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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 16-18, 20-21, 24-25, 27, 29-30 and 56-59 are pending under examination. Claims 1-15, 19, 22-23, 26, 28 and 31-55 are canceled. The arguments presented in the pre-appeal brief were fully considered and the pre-appeal conference decision has been mailed to the Applicant on April 10, 2026. The Applicant’s arguments over the rejection of claims under 35 USC 103 as being obvious over Peterson et al. in view of Hodge et al. have been found unpersuasive for the reasons as presented in the previous office action and the rejection has been maintained and restated as follows.
Claim Rejections - 35 USC § 103-Maintained
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.
Claims 16-18, 20-21, 24-25, 27, 29-30 and 56-59 are rejected under 35 U.S.C. 103 as being unpatentable over Peterson et al. (US 2018/0163259) in view of Hodge et al. (US 2010/0105050).
Peterson et al. teach a method of claim 16, 56, for determining which of two different FRET cassettes in a reaction mixture cleaved to generate a fluorescent signal, comprising: (a) performing a multiplex invasive cleavage reaction in the reaction mixture to cleave one or both of a first and a second FRET to produce two different fluorescent cleavage products, if cleavage of the first FRET cassette and the second FRET cassette occurred, the first FRET cassette comprises a 5’ terminus and comprises a first
5’ flap portion having a fluorophore attached thereto and cleavage of the FRET cassette by a FEN-1 endonuclease in the multiplex invasive reaction produces a first cassette cleaved flap comprising the 5’ flap the fluorophore (0008-0014, 0027, 0054-0056, 0066-0078, 0043);
the reaction comprises primary probes or cycling probes comprising a 3’ target binding sequence and a 5’ flap sequence that stably hybridizes to the first cassette cleaved flap to form a first duplex at a temperature below a first Tm, but not at a temperature above the first Tm, wherein fluorescence emission from the fluorophore of the first cassette cleaved flap of the first duplex is quenched, and each of the two different fluorescent cleavage products produced in the multiplex invasive cleavage reaction being characterized by different temperature -dependent profiles in the rection mixture (para
0009-0014, 0020-0025, 0054-0056, 0066-0089, 0092-0093, 0101-0116, 0159-0145);
(b) measuring fluorescent signal produced in the rection mixture using a single channel of a fluorescence monitoring apparatus under temperature conditions that differentially quench fluorescence produced by the different fluorescent cleavage products of the multiplex invasive cleavage reaction (para 0066-0089, 0009-0014, 0101-0116); and
(c) determining from the results of step (b) which of the different FRET cassettes was cleaved in the multiplex invasive cleavage reaction (para 0066-0083, 0009-0012, 0101-0116).
With reference to claim 17, Peterson et al. teach that the second FRET cassette in step (a) if cleaved, produces a fluorescent cleavage product (para 0078-0089,0101-0102).
With reference to claim 18, Peterson et al. teach that the step (c) comprises comparing fluorescence signals measured at the temperature below the first Tm and the temperature above the first Tm (para 0011-0014).
With reference to claim 20, Peterson et al. teach that step (b) comprises measuring any of the fluorescent signal at the temperature below the first Tm, where fluorescence emission from the fluorophore of the first cassette cleaved flap of the first duplex is quenched, and wherein step (c) comprises determining that the second FRET cassette cleaved in the reaction mixture if measurable fluorescence was detected in step (b) (para 0088-0089, 0101-0104, 0109).
With reference to claim 21, Peterson et al. teach that step (b) comprises measuring any of the fluorescent signal at each of the temperature below the first Tm and the temperature above the first Tm, and wherein step (c) comprises determining that the first FRET cassette cleaved in the reaction mixture if the fluorescent signal measured at the temperature above the first Tm is greater than the fluorescent signal measured at the temperature below the first Tm (para 0101-0116).
With reference to claim 24, Peterson et al. teach the second FRET cassette comprises a second 5’ flap sequence having a fluorophore attached thereto, attachment of the fluorophore being arranged so that cleavage of the second FRET cassette by the FEN-1 endonuclease in the multiplex invasive cleavage reaction produces a second cassette cleaved flap comprising the fluorophore, wherein the reaction mixture comprises a second probe that hybridizes to the second cassette cleaved flap to form a second
duplex at a temperature below a second Tm, but not at a temperature above the second Tm, wherein fluorescence emission from the fluorophore of the second cassette cleaved flap of the second duplex is quenched, and wherein the first Tm and the second Tm differ by at least 5°C (para 0101-0116).
With reference to claim 25, Peterson et al. teach the first Tm is greater than the second Tm, wherein step (b) comprises measuring any of the fluorescent signal at the temperature below the second Tm and at the temperature above the first Tm, and wherein step (c) comprises determining that at least one of the first FRET cassette and the second FRET cassette cleaved in the reaction mixture if the fluorescent signal measured at the temperature above the first Tm is greater than the fluorescent signal measured at the temperature below the second Tm (para 0009-0014, 0096).
With reference to claims 27, 57-59, Peterson et al. teach step (b) comprises measuring any of the fluorescent signal produced in the reaction mixture as a function of temperature to generate a melting/annealing curve, and wherein step (c) comprises calculating a derivative of the melting/ annealing curve, and then (i) determining from the calculated derivative whether the reaction mixture comprises the first duplex characterized by the first Tm as an indicator that the first FRET cassette cleaved in the reaction mixture, and/or (ii) determining from the calculated derivative whether the
reaction mixture comprises the second duplex characterized by the second Tm as an indicator that the second FRET cassette cleaved in the reaction mixture (para 0055, 0066-0089, 0196).
With reference to claims 29-30, Peterson teach the first and second FRET cassettes are labeled with identical fluorophores, not labeled with identical fluorophores (para 0097-0098).
However, Peterson et al. did not teach a masking oligonucleotide comprising a quencher moiety to bind with the cleaved 5’ flap having fluorophore.
Hodge 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 during amplification the denatured (cleaved) 5’ flaps comprising
fluorophore from the FRET cassette is captured using a quencher oligonucleotide having a quencher moiety (masking oligonucleotide) and quenches fluorescence emission from the fluorophore during amplification, allowing quantitation of the signal in real-time and also recreates the FRET cassette (para 0057-0064, 0075-0077).
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 quencher oligonucleotides having quencher moiety to capture cleaved 5’ flaps comprising fluorophore from a FRET cassette as taught by Hodge 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 modify the method of Peterson et al. with quencher oligonucleotides comprising a quencher moiety as taught by Hodge and have a reasonable expectation of success that the combination would improve the sensitivity by reducing the background signal noise because Hodge et al. explicitly taught use of quencher oligonucleotides having quencher moiety to capture 5’ flaps comprising fluorophore, for temperature dependent quantitative amplification, reducing background nonspecific amplification and recreating FRET cassette (para 0062-0064) and such a modification of the method is considered obvious over the cited prior art.
Response to Arguments:
With reference to the rejection of claims under 35 USC 103 as being unpatentable over Peterson et al. in view of Hodge, the Applicant’s arguments were found unpersuasive. Applicant’s arguments drawn to the definition of FRET cassette of the instant specification and the assertions drawn to no teaching of said FRET cassette by Peterson or Hodge have been fully considered and found unpersuasive because
Peterson et al. defined FRET cassette in para 0034-0035, comprising a stem-loop structure that includes interactive label-pair, a fluorophore and a quencher at 5’ end and 3’ end, wherein 5’ end is a flap region. Hodge et al. teach signal generating molecule or FRET cassette (para 0057), wherein signal generation molecule comprises a stem-loop structure (single nucleotide loop) (Fig. 19). With reference to the Applicant’s arguments drawn to para 0064 of Hodge et al., the arguments were found unpersuasive because the paragraph discusses about biparate probe and reverse primer oligonucleotides, not
FRET cassette. As discussed in the rejection, Hodge et al. teach quencher oligonucleotide that quenches fluorescence signal of the signal generating molecule which reduces background noise (abstract, para 0062). For all the above the rejection has been maintained.
Claim Rejections - 35 USC § 103
4. 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 16-18, 20-21, 24-25, 27, 29-30 and 56-59 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 16, 56, for determining which of two different FRET cassettes in a reaction mixture cleaved to generate a fluorescent signal, comprising: (a) performing a multiplex invasive cleavage reaction in the reaction mixture to cleave one or both of a first and a second FRET to produce two different fluorescent cleavage products, if cleavage of the first FRET cassette and the second FRET cassette occurred, the first FRET cassette comprises a 5’ terminus and comprises a first
5’ flap portion having a fluorophore attached thereto and cleavage of the FRET cassette by a FEN-1 endonuclease in the multiplex invasive reaction produces a first cassette cleaved flap comprising the 5’ flap the fluorophore (page 8, line 26 to line 2 on page 10, page 41, line 3 to line 7 on page 42, page 44, line 19 to page 50);
the reaction comprises primary probes or cycling probes comprising a 3’ target binding sequence and a 5’ flap sequence that stably hybridizes to the first cassette cleaved flap to form a first duplex at a temperature below a first Tm, but not at a temperature above the first Tm, wherein fluorescence emission from the fluorophore of the first cassette cleaved flap of the first duplex is quenched, and each of the two different fluorescent cleavage products produced in the multiplex invasive cleavage reaction being characterized by different temperature -dependent profiles in the rection mixture (page 8, line 26 to line 2 on page 10, page 41, line 3 to line 7 on page 42, page 44, line 19 to page 50);
(b) measuring fluorescent signal produced in the rection mixture using a single channel of a fluorescence monitoring apparatus under temperature conditions that differentially quench fluorescence produced by the different fluorescent cleavage products of the multiplex invasive cleavage reaction (page 8, line 26 to line 2 on page 10, page 41, line 3 to line 7 on page 42, page 44, line 19 to page 50); and
(c) determining from the results of step (b) which of the different FRET cassettes was cleaved in the multiplex invasive cleavage reaction (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 50, page 39, line 18-26).
With reference to claim 17, Gagrat et al. teach that the second FRET cassette in step (a) if cleaved, produces a fluorescent cleavage product (page 42, line 15-30, page 48, line 1 to line 19 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 18, Gagrat et al. teach that the step (c) comprises comparing fluorescence signals measured at the temperature below the first Tm and the temperature above the first Tm (page 8, line 26 to line 2 on page 10).
With reference to claim 20, Gagrat et al. teach that step (b) comprises measuring any of the fluorescent signal at the temperature below the first Tm, where fluorescence emission from the fluorophore of the first cassette cleaved flap of the first duplex is quenched, and wherein step (c) comprises determining that the second FRET cassette cleaved in the reaction mixture if measurable fluorescence was detected in step (b) (page 8, line 26 to line 2 on page 10, page 44, line 19 to page 50, page 39, line 18-26).
With reference to claim 21, Gagrat et al. teach that step (b) comprises measuring any of the fluorescent signal at each of the temperature below the first Tm and the temperature above the first Tm, and wherein step (c) comprises determining that the first FRET cassette cleaved in the reaction mixture if the fluorescent signal measured at the temperature above the first Tm is greater than the fluorescent signal measured at the temperature below the first Tm (page 8, line 26 to line 2 on page 10, page 44, line 19 to page 50, page 39, line 18-26).
With reference to claim 24, Gagrat et al. teach the second FRET cassette comprises a second 5’ flap sequence having a fluorophore attached thereto, attachment of the fluorophore being arranged so that cleavage of the second FRET cassette by the FEN-1 endonuclease in the multiplex invasive cleavage reaction produces a second cassette cleaved flap comprising the fluorophore, wherein the reaction mixture comprises a second probe that hybridizes to the second cassette cleaved flap to form a second
duplex at a temperature below a second Tm, but not at a temperature above the second Tm, wherein fluorescence emission from the fluorophore of the second cassette cleaved flap of the second duplex is quenched, and wherein the first Tm and the second Tm differ by at least 5°C (page 48, line 1 to line 6 on page 50).
With reference to claim 25, Gagrat et al. teach the first Tm is greater than the second Tm, wherein step (b) comprises measuring any of the fluorescent signal at the temperature below the second Tm and at the temperature above the first Tm, and wherein step (c) comprises determining that at least one of the first FRET cassette and the second FRET cassette cleaved in the reaction mixture if the fluorescent signal measured at the temperature above the first Tm is greater than the fluorescent signal measured at the temperature below the second Tm (page 48, line 1 to line 19 on page 50).
With reference to claims 27, 57-59, Gagrat et al. teach step (b) comprises measuring any of the fluorescent signal produced in the reaction mixture as a function of temperature to generate a melting/annealing curve, and wherein step (c) comprises calculating a derivative of the melting/ annealing curve, and then (i) determining from the calculated derivative whether the reaction mixture comprises the first duplex characterized by the first Tm as an indicator that the first FRET cassette cleaved in the reaction mixture, and/or (ii) determining from the calculated derivative whether the
reaction mixture comprises the second duplex characterized by the second Tm as an indicator that the second FRET cassette cleaved in the reaction mixture (page 48, line 1 to line 19 on page 50).
With reference to claims 29-30, Gagrat et al. teach the first and second FRET cassettes are labeled with identical fluorophores, not labeled with identical fluorophores (page 48, line 1 to line 19 on page 50).
However, Gagrat et al. did not teach a masking oligonucleotide comprising a quencher moiety to bind with the cleaved 5’ flap having fluorophore.
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 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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