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
The amendments filed on 03/20/2026 and 04/29/2026 has been entered.
Excess claim fee paid on 04/29/2026 is acknowledged.
Claims 68, 71, 75, 80, and 89 were amended in the claim set filed on 03/20/2026.
Applicant's election with traverse of Group I, claims 68 to 89, drawn to a method for quantitating a plurality of nucleic acid molecules, in the reply filed on 07/15/2025 is acknowledged.
Applicant is reminded that upon the cancelation of claims to a non-elected invention, the inventorship must be corrected in compliance with 37 CFR 1.48(a) if one or more of the currently named inventors is no longer an inventor of at least one claim remaining in the application. A request to correct inventorship under 37 CFR 1.48(a) must be accompanied by an application data sheet in accordance with 37 CFR 1.76 that identifies each inventor by his or her legal name and by the processing fee required under 37 CFR 1.17(i).
Claims 90-91 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected Group II, drawn to a mixture and a kit comprising a plurality of detectably labeled probes and extensions primers.
Claims 92-93 were added in the claim set filed on 03/20/2026. No new matter was added.
Claim 69 was canceled in the claim set filed on 03/20/2026.
Claims 90-91 were canceled in the claim set filed on 04/29/2026.
Claims 68, 70-89 and 92-93 in the claim set filed on 04/29/2026 are currently pending and under examination.
Response to the Arguments
Objections to the Claims and Specification in the previously mailed non-final have been withdrawn in light of applicants Claim and Specification amendments. However, new grounds of objections are made below on Pg. 4.
Previous rejection(s) of claim(s) 69 under 35 U.S.C. 101 and 102 are withdrawn due to cancellation of claim.
Applicant’s arguments regarding previous rejection(s) of claim(s) 68 and 70-89 under 35 U.S.C. 101 have been fully considered and are persuasive. The 35 U.S.C. 101 rejections documented in the previously mailed non-final have been withdrawn in light of applicants claim amendments and arguments directed to the Subject Matter Eligibility Examples document of May 2016 example 31 claims 70, 75, 80, and 85 and arguments on Pg 16-19 of the Remarks filed 03/20/2026.
As necessitated by amendment, the 35 U.S.C. 102 rejections of claim(s) 68 and 70-89 documented in the previously mailed non-final have been withdrawn in light of applicants claim amendments. Applicant' s argument on Pg. 8, states that “White et al. does not disclose or suggest this element now recited by amended independent claim 68.” However, upon further consideration and search, new grounds of rejection are made as documented below in the 35 U.S.C. 103 rejection in this office action on Pg. 5-20.
As necessitated by amendment, the 35 U.S.C. 103 rejections of claim(s) 68, 73-75, 80, and 84 documented in the previously mailed non-final have been withdrawn in light of applicants claim amendments and arguments on Pg. 9 However, upon further consideration and search, new grounds of rejection are made as documented below in the 35 U.S.C. 103 rejection in this office action on Pg. 5-20.
The new grounds of objections and rejections for claims 68, 70-89 and 92-93 are documented below in this Final Office Action are necessitated by claim amendments filed on 03/20/2026.
Priority
This application is a 371 of PCT/US21/15859, filed on January 29,2021 which claims priority to U.S. Provisional Patent Application No. 62/968,898, filed on. January 31, 2020. The priority date of claim set filed on April 01, 2023, is determined to be January 31, 2020.
Claim Objections
Claim 92 is objected to under 37 CFR 1.75 as being a substantial duplicate of claim 74. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m).
Claim 93 is objected to under 37 CFR 1.75 as being a substantial duplicate of claim 85. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m).
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 75 and 92 are indefinite. Claim 75 is indefinite over the limitation "hybridizes to the contiguous domain of the respective one of the plurality of adapter molecules" in lines 18-19 and “wherein the contiguous domain comprises a portion of the first adapter sequence and a portion of the second adapter sequence directly adjacent to each other” in lines 24-25. It is unclear as to what molecule the plurality of adapter molecules are adapted to besides each other. Furthermore, claim 75 was amended to be an independent claim and thus “the plurality of adapter molecules” (ln 18-19), “the first adapter sequence” (ln 24), and “the second adapter sequence” (ln 25) are not previously referenced in the claims. There is insufficient antecedent basis for this limitation in the claim. Claim 92 depends on claim 75.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
Claims 68 and 70-71 are rejected under 35 U.S.C. 103 as being unpatentable over Lee et al. (“Lee”; Patent App. Pub. WO 2012106288 A2, Aug. 09, 2013).
Lee discloses “approaches for the identification, detection, and quantification of nucleic acids in a biological sample.” (Abstract).
Regarding claim 68, Lee teaches a method comprising “the method permits the detection of a plurality of target nucleic acids in the same nucleic acid sample, such that step (a) comprises contacting the nucleic acid sample with a different oligonucleotide primer and detectably labeled oligonucleotide probe member for each of the plurality of target nucleic acids” (Para. 9). Lee also teaches a method comprising “extending annealed oligonucleotide primers” (Para. 20). Thus, Lee suggests a method for quantitating a plurality of nucleic acid molecules comprising a plurality of target nucleic acid fragments comprising: contacting a plurality of detectably-labeled probes and a plurality of extension primers with the plurality of nucleic acid molecules.
Regarding claim 68, Lee teaches a method comprising “wherein when the fluorescently labeled probe is FAM-labeled… where the FAM label on the labeled probe is attached to the 5' terminal nucleotide of the probe” (Para. 20). Thus, Lee suggests a method wherein each of the plurality of detectably-labeled probes comprises a first labeled nucleic acid domain comprising a first label.
Regarding claim 68, Lee teaches a method comprising “extending annealed oligonucleotide primers using a nucleic acid polymerase having 5' to 3' exonuclease activity, under conditions sufficient to permit primer extension of annealed oligonucleotide primers and to permit the 5' to 3' nuclease activity to cleave annealed oligonucleotide probes and thereby release a labeled detection molecule specific for each of the plurality of target nucleic acids present in said sample” (Para. 20). Lee teaches a method comprising “"amplifying" as used herein can also refer to linear” (Para. 50) and “These methods employ a labeled oligonucleotide probe, which can be used in a replication and/or amplification reaction for a target nucleic acid species” (Para. 65). Lee also teaches “In such aspects, the nucleic acid polymerase binds to the 3' end of the upstream oligonucleotide primer and extends the primer sequence by generating a polynucleotide sequence complementary to the target nucleic acid to which the upstream primer is bound. As the polymerization or extension continues, the polymerase encounters the downstream labeled oligonucleotide probe and progressively cleaves mononucleotides or small oligonucleotides from its 5' end, thus producing labeled detection molecules of a specific size. This cleaving continues until the remainder of the downstream oligonucleotide has been destabilized to the extent that it dissociates from the template molecule.” (Para. 67). Thus, Lee suggests a method comprising producing in a linear reaction an extension product of each of the plurality of target nucleic acid fragments by extending a respective one of the plurality of extension primers with a polymerase.
Regarding claim 68, Lee teaches a method comprising “polymerase further possesses 5' to 3' exonuclease activity, whereby a polynucleotide (e.g., a oligonucleotide probe), annealed downstream of and on the same strand as an extending oligonucleotide primer, is hydrolyzed or cleaved by the polymerase's 5' to 3' activity, releasing probe fragments, including labeled fragments or nucleotides, as hydrolysis continues” (Para. 40) and “extending annealed oligonucleotide primers using a thermostable nucleic acid polymerase having 5' to 3' exonuclease activity, under conditions sufficient to permit primer extension of annealed oligonucleotide primers and to permit the 5' to 3' nuclease activity to cleave annealed oligonucleotide probe to thereby release a labeled detection molecule” (Para. 15). Thus, Lee suggests a method comprising hydrolyzing each of the plurality of detectably-labeled probes hybridized to the respective one of the target nucleic acid fragments during extending the respective one of the plurality of extension primers with the polymerase.
Regarding claim 68, Lee teaches a method comprising “detecting a released labeled detection molecule in the separated nucleic acids to thereby detect the presence and/or amount of the target nucleic acid present in the sample” (Para. 15). Thus, Lee suggests a method comprising detecting a first signal produced as a result of hydrolyzing the plurality of detectably-labeled probes to quantitate a number of the plurality of target nucleic acid fragments.
Regarding claim 68, Lee teaches a method comprising “"replication reaction" refers to an in vitro means for making a single copy of a target sequence of nucleic acid, i.e., where amplification of a target nucleic acid sequence does not occur” (Para. 49) and “The methods described herein provide novel ways of quantifying and detecting nucleic acids… which can be used in a replication and/or amplification reaction for a target nucleic acid species” (Para. 65). Lee also teaches “These methods can be used on unamplified or amplified target nucleic acid species, and in singleplex or multiplex formats… detection by, for example, fluorescence detection” (Para. 62). Thus, Lee suggests a method wherein the method does not comprise template amplification and does not comprise successive cycles of amplification.
The teachings of Lee are documented above in the rejection of claim 68 under 35 U.S.C. 103. Claims 70-71 depend on claim 68. Rejection of dependent claims 70-71 under 35 U.S.C. 103 are documented below.
Regarding claim 70, Lee teaches a method wherein “A 5' to 3' exonuclease activity useful in the methods described herein does not catalyze the hydrolysis of oligonucleotide probe molecules that are not annealed to a target nucleic acid” (Para. 42). Lee teaches a method wherein “quantification of target nucleic acids in a sample by detecting and quantifying a labeled product generated by the 5' to 3' nuclease activity of a nucleic acid polymerase on a detectably labeled oligonucleotide probe hybridized to a target nucleic acid” (Para. 62). Thus, Lee suggests a method wherein a total number of hydrolyzed detectably-labeled probes is substantially same as a total number of the plurality of target nucleic acid fragments prior to contacting the plurality of detectably-labeled probes and the plurality of extension primers with the plurality of nucleic acid molecules.
Regarding claim 71, Lee teaches a method comprising “extending annealed oligonucleotide primers using a nucleic acid polymerase having 5' to 3' exonuclease activity, under conditions sufficient to permit primer extension of annealed oligonucleotide primers and to permit the 5' to 3' nuclease activity to cleave annealed oligonucleotide probes and thereby release a labeled detection molecule specific for each of the plurality of target nucleic acids present in said sample” (Para. 20). Lee teaches a method comprising “"amplifying" as used herein can also refer to linear” (Para. 50) and “These methods employ a labeled oligonucleotide probe, which can be used in a replication and/or amplification reaction for a target nucleic acid species” (Para. 65). Lee also teaches “In such aspects, the nucleic acid polymerase binds to the 3' end of the upstream oligonucleotide primer and extends the primer sequence by generating a polynucleotide sequence complementary to the target nucleic acid to which the upstream primer is bound. As the polymerization or extension continues, the polymerase encounters the downstream labeled oligonucleotide probe and progressively cleaves mononucleotides or small oligonucleotides from its 5' end, thus producing labeled detection molecules of a specific size. This cleaving continues until the remainder of the downstream oligonucleotide has been destabilized to the extent that it dissociates from the template molecule.” (Para. 67). Thus, Lee suggests a method wherein subsequent to contacting the plurality of detectably-labeled probes and the plurality of extension primers with the plurality of nucleic acid molecules, and prior to detecting the first signal, no additional cycle of extension reactions is performed other than a single cycle of extension reactions.
Therefore, the invention as recited in claims 68 and 70-71 is prima facie obvious over the prior art Lee et al. One of ordinary skill in the art would have had a reasonable expectation of success given the obviousness of the claim limitations. It would have been obvious to provide a method for quantitating a plurality of nucleic acid molecules according to the limitations of the instant application claims 68 and 70-71 based on Lee et al. (Patent App. Pub. No. WO 2012106288 A2).
Response to Arguments
Applicant's arguments filed 03/20/2026 (Pg. 19-26) with respect to claims 68, 70-89 and 92-93 have been fully considered but do not apply to the new grounds of rejection in view of Lee et al..
Claims 72-89 and 92-93 are rejected under 35 U.S.C. 103 as being unpatentable over Lee et al. (“Lee”; Patent App. Pub. WO 2012106288 A2, Aug. 09, 2013) in view of So et al. (“So”; Patent App. Pub. WO 2014130890 A1, Aug. 28, 2018).
The teachings of Lee are documented above in the rejection of claims 68 and 70-71 under 35 U.S.C. 103. Claim 72 depends on claim 68.
However, Lee does not explicitly teach the limitations of claim 72.
So discloses “Aspects of the invention relate to methods and kits for assessing cancer. Some aspects of the invention relate to methods and kits for preparing a sample library for sequencing… Some aspects of the invention relate to sensitive detection of amplicons” (Abstract).
So teaches a method wherein “Adaptor 1 -template nucleic acid. The ligation products may then be collected and optionally further processed … by ligation of a second adaptor sequence to a 3' end (as described in, e.g., FIG. 14A), followed by… by target-selective library preparation” (Para. 375). So teaches a method wherein “the oligonucleotide probes may be hybridizable to one or more adaptor sequences” (Para. 390). So teaches a method wherein “the first or second adaptor sequence comprises a binding site for a sequencing primer” (Para. 37). So teaches a method wherein “hybridizing primers to the pdo's, wherein the primers comprise a sequence complementary to the adaptor oligonucleotide sequence and comprise a first adaptor sequence” (Para. 40). So teaches a method wherein “a primer annealing step, and a synthesis step, whereby cleavage and displacement occurs simultaneously with primer-dependent template extension… In some embodiments, one cycle of amplification is performed” (Para. 246). So teaches a method wherein “the invention provides a method of preparing a nucleic acid library, comprising ligating an oligonucleotide sequence to a first end… the oligonucleotide sequence is an adaptor sequence… the oligonucleotide sequence comprises a detectable label” (Para. 50). So also teaches “In some embodiments, preparing does not require exponential PCR amplification prior to sequencing of the library. In some embodiments the preparing comprises a linear amplification step. In some embodiments the preparing does not require amplification” (Para. 27) and “In some embodiments, the amplifying comprises linear amplification” (Para. 37).
Thus, Lee and So suggest a method wherein each of the plurality of target nucleic acid fragments comprises a first adapter sequence, a target insert sequence, and a second adapter sequence; wherein the first adapter sequence is linked to the second adapter sequence through the target insert sequence; wherein hydrolyzing each of the plurality of detectably-labeled probes comprises hydrolyzing one of the plurality of detectably-labeled probes hybridized to at least a portion of the first adapter sequence; and wherein extending the respective one of the plurality of extension primers with the polymerase comprises extending an extension primer hybridized to at least a portion of the second adapter sequence.
Lee and So are both considered to be analogous to the claimed invention because they are in the same field of nucleic acid analysis. Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the methods of quantitating a plurality of nucleic acid molecules as taught by Lee to incorporate the method wherein the first adapter sequence is linked to the second adapter sequence through the target insert sequence; wherein the plurality of detectably-labeled probes comprises hydrolyzing one of the plurality of detectably-labeled probes hybridized to at least a portion of the first adapter sequence; and wherein extending the respective one of the plurality of extension primers with the polymerase comprises extending an extension primer hybridized to at least a portion of the second adapter sequence as taught by So and provide a method for according to the limitations of claim 72. These claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome according to the limitations of claim 72. Doing so would allow for quantitation of the library of each prepared nucleic molecule.
Claims 72-73, 76-79, 87 and 89 depend on claim 68. Claims 74 and 85-86 depend on claim 73, which depends on claim 68. Claim 88 depends on claim 87, which depends on claim 68. Claims 75 and 80 are independent claims. Claim 92 depends on claim 75. Claims 81-84 and 93 depend on claim 80. Rejection of dependent claims 70-74, 76-79 and 85-89 and independent claims 75 and 80 under 35 U.S.C. 103 are documented below.
Regarding claim 73, So teaches a method wherein “serially stitching adaptors together. The number of adaptors that can be stitched can be 1, 2, 3, 4 or more” (Para. 199). So also teaches a method wherein “Such oligonucleotides comprise a sequence for hybridizing to a first and second Illumina-specific adaptor oligonucleotide” (Para. 200). Thus, Lee and So suggest a method wherein the plurality of nucleic acid molecules further comprise a plurality of adapter molecules; wherein each of the plurality of adapter molecules comprises a first adapter sequence directly linked to a second adapter sequence; wherein the method further comprises contacting a plurality of hybridizing oligonucleotides with the plurality of nucleic acid molecules; wherein each of the plurality of hybridizing oligonucleotides comprises a sequence complementary to a contiguous domain of a respective one of the plurality of adapter molecules.
Regarding claims 74-75, 86 and 92, Lee teaches a method wherein “The probe is preferably detectably labeled… The probe, preferably, does not contain a sequence complementary to the region of a target nucleic acid sequence(s) to which an oligonucleotide primer binds to in the same reaction. Generally, the 3' terminus of an oligonucleotide probe will be "blocked" to prohibit incorporation of the probe into a primer extension product. "Blocking" can be achieved by using non-complementary bases, as described herein, or by adding a chemical moiety, such as biotin or a phosphate group, to the 3' hydroxyl of the last nucleotide of the oligonucleotide probe, which can, depending upon the selected moiety, serve a dual purpose by also acting as a label for subsequent detection or capture of the nucleic acid attached to the label. Blocking can also be achieved by removing the 3'-OH or by using a nucleotide that lacks a 3'-OH such as a dideoxynucleotide.” (Para. 37). Thus, Lee and So suggest a method of: (claim 74) wherein each of the plurality of hybridizing oligonucleotides comprises a blocker nucleic acid domain; subsequent to contacting the plurality of hybridizing oligonucleotides with the plurality of nucleic acid molecules, each of the plurality of hybridizing oligonucleotides hybridizes to the contiguous domain of the respective one of the plurality of adapter molecules, but not to the plurality of target nucleic acid fragments; and hydrolyzation of each of the plurality of hybridizing oligonucleotides hybridized to the contiguous domain and hydrolyzation of each of the plurality of detectably-labeled probes hybridized to the first adapter sequence of a respective one of the plurality of adapter molecules are blocked by a blocking moiety in the blocker nucleic acid domain; and wherein each of the plurality of hybridizing oligonucleotides comprises a blocker nucleic acid domain; (claim 75) for quantitating a plurality of nucleic acid molecules comprising a plurality of target nucleic acid fragments, which comprises: contacting a plurality of detectably-labeled probes and a plurality of extension primers with the plurality of nucleic acid molecules, wherein each of the plurality of detectably- labeled probes comprises a first labeled nucleic acid domain comprising a first label; producing an extension product of each of the plurality of target nucleic acid fragments by extending a respective one of the plurality of extension primers with a polymerase; hydrolyzing each of the plurality of detectably-labeled probes hybridized to the respective one of the target nucleic acid fragments during extending the respective one of the plurality of extension primers with the polymerase; and detecting a first signal produced as a result of hydrolyzing the plurality of detectably- labeled probes to determine a number of the plurality of target nucleic acid fragments, wherein each of the plurality of hybridizing oligonucleotides comprises a blocker nucleic acid domain; wherein subsequent to contacting the plurality of hybridizing oligonucleotides with the plurality of nucleic acid molecules, each of the plurality of hybridizing oligonucleotides hybridizes to the contiguous domain of the respective one of the plurality of adapter molecules, but not to the plurality of target nucleic acid fragments; and hydrolyzation of each of the plurality of hybridizing oligonucleotides hybridized to the contiguous domain and hydrolyzation of each of the plurality of detectably-labeled probes hybridized to the first adapter sequence of a respective one of the plurality of adapter molecules are blocked by a blocking moiety in the blocker nucleic acid domain, and wherein the contiguous domain comprises a portion of the first adapter sequence and a portion of the second adapter sequence directly adjacent to each other; (Claim 86) wherein each of the plurality of hybridizing oligonucleotides comprises a blocker nucleic acid domain; and (Claim 92) wherein producing an extension product of each of the plurality of target nucleic acid fragments is by way of a linear reaction, and wherein the method does not comprise template amplification and does not comprise successive cycles of amplification.
Regarding claim 76, Lee teaches a method wherein “the first oligonucleotide primer and the fluorescently labeled oligonucleotide probe for each nucleic acid target hybridize to the same strand of the target nucleic acid; and iv) at least one fluorescently labeled probe is FAM-labeled… extending annealed oligonucleotide primers using a nucleic acid polymerase having 5' to 3' exonuclease activity, under conditions sufficient to permit primer extension of annealed oligonucleotide primers and to permit the 5' to 3' nuclease activity to cleave annealed oligonucleotide probes and thereby release a labeled detection molecule” (Para. 20). So teaches a method wherein “Cleavage of the reporter probe, e.g., by hydrolysis, can separate the detectable moiety from the quencher moiety. The separation can enable the fluorescent moiety to produce a detectable fluorescent signal” (Para. 271). Thus, Lee and So suggest a method wherein the first signal is a first fluorescent signal, and the signals detected upon the extension reactions are fluorescent signals comprising the first fluorescent signal.
Regarding claims 77-79, So teaches a method wherein “The reporter probe can comprise a detectable moiety and a quencher moiety” (Para. 271). So teaches a method wherein “Quenching can involve a transfer of energy between the fluorophore and the quencher. The emission spectrum of the fluorophore and the absorption spectrum of the quencher can overlap. When the probe is intact, the fluorescent signal from the detectable moiety can be substantially suppressed by the quencher. Cleavage of the reporter probe, e.g., by hydrolysis, can separate the detectable moiety from the quencher moiety. The separation can enable the fluorescent moiety to produce a detectable fluorescent signal” (Para. 271). Lee teaches a method wherein “base-linked fluorophores and quenchers are well-known in the art” (Para. 105). Thus, Lee and So suggest a method of (claim 79) wherein each of the plurality of detectably-labeled probes further comprises a first and second labeled nucleic acid domain; wherein the first labeled nucleic acid domain is a first reporter domain; wherein the second labeled nucleic acid domain is a first quencher domain; and wherein the plurality of detectably-labeled probes are hydrolyzed to release at least one of the first label or the second label; (Claim 78) wherein each of the plurality of detectably-labeled probes further comprises a second labeled nucleic acid domain comprising a second label; wherein the first label and the second label are two spectrally similar or identical reporters; and wherein the plurality of detectably-labeled probes are hydrolyzed to release at least one of the first label or the second label; and (Claim 79) wherein the first labeled nucleic acid domain is a first reporter domain; wherein each of the plurality of detectably-labeled probes comprises a quenching nucleotide that quenches an energy from the first label in an excited state; and wherein the plurality of detectably-labeled probes are hydrolyzed to release at least one of the quenching nucleotide or the first label.
Regarding claims 85, 80-84 and 93, Lee teaches a method wherein “multiplex formats and approaches for the detection and quantification of multiple target nucleic acids in a sample, using, for example, multiple detectably labeled oligonucleotide probes having different sizes and/or labels” (Para. 4). Lee also teaches a method wherein “respective ones of the labeled oligonucleotide probes comprise different distinguishable fluorescent labels” (Para. 21). Furthermore, So teaches a method wherein ''the oligonucleotide probes may be hybridizable to one or more adaptor sequences. The amount of detectable signal at a certain addressable location can indicate the amount of nucleic acids containing the target region in the sample” (Para. 390). So teaches a method wherein “The reporter probe can comprise a detectable moiety and a quencher moiety” (Para. 271). So teaches a method wherein “Quenching can involve a transfer of energy between the fluorophore and the quencher. The emission spectrum of the fluorophore and the absorption spectrum of the quencher can overlap. When the probe is intact, the fluorescent signal from the detectable moiety can be substantially suppressed by the quencher. Cleavage of the reporter probe, e.g., by hydrolysis, can separate the detectable moiety from the quencher moiety. The separation can enable the fluorescent moiety to produce a detectable fluorescent signal” (Para. 271). Thus, Lee and So suggest a method of (Claim 85) wherein each of the plurality of hybridizing oligonucleotides comprises a third labeled nucleic acid domain comprising a third label; and subsequent to contacting the plurality of hybridizing oligonucleotides with the plurality of nucleic acid molecules, each of the plurality of hybridizing oligonucleotides hybridizes to the contiguous domain of the respective one of the plurality of adapter molecules, but not to the plurality of target nucleic acid fragments; the method further comprising: producing an extension product of each of the plurality of adapter molecules by extending a respective one of the plurality of extension primers in the extension reactions with the polymerase; hydrolyzing each of the plurality of hybridizing oligonucleotides hybridized to the contiguous domain and hydrolyzing each of the plurality of detectably-labeled probes hybridized to the respective one of the adapter molecules, during producing an extension product of each of the plurality of adapter molecules; detecting a second signal produced as a result of hydrolyzing the plurality of hybridizing oligonucleotides, wherein the first signal and the second signal are distinguishably detected; (Claim 80) comprising a plurality of target nucleic acid fragments, which comprises: contacting a plurality of detectably-labeled probes and a plurality of extension primers with the plurality of nucleic acid molecules, wherein each of the plurality of detectably- labeled probes comprises a first labeled nucleic acid domain comprising a first label; producing an extension product of each of the plurality of target nucleic acid fragments by extending a respective one of the plurality of extension primers with a polymerase; hydrolyzing each of the plurality of detectably-labeled probes hybridized to the respective one of the target nucleic acid fragments during extending the respective one of the plurality of extension primers with the polymerase; and detecting a first signal produced as a result of hydrolyzing the plurality of detectably- labeled probes to determine a number of the plurality of target nucleic acid fragments; wherein the plurality of nucleic acid molecules further comprises a plurality of adapter molecules; wherein each of the plurality of adapter molecules comprises a first adapter sequence directly linked to a second adapter sequence; wherein the method further comprises contacting a plurality of hybridizing oligonucleotides with the plurality of nucleic acid molecules; wherein each of the plurality of hybridizing oligonucleotides comprises a sequence complementary to a contiguous domain of a respective one of the plurality of adapter molecules, wherein each of the plurality of hybridizing oligonucleotides further comprises a third labeled nucleic acid domain comprising a third label; wherein subsequent to contacting the plurality of hybridizing oligonucleotides with the plurality of nucleic acid molecules, each of the plurality of hybridizing oligonucleotides hybridizes to the contiguous domain of the respective one of the plurality of adapter molecules, but not to the plurality of target nucleic acid fragments; and the method further comprising: detecting a second signal produced as a result of partially hydrolyzing the plurality of hybridizing oligonucleotides, wherein the first signal and the second signal are distinguishably detected; (Claim 81) wherein each of the plurality of hybridizing oligonucleotides further comprises a fourth labeled nucleic acid domain comprising a fourth label; wherein the third labeled nucleic acid domain is a second reporter domain; wherein the fourth labeled nucleic acid domain is a second quencher domain; wherein the second reporter domain and the second quencher domain are linked by the blocker nucleic acid domain; and wherein the plurality of hybridizing oligonucleotides are partially hydrolyzed to release at least one of the third label or the fourth label; (Claim 82) ; (Claim 83) wherein the third labeled nucleic acid domain is a third reporter domain; wherein each of the plurality of hybridizing oligonucleotides comprises a quenching nucleotide that quenches an energy from the third label in an excited state; and wherein the plurality of hybridizing oligonucleotides are partially hydrolyzed to release at least one of the quenching nucleotide or the third label; and (Claim 84) wherein the first signal is a first fluorescent signal, wherein the second signal is a second fluorescent signal, and wherein the signals detected upon the extension reactions are fluorescent signals comprising the first fluorescent signal and the second fluorescent signal.
Regarding claim 87, Lee teaches a method wherein “The nucleic acid fragments or DNA are typically visualized with stains, UV shadowing, intercalating dyes, such as ethidium bromide” (Para. 132). Lee teaches a method wherein “each of the labeled detection molecules specific for each of the target molecules present in the sample differs from each other in one or both of a) the type or identity of label on the released detection molecule and b) the separation characteristics of the released detection molecule. In some such embodiments, the separation characteristics comprise mass, length, charge, or a combination thereof” (Para. 25). Lee teaches a method wherein “Hybridization of the probe, in conjunction with replication and/or amplification of the target sequence with oligonucleotide primers to replicate or amplify the template nucleic acid sequence, can be used to provide a determination of the presence and/or amount of a target nucleic acid sequence in a sample” (Para. 89). Thus, Lee and So suggest a method further comprising: contacting a plurality of extension primers and a plurality of nucleic acid binding dye molecules with the plurality of nucleic acid molecules; subsequent to producing the extension product of each of the plurality of target nucleic acid fragments, measuring a signal produced by the plurality of nucleic acid binding dye molecules; and estimating an average size of the plurality of target nucleic acid fragments based on a number of the plurality of target nucleic acid fragments and the signal produced by the plurality of nucleic acid binding dye molecules.
Regarding claim 88, Lee teaches a method wherein “different dyes cause anomalous migration of small cleavage products to different extents, the ordinarily skilled artisan can develop a key for each dye label of interest by capillary electrophoretic separation of fragments of known sizes labeled with the dye of choice… The relative migration and inflection point at which increasing size begins to correlate with decreased mobility for a single dye provide the keys to peak alignments for fragments smaller than the size at the inflection point” (Para. 71). Thus, Lee and So suggest a method wherein estimating an average size of the plurality of target nucleic acid fragments comprises: determining a correlation factor using signals respectively produced by a plurality of reference nucleic acid libraries; estimating the average size of the plurality of target nucleic acid fragments according to the following Equation: Average size = (S/ (correlation factor x N)); wherein S stands for the signal produced by the plurality of nucleic acid binding dye molecules, and N stands for the number of the plurality of target nucleic acid fragments.
Regarding claim 89, So teaches a method wherein “In some embodiments of any of the methods herein, the determining comprises the step of diluting nucleic acid molecules from the sample into discrete reaction volumes, wherein the discrete reaction volumes contain on average less than 10, 5, 4, 3, 2, or 1 nucleic acid molecule from the sample. In some embodiments the discrete reaction volumes contain 0-10 molecules of the nucleic acid from the sample” (Para. 26). So teaches a method wherein “The step of targeted sequencing can comprise preparing a DNA library … In some embodiments, preparing does not require exponential PCR amplification prior to sequencing of the library. In some embodiments the preparing comprises a linear amplification step” (Para. 27). Thus, Lee and So suggest a method further comprising: generating a plurality of nucleic acid molecules comprising a plurality of target nucleic acid fragments from a nucleic acid sample by incubation of the target nucleic acid fragments with a DNA polymerase under conditions in which the DNA polymerase catalyzes polymerization; diluting the plurality of target nucleic acid fragments to a predetermined concentration, and sequencing at least one portion of the plurality of target nucleic acid fragments.
Response to Arguments
Applicant's arguments filed 03/20/2026 (Pg.19-26) with respect to claims 68, 70-89 and 92-93 have been fully considered but do not apply to the new grounds of rejection in view of Lee et al and So et al..
Conclusion of Response to Arguments
In view of the amendments, new grounds of rejections and above responses to arguments have been made, no claims are in condition for allowance.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. Fekete, R. A., Miller, M. J., & Chattoraj, D. K. (2003). Fluorescently labeled oligonucleotide extension: a rapid and quantitative protocol for primer extension. Biotechniques, 35(1), 90-98. (entire document- Fluorescently labeled oligonucleotide extension: a rapid and quantitative protocol for primer extension) Claims 68, 70-89 and 92-93
Lee, J. H., Daugharthy, E. R., Scheiman, J., Kalhor, R., Ferrante, T. C., Terry, R., ... & Church, G. M. (2015). Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues. Nature protocols, 10(3), 442-458) Figure 4- adapter specific fluorescent probe hybridization) Claims 73-74, 80 and 85-86
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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 KENDRA R VANN-OJUEKAIYE whose telephone number is (571)270-7529. The examiner can normally be reached M-F 9:00 AM- 5:00 PM.
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/KENDRA R VANN-OJUEKAIYE/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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