), or the sample does not comprise the first target nucleic acid if fluorescence emitted from the second fluorophore moiety is not detected in step (b) (para 0013-0014, 0066-0070, 0107-0110).
With reference to claim 4, Peterson et al. teach that the step (b) comprises detecting with a single channel of a fluorescence monitoring device any fluorescence emitted from the first and second fluorophore moieties (para 0049, 0090).
With reference to claim 5, Peterson et al. teach that step (b) is performed while a nucleic acid amplification reaction is occurring in the reaction mixture, and wherein products of the nucleic acid amplification reaction comprise the first target nucleic acid and the second target nucleic acid (para 0009. 0013-0014, 0070-0073).
With reference to claim 6, Peterson et al. teach that the nucleic acid amplification reaction comprises steps for thermocycling, and wherein the reaction mixture further comprises a thermostable DNA polymerase (para 0124, 0165).
With reference to claim 7, Peterson et al. teach that step (b) is performed as the temperature of the reaction mixture decreases complementary cassette cleaved flaps (para 0103-0104).
With reference to claim 8, Peterson et al. teach that the first and second fluorophore moieties are the same as each other (para 0075, 0086).
With reference to claims 9-10, 14, Peterson et al. teach that the step of (b) detecting any fluorescence comprises measuring any fluorescence, further comprising the step of either detecting or measuring fluorescence at the first temperature and fluorescence from the second fluorophore is detected and/or measured at the first temperature (para 0090-0091, 0192).
With reference to claims 11-12, 15, Peterson et al. teach that the first fluorophore moiety and the second fluorophore moiety are both detectable in the same fluorescence detection channel of an energy sensor device, wherein the second fluorophore moiety is the same as the first fluorophore moiety or the second fluorophore moiety is not the same as the first fluorophore moiety and the third quencher moiety is the same as the first quencher moiety and/or the second quencher moiety (para 0090, 0097-0100).
With reference to claim 16, Peterson et al. teach that the reaction mixture comprises primer oligonucleotides that amplify target nucleic acids, wherein at least one primer oligonucleotide acts as an invasive oligonucleotide in the presence of a primary probe oligonucleotide and target nucleic acid and/or target amplicon to form an invasive cleavage structure that is cleaved by the FEN-1 endonuclease (para 0071-0084, 0124).
However, Peterson et al. did not teach a masking oligonucleotide to bind with the cleaved flap.
Crothers et al. teach a method for multiplex detection of target nucleic acids using invasive cleavage reaction wherein FRET cassette comprises 5’ terminus and a 5’-flap portion having a fluorophore and upon cleavage by FEN-1, 5’-flaps comprising fluorophore from the FRET cassette are released and captured using a capture probe or quencher oligonucleotide having a quencher moiety (masking oligonucleotide) and quenches fluorescence emission from the fluorophore, allowing quantitation of the signal in real-time and preventing interaction with another target (para 0216-0225, 0275-0277, Fig. 1 and 6).
It would be prima facie obvious to an ordinary person having skilled in the art before the effective filing date of the invention to modify the method of Peterson et al. with capture oligonucleotides comprising a quencher moiety to detect cleaved flaps as taught by Crothers et al. to improve the sensitivity of the method for detecting target nucleic acids in a sample. The ordinary person skilled in the art would have motivated to combine the method of Peterson et al. with capture probes as taught by Crothers et al. and have a reasonable expectation of success that the combination would improve the sensitivity of the method because Crothers et al. explicitly taught use of capture probes to bind with the cleaved flaps to reduce the background noise (0216-0225, 0275-0277) and such a modification of the method is considered obvious over the cited prior art.
B. Claims 1-12 and 14-16 are rejected under 35 U.S.C. 103 as being unpatentable over Gagrat et al. (WO 2021/055508) in view of Crothers et al. (US 2007/0092880).
Gagrat et al. teach a method of claim 1, analyzing a sample comprising target nucleic acids, the method comprising the steps of: (a) contacting, in a reaction mixture, any of a first target nucleic acid of the sample with a first primary probe oligonucleotide (invasive oligonucleotide or test substrate) comprising a sequence complementary thereto, and a FEN-1 endonuclease under conditions such that if the first primary probe oligonucleotide (invasive oligonucleotide) is hybridized to the first target nucleic acid, the
first primary probe is cleaved by the FEN-1 endonuclease to generate a first primary cleaved flap, wherein the first primary cleaved flap hybridizes to a cleaved flap-hybridizing sequence of a first FRET cassette oligonucleotide (first FRET reporter contained in the reaction mixture, to form an invasive cleavage structure, wherein the first FRET cassette comprises a first 5’ flap portion having a fluorophore moiety attached there to, wherein the invasive cleavage structure is cleaved by the FEN-1 endonuclease at a cleavage site between the first fluorophore moiety and a first quencher moiety of the first FRET cassette oligonucleotide to release a first cassette cleaved flap comprising the first fluorophore moiety wherein at a first temperature that is below a first Tm of the cleaved flap comprising the fluorophore emits fluorescence, wherein at a second temperature that is above the first Tm, the first cassette cleaved flap do not form a stable structure (page 8, line 26 to line 2 on page 10, page 41, line 3
to line 7 on page 42, page 44, line 16 to page 47);
(b) detecting any fluorescence emitted from the first fluorophore moiety at the second temperature (page 8, line 26 to line 2 on page 10, page 41, line 3 to line 7 on page 42, page 44, line 16 to page 47); and
(c) determining either that the sample comprises the first target nucleic acid if fluorescence emitted from the first fluorophore moiety is detected in step (b), or the sample does not comprise the first target nucleic acid if fluorescence emitted from the first fluorophore moiety is not detected in step (b) (page 8, line 26 to line 2 on page 10, page 41, line 3 to line 7 on page 42, page 44, line 16 to page 47, page 39, line 18-26).
With reference to claims 2-3, Gagrat et al. teach that step (a) further comprises contacting, in the reaction mixture, any of a second target nucleic acid of the sample with a second primary probe oligonucleotide, a second FRET cassette oligonucleotide comprising a 5’ flap portion labeled with a fluorophore contained in the reaction mixture to form an invasive cleavage structure that is cleaved by the FEN-1 endonuclease at a cleavage site between a second fluorophore moiety and a third quencher moiety of the second FRET cassette oligonucleotide to release a second cassette cleaved flap comprising the second fluorophore moiety; wherein step (b) further comprises detecting any fluorescence emitted from the second fluorophore moiety at the fourth temperature ;
and wherein step (c) further comprises determining either that the sample comprises the second target nucleic acid if fluorescence emitted from the second fluorophore moiety of a 5’ flap cleavage product of the second FRET cassette oligonucleotide is detected in step (b), or the sample does not comprise the first target nucleic acid if fluorescence emitted from the second fluorophore moiety of the 5' flap cleavage product of the second FRET cassette oligonucleotide is not detected in step (b) (page 42, line 15-30, page 48, line 1 to line 6 on page 50, page 8, line 26 to line 2 on page 10, page 41, line 3
to line 7 on page 42);
With reference to claim 4, Gagrat et al. teach that the step (b) comprises detecting with a single channel of a fluorescence monitoring device any fluorescence emitted from the first and second fluorophore moieties (page 39, line 27 to line 13 on page 40, page 52, line 12-14).
With reference to claim 5, Gagrat et al. teach that step (b) is performed while a nucleic acid amplification reaction is occurring in the reaction mixture, and wherein products of the nucleic acid amplification reaction comprise the first target nucleic acid 16 to page 47, page 48, line 1 to line 6 on page 50).
With reference to claim 6, Gagrat et al. teach that the nucleic acid amplification reaction comprises steps for thermocycling, and wherein the reaction mixture further comprises a thermostable DNA polymerase (page 41, line 3 to line 7 on page 42, page 44, line 16 to page 47).
With reference to claim 7, Gagrat et al. teach that step (b) is performed as the temperature of the reaction mixture decreases complementary cassette cleaved flaps (page 8, line 26 to line 2 on page 10).
With reference to claim 8, Gagrat et al. teach that the first and second fluorophore moieties are the same as each other (page 48, line 1 to line 6 on page 50).
With reference to claims 9-10, 14, Gagrat et al. teach that the step of (b) detecting any fluorescence comprises measuring any fluorescence, further comprising the step of either detecting or measuring fluorescence at the first temperature and fluorescence from the second fluorophore is detected and/or measured at the first temperature (page 48, line 1 to line 6 on page 50).
With reference to claims 11-12, 15, Gagrat et al. teach that the first fluorophore moiety and the second fluorophore moiety are both detectable in the same fluorescence detection channel of an energy sensor device, wherein the second fluorophore moiety is the same as the first fluorophore moiety or the second fluorophore moiety is not the same as the first fluorophore moiety and the third quencher moiety is the same as the first quencher moiety and/or the second quencher moiety (page 48, line 1 to line 6 on page 50).
With reference to claim 16, Gagrat et al. teach that the reaction mixture comprises primer oligonucleotides that amplify target nucleic acids, wherein at least one primer oligonucleotide acts as an invasive oligonucleotide in the presence of a primary probe oligonucleotide and target nucleic acid and/or target amplicon to form an invasive cleavage structure that is cleaved by the FEN-1 endonuclease (page 8, line 26 to line 2 on page 10).
However, Gagrat et al. did not teach use of masking oligonucleotide comprising a quencher to bind with the cleaved 5’ labeled flap.
Crothers et al. teach a method for detecting target nucleic acids using invasive cleavage reaction wherein FRET cassette comprises 5’ terminus and a 5’ flap portion having a fluorophore and upon cleavage by FEN-1 5’ flaps comprising fluorophore from the FRET cassette are released and captured using a capture probe or quencher oligonucleotide having a quencher moiety (masking oligonucleotide) and quenches fluorescence emission from the fluorophore, allowing quantitation of the signal in real-time and preventing interaction with another target (para 0216-0225, 0275-0277 Fig. 1 and 6).
It would be prima facie obvious to an ordinary person having skilled in the art before the effective filing date of the invention to modify the method of Gagrat et al. with capture oligonucleotides comprising a quencher moiety to detect cleaved flaps as taught by Crothers et al. to improve the sensitivity of the method for detecting target nucleic acids in a sample. The ordinary person skilled in the art would have motivated to combine the method of Gagrat et al. with capture probes as taught by Crothers et al. and have a reasonable expectation of success that the combination would improve the sensitivity of the method because Crothers et al. explicitly taught use of capture probes to bind with the cleaved flaps to reduce the background noise (0216-0225, 0275-0277) and such a modification of the method is considered obvious over the cited prior art.
Conclusion
No claims are allowable.
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Suryaprabha Chunduru
Primary Examiner
Art Unit 1681
/SURYAPRABHA CHUNDURU/Primary Examiner, Art Unit 1681
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