DETAILED ACTION
The amendment filed on 09/17/2025 has been entered.
Claims 1 and 8-9 were amended, claim 16 was canceled, and claim 21 was added in the claim set filed on 09/17/2025.
Claims 1-15 and 17-21 in the claim set filed on 09/17/2025 are currently under examination.
Response to the Arguments
Objections to the Drawings and Specification, in the previously mailed non-final have been withdrawn in light of applicants Drawings amendments and argument. Note: Further amendment to the drawings may be required if the claims become allowable.
Objections to the Claims, in the previously mailed non-final have been withdrawn in light of applicants claim amendments filed on 09/17/2025.
As necessitated by Applicant’s claim amendments and arguments filed 09/17/2025 (Pg. 8), previous rejection(s) of claim(s) 1, 8-9, and 16 under 35 U.S.C. 112 have been fully considered and are persuasive. The previous rejection of claim(s) 1, 8-9, and 16 under 35 U.S.C. 112 mailed on 06/17/2025 in the Non-final office action has been withdrawn.
As necessitated by Applicant’s claim amendments and arguments filed 09/17/2025 (Pg. 8-10), previous rejection(s) of claim(s) 1-3, 5-11, 13-17 and 20 under 35 U.S.C. 103 have been fully considered and are persuasive. Applicant’s argument on Pg. 8-10, states that “Liu, as well as the secondary references, consistently discloses real-time PCR methods in which fluorescence is monitored continuously throughout thermal cycling, with baseline correction performed computationally after the run. None of the cited references discloses or suggests suppressing fluorescence detection during the early PCR cycles or initiating optical detection only after a defined number of cycles. By contrast, the amended claims recite a method in which fluorescence detection is not merely filtered or corrected after the fact, but is intentionally delayed until after a predetermined cycle count has elapsed...Reconsideration and withdrawal of all rejections are respectfully requested.” Claims 1-20 35 under U.S.C. 103 rejections documented in the previously mailed non-final have been withdrawn in light of applicants claim amendments and arguments on Pg. 8-10. 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. 4-20.
As necessitated by Applicant’s claim amendments and arguments, filed 09/17/2025 (Pg.11) regarding previous rejection(s) of claim(s) 1-15 and 17-21 under Double Patenting have been fully considered but they are persuasive. However, upon further consideration and search, new grounds of rejection are made as documented below in the Double Patenting rejection in this office action on Pg. 20-25.
The rejections for claims 1-15 and 17-21, necessitated by claim amendments filed on 09/17/2025, are documented below in this Final Office Action.
Priority
This application is a 371 of PCT/JP2021/006319 filed on 02/19/2021.
Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d). The certified copy of JAPAN 2020-028303 filed on 02/21/2020 has been submitted of the record on 08/19/2020.
An English translation of the foreign application JAPAN 2020-028303 is required for the record to be considered for the priority date of 02/21/2020.
Accordingly, the priority date of instant claims is determined to be 02/19/2021, the filing date of PCT/JP2021/006319.
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.
Claims 1-3, 5, 8, 17 and 20-21 are rejected under 35 U.S.C. 103 as being unpatentable over Liu et al. (“Liu”; Patent App. Pub. WO 2015057330 A1, April 2013, 2015) in view of Masataka et al. (“Masataka”; Patent App. Pub. JP 4217318 B2, Jan. 28, 2009, English translation provided).
Liu discloses a workflow for direct qPCR quantification of unprocessed forensic casework samples is disclosed herein. 13 pg of DNA has been detected by direct amplification from a paper substrate. Direct qPCR quantification of unprocessed forensic casework samples and direct STR amplification of unprocessed forensic casework samples collected on the same PE-swab will greatly increase forensic laboratory's efficiency and capability (Abstract).
Regarding claim 1, Liu teaches a method wherein “Nucleic acid can be quantified using Polymerase Chain Reaction (PCR) by the detection of amplification products present at the end of PCR, endpoint quantitative PCR, or during PCR, real-time PCR (rtPCR)” (Para. 21). Dry blood stain samples were prepared … de-ionized water was applied to each dried blood stain, and the moisten stain was then swabbed with a 20 mm PE-swab. After swabbing, the filter paper stripe was detached from the swab holder and air dried before taking punches” (Para. 112) Thus, Liu teaches a method wherein a method for detecting a target nucleic acid in dry blood filter paper by real- time PCR.
Regarding claim 1, Liu teaches a method wherein “rtPCR, fluorescent dyes are generally used to label PCR products during thermal cycling” (Para. 21). Liu teaches a method wherein “filter paper, can be successfully incorporated into rtPCR” (Para. 23). Liu also teaches “performing a PCR within the vessel and detecting the level of fluorescence emitted from the vessel” (Para. 8) Thus, Liu teaches a method wherein (1) amplifying the target nucleic acid in the dry blood filter paper by applying thermal cycles to a sample solution containing a dry blood filter paper punch piece and a PCR reagent, wherein the PCR reagent contains a fluorescently labeled probe.
Regarding claim 1, Liu teaches a method wherein “background florescent signal increases after each thermal cycle” (Para. 105). “increases after each thermal cycle” is interpreted as the fluorescence intensity having been determined after a predetermined number of initial cycles. Liu further teaches Figures 3A-3D, in which fluorescence is depicted as being detected after a predetermined cycle (Figures 3A-3D). Thus, Liu suggests a method wherein (2) optically detecting the fluorescence intensity of the sample solution starting after a predetermined number of initial cycles.
Regarding claim 1, Liu teaches a method wherein “To determine the threshold cycle (CT) value, the SDS software first determines the Rn (normalized florescent signal) by dividing the florescent signal in each dye channel by the florescent signal of the passive reference (ROX.TM.). The SDS software then uses the Rn values collected from a predefined range of PCR cycles to serve as baseline. After generating a baseline-subtracted amplification plot of ΔRn versus cycle number, an algorithm defines the cycle number at which the ΔRn value crosses the threshold setting as the threshold cycle (CT)” (Para. 106). Liu further teaches Figures 3A-3D, in which fluorescence is depicted as being detected after a single predetermined cycle (Figures 3A-3D). Thus, Liu teaches a method wherein (3) performing quantitative analysis of the target nucleic acid using data after a predetermined number of cycles of the optically detected data.
However, Liu does not explicitly teach the limitation “optically detecting the fluorescence intensity of the sample solution starting after a predetermined number of initial cycles”.
Masataka discloses “The present invention relates to a technique for analyzing a nucleic acid molecule present in a sample, and more particularly to an analysis method and apparatus for detecting a target nucleic acid molecule based on a reaction result by a nucleic acid amplification reaction. The present invention is applied to a detection method in which the presence / absence of an amplification reaction and / or the reaction amount is obtained by effectively using tracking data relating to a single nucleic acid molecule. Therefore, the present invention can be effectively applied to any purpose for obtaining information related to the number of target nucleic acid molecules.” (Abstract)
Regarding claim 1, Masataka teaches “the method of obtaining multiple data within the elapsed time in a PCR cycle is either when obtaining multiple data continuously or intermittently within a certain time after a predetermined cycle, or when obtaining an appropriate number of measurement data for different cycles” (Para. 33). Thus, Liu and Masataka teach (2) optically detecting the fluorescence intensity of the sample solution starting after a predetermined number of initial cycles.
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 provided a method for detecting target nucleic acids in dry blood filter paper by real-time PCR comprising amplification, detection of fluorescence, and quantitative analysis of the target as taught by Liu to incorporate the method of obtaining multiple data within a certain time after a predetermined cycle as taught by Masataka. Doing so would allow for the avoidance of collection of noise in initial cycle(s), quantification of target nucleic acid in a biological sample by real-time PCR along with the optimization of predetermine cycles to be used for baseline correction. It would be obvious to the ordinary artisan to utilize the methods of Liu in view of Masataka with the reasonable expectation of detecting target nucleic acids in dry blood filter paper by real-time PCR with less baseline to correct for during quantitative analysis.
The teachings of Liu are documented above in the rejection of claim 1 under 35 U.S.C. 103. Claim 2-3, 5 and 21 depend on claim 1. Claims 17 and 20 depend on claim 2, which depends on claim 1.
Regarding claim 2, Liu teaches a method wherein fluorescence of fluorescent dyes was collected during an rtPCR with circular filter paper punches over the 42 cycles with and without filter paper (Figure 3A, 3B). 42 cycles are interpreted as encompassing 10-25 cycles. Liu also suggests a method wherein cycles 1-25 or 15-25 is used for a baseline (Figure 4) and “optimal baseline setting was set at 15 and 19” (Para. 109). 1-25 is interpreted as encompassing 10-25 cycles. Liu teaches a method wherein “To determine the threshold cycle (CT) value, the SDS software first determines the Rn (normalized florescent signal) by dividing the florescent signal in each dye channel by the florescent signal of the passive reference (ROX.TM.). The SDS software then uses the Rn values collected from a predefined range of PCR cycles to serve as baseline. After generating a baseline-subtracted amplification plot of ΔRn versus cycle number, an algorithm defines the cycle number at which the ΔRn value crosses the threshold setting as the threshold cycle (CT)” (Para. 106). As stated above in the 112 rejection, the limitation “predetermined cycles” is indefinite. Furthermore, the MPEP states, "Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." (MPEP 2144.05). Thus, Liu suggests a method wherein the predetermined number of cycles in (3) is 10 cycles or more and 25 cycles or less.
Regarding claims 3 and 17, Liu teaches a method wherein “Nucleic acid can be quantified using Polymerase Chain Reaction (PCR) by the detection of amplification products present at the end of PCR, endpoint quantitative PCR, or during PCR, real-time PCR (rtPCR). (Para.21). Liu teaches a method wherein “detecting the level of fluorescence emitted from the vessel while rtPCR is on-going, wherein the level of fluorescence is detected by a charge-coupled device” (Para. 40). “While rtPCR is on-going” is interpreted as during the thermal cycle and after the cycle as it continues to the next thermal cycle. Thus, Liu teaches a method wherein the data after the predetermined number of cycles in (3) is data obtained by optical detection started later than the start of the thermal cycles.
Regarding claims 5 and 20, Liu teaches a method wherein “The 5'-exonuclease (TaqMan.TM.) probes are oligonucleotides that contain fluorophore and quencher moieties preferably located on 5' and 3' termini … during PCR amplification, the probe specifically hybridizes to its target sequence and the 5'-3'-exonuclease activity of Taq polymerase cleaves the probe between fluorophore and quencher moieties. Enzymatic cleavage of TaqMan.TM. probes spatially separates fluorophore and quencher components, causing significant increases in fluorescence emission correlated with target amplification” (Para. 84). Thus, Liu teaches a method wherein the fluorescently labeled probe comprises a fluorescent substance and a quencher and comprises a partial sequence complementary to the template of the nucleic acid amplification reaction, and the amplified target nucleic acid is detected by detection of the fluorescence generated by irradiation of the fluorescent substance with excitation light.
Regarding claim 8, Liu teaches a method wherein cycles 1-25 or 15-25 is used for a baseline (Figure 4) and “optimal baseline setting was set at 15 and 19” (Para. 109). Setting the optimal baseline is interpreted as setting unphotometric parameters from the start of thermal cycles to the predetermined number of cycles. Setting photometric parameters after the predetermined number of cycles is broadly interpreted as setting a real-time PCR instrument program to detect fluorescence, which is needed before and after baseline detection. Thus, Liu teaches a method wherein the step of optically detecting the fluorescence intensity of the sample solution for each of the thermal cycles in (2) is a step based on photometric parameters set in a thermal cycler and is a step in which optical detection is started later than the start of the thermal cycles by setting unphotometric parameters from the start of thermal cycles to the predetermined number of cycles and setting photometric parameters after the predetermined number of cycles.
Regarding claim 21, Liu teaches a method wherein ”2 mm punches” (Para. 11). Liu also teaches a method wherein “the solid support has a surface area approximately the same as that of a circle with a diameter of about 2 mm” (Para. 28) Thus, Liu teaches a method wherein the size of the punch piece is 1.2 to 2.0 mm in diameter.
Response to Arguments
Applicant' s arguments filed 9/17/2025 (Pg.8-11) with respect to claims 1-15 and 17-21 have been fully considered but do not apply to the new grounds of rejections. To clarify some instances argued in the response filed 9/15/2025 see responses to each argument made by Applicant below:
Applicants’ argument: “In view of the amendment to claim 1, Applicant respectfully submits that the cited art does not disclose or suggest the newly introduced limitation-namely, that optical detection is initiated only after a predetermined number of initial thermal cycles.” (Pg. 10)
Response: In response to applicant's arguments, Liu does suggest optical detection is initiated only after a predetermined number of initial thermal cycles (see Figures 3A-3D). Furthermore, Masataka teaches “obtaining multiple data within the elapsed time in a PCR cycle is either when obtaining multiple data continuously or intermittently within a certain time after a predetermined cycle, or when obtaining an appropriate number of measurement data for different cycles” (Para. 33). Thus, the limitation “optical detection after a predetermined number of thermal cycles” is made obvious over Liu in view of Masataka.
Claims 9-11 and 13 are rejected under 35 U.S.C. 103 as being unpatentable over Liu et al. (“Liu”; Patent App. Pub. WO 2015057330 A1, April 2013, 2015) in view of Masataka et al. (“Masataka”; Patent App. Pub. JP 4217318 B2, Jan. 28, 2009, English translation provided).
Liu discloses a workflow for direct qPCR quantification of unprocessed forensic casework samples is disclosed herein. 13 pg of DNA has been detected by direct amplification from a paper substrate. Direct qPCR quantification of unprocessed forensic casework samples and direct STR amplification of unprocessed forensic casework samples collected on the same PE-swab will greatly increase forensic laboratory's efficiency and capability (Abstract).
Regarding claim 9, Liu teaches a method wherein “Nucleic acid can be quantified using Polymerase Chain Reaction (PCR) by the detection of amplification products present at the end of PCR, endpoint quantitative PCR, or during PCR, real-time PCR (rtPCR)” (Para. 21). Dry blood stain samples were prepared … de-ionized water was applied to each dried blood stain, and the moisten stain was then swabbed with a 20 mm PE-swab. After swabbing, the filter paper stripe was detached from the swab holder and air dried before taking punches” (Para. 112) Thus, Liu teaches a method wherein a method for detecting a target nucleic acid in dry blood filter paper by real- time PCR, the method comprising
Regarding claim 9, Liu teaches a method wherein “rtPCR, fluorescent dyes are generally used to label PCR products during thermal cycling” (Para. 21). Liu teaches a method wherein “filter paper, can be successfully incorporated into rtPCR” (Para. 23). Liu also teaches “performing a PCR within the vessel and detecting the level of fluorescence emitted from the vessel” (Para. 8) Thus, Liu teaches a method wherein (1) amplifying the target nucleic acid in the dry blood filter paper by applying thermal cycles to a sample solution containing a dry blood filter paper punch piece and a PCR reagent, wherein the PCR reagent contains a fluorescently labeled probe.
Regarding claim 9, Liu teaches a method wherein “background florescent signal increases after each thermal cycle” (Para. 105). “increases after each thermal cycle” is interpreted as the fluorescence intensity having been determined after each cycle. Florescence is interpreted as being determined continuously in real-time PCR. Liu further teaches Figures 3A-3D, in which fluorescence is depicted as being detected after a single predetermined cycle (Figures 3A-3D). Thus, Liu suggests a method wherein (2) optically detecting the fluorescence intensity of the sample solution for each of the thermal cycles;
Regarding claim 9, Liu teaches a method wherein “To determine the threshold cycle (CT) value, the SDS software first determines the Rn (normalized florescent signal) by dividing the florescent signal in each dye channel by the florescent signal of the passive reference (ROX.TM.). The SDS software then uses the Rn values collected from a predefined range of PCR cycles to serve as baseline. After generating a baseline-subtracted amplification plot of ΔRn versus cycle number, an algorithm defines the cycle number at which the ΔRn value crosses the threshold setting as the threshold cycle (CT)” (Para. 106). Thus, Liu teaches a method wherein (3) performing quantitative analysis of the target nucleic acid using data after a predetermined number of cycles of the optically detected data.
Regarding claim 9, Liu teaches a method wherein cycles 1-25 or 15-25 is used for a baseline (Figure 4) and “optimal baseline setting was set at 15 and 19” (Para. 109). Setting the optimal baseline is interpreted as setting unphotometric parameters from the start of thermal cycles to the predetermined number of cycles. Setting photometric parameters after the predetermined number of cycles is broadly interpreted as setting a real-time PCR instrument program to detect fluorescence, which is needed before and after baseline detection. Thus, Liu teaches a method wherein the step of optically detecting the fluorescence intensity of the sample solution for each of the thermal cycles in (2) is a step based on photometric parameters set in a thermal cycler and is a step in which optical detection is started later than the start of the thermal cycles by setting unphotometric parameters from the start of thermal cycles to the predetermined number of cycles and setting photometric parameters after the predetermined number of cycles.
However, Liu does not explicitly teach the limitation “optically detecting the fluorescence intensity of the sample solution starting after a predetermined number of initial cycles”.
Masataka discloses “The present invention relates to a technique for analyzing a nucleic acid molecule present in a sample, and more particularly to an analysis method and apparatus for detecting a target nucleic acid molecule based on a reaction result by a nucleic acid amplification reaction. The present invention is applied to a detection method in which the presence / absence of an amplification reaction and / or the reaction amount is obtained by effectively using tracking data relating to a single nucleic acid molecule. Therefore, the present invention can be effectively applied to any purpose for obtaining information related to the number of target nucleic acid molecules.” (Abstract)
Regarding claim 9, Masataka teaches “the method of obtaining multiple data within the elapsed time in a PCR cycle is either when obtaining multiple data continuously or intermittently within a certain time after a predetermined cycle, or when obtaining an appropriate number of measurement data for different cycles” (Para. 33). Thus, Liu and Masataka teach (2) optically detecting the fluorescence intensity of the sample solution starting after a predetermined number of initial cycles.
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 provided a method for detecting target nucleic acids in dry blood filter paper by real-time PCR comprising amplification, setting of photometric and unphotometric parameters, detection of fluorescence, and quantitative analysis of the target as taught by Liu to incorporate the method of obtaining multiple data within a certain time after a predetermined cycle as taught by Masataka. Doing so would allow for quantification of target nucleic acid in a biological sample by real-time PCR along with the optimization of predetermine cycles to be used for baseline correction to avoid noise collected in the initial cylce(s). It would be obvious to the ordinary artisan to utilize the methods of Liu in view of Masataka with the reasonable expectation of detecting target nucleic acids in dry blood filter paper by real-time PCR with less baseline to correct for during quantitative analysis.
The teachings of Liu are documented above in the rejection of claim 9 under 35 U.S.C. 103. Claim 10-11 and 13 depend on claim 9.
Regarding claim 10, Liu teaches a method wherein fluorescence of fluorescent dyes was collected during an rtPCR with circular filter paper punches over the 42 cycles with and without filter paper (Figure 3A, 3B). 42 cycles are interpreted as encompassing 10-25 cycles. Liu also suggests a method wherein cycles 1-25 or 15-25 is used for a baseline (Figure 4) and “optimal baseline setting was set at 15 and 19” (Para. 109). 1-25 is interpreted as encompassing 10-25 cycles. Liu teaches a method wherein “To determine the threshold cycle (CT) value, the SDS software first determines the Rn (normalized florescent signal) by dividing the florescent signal in each dye channel by the florescent signal of the passive reference (ROX.TM.). The SDS software then uses the Rn values collected from a predefined range of PCR cycles to serve as baseline. After generating a baseline-subtracted amplification plot of ΔRn versus cycle number, an algorithm defines the cycle number at which the ΔRn value crosses the threshold setting as the threshold cycle (CT)” (Para. 106). As stated above in the 112 rejection, the limitation “predetermined cycles” is indefinite. Furthermore, the MPEP states, "Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." (MPEP 2144.05). Thus, Liu suggests a method wherein the predetermined number of cycles in (3) is 10 cycles or more and 25 cycles or less.
Regarding claim 11, Liu teaches a method wherein “Nucleic acid can be quantified using Polymerase Chain Reaction (PCR) by the detection of amplification products present at the end of PCR, endpoint quantitative PCR, or during PCR, real-time PCR (rtPCR). (Para.21). Liu teaches a method wherein “detecting the level of fluorescence emitted from the vessel while rtPCR is on-going, wherein the level of fluorescence is detected by a charge-coupled device” (Para. 40). “While rtPCR is on-going” is interpreted as during the thermal cycle and after the cycle as it continues to the next thermal cycle. Thus, Liu teaches a method wherein the data after the predetermined number of cycles in (3) is data obtained by optical detection started later than the start of the thermal cycles.
Regarding claim 13, Liu teaches a method wherein “The 5'-exonuclease (TaqMan.TM.) probes are oligonucleotides that contain fluorophore and quencher moieties preferably located on 5' and 3' termini … during PCR amplification, the probe specifically hybridizes to its target sequence and the 5'-3'-exonuclease activity of Taq polymerase cleaves the probe between fluorophore and quencher moieties. Enzymatic cleavage of TaqMan.TM. probes spatially separates fluorophore and quencher components, causing significant increases in fluorescence emission correlated with target amplification” (Para. 84). Thus, Liu teaches a method wherein the fluorescently labeled probe comprises a fluorescent substance and a quencher and comprises a partial sequence complementary to the template of the nucleic acid amplification reaction, and the amplified target nucleic acid is detected by detection of the fluorescence generated by irradiation of the fluorescent substance with excitation light.
Response to Arguments
Applicant' s arguments filed 9/17/2025 (Pg.8-11) with respect to claims 9-11 and 13 have been fully considered but do not apply to the new grounds of rejections. See response to argument as discussed above.
Claims 1, 4 and 18-19 are rejected under 35 U.S.C. 103 as being unpatentable over Liu et al. (“Liu”; Patent App. Pub. WO 2015057330 A1, April 2013, 2015) in view of Masataka et al. (“Masataka”; Patent App. Pub. JP 4217318 B2, Jan. 28, 2009, English translation provided) and Taylor et al. (“Taylor”; (2011). Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples. Malaria journal, 10, 1-8).
The teachings of Liu and Masataka are documented above in the rejection of claims 1-3, 5, 8-11, 13, 17 and 20-21 under 35 U.S.C. 103. Claims 4 depends on claim 1. Claims 18 and 19 depend on claims 2 and 3, respectively, which depend on claim 1. Liu and Masataka do not explicitly teach the limitations of claims 4 and 18-19.
Taylor discloses the methodology described facilitates high-throughput testing of blood samples collected in the field by fluorescence-based real-time PCR. This method can be applied to a broad range of clinical studies with the advantages of immediate sample testing, lower experimental costs and time-savings (Abstract-Conclusions).
Regarding claims 4 and 18-19, Taylor teaches a method wherein “PCR was performed … polymerase … 200 µM dNTPs, 0-40× SYB Green” and “To detect the … gene … primers … were used (Pg. 2, Real-time PCR and melt curve analysis from blood, (Para. 1). Taylor also teaches a method wherein “blood spots were prepared from a clinical sample ... tested under the same reaction conditions used for whole blood” (Pg. 3, Detection from dried blood spots, Para. 1). Thus, Taylor teaches a method wherein the PCR reagent comprises at least primers, polymerase, and dNTPs besides the fluorescently labeled probe.
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 method for detecting target nucleic acids in dry blood filter paper by real-time PCR comprising amplification, detection of fluorescence after a number of predetermined cycles set as baseline, and quantitative analysis of the target as taught by Liu and Masataka to incorporate the method comprising PCR reagents as taught by Taylor and provide PCR reagent comprises at least primers, polymerase, and dNTPs besides the fluorescently labeled probe. One of ordinary skill in the art would be motivated to do so because the reagents are common necessary components for a real time PCR amplification reaction. It would be obvious to the ordinary artisan to include the reagents of Taylor to the method of Liu with the reasonable expectation of having the common reagents needed for PCR amplification.
Response to Arguments
Applicant' s arguments filed 9/17/2025 (Pg.8-11) with respect to claims 1, 4 and 18-19 have been fully considered but do not apply to the new grounds of rejections. See response to argument as discussed above.
Claims 9 and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Liu et al. (“Liu”; Patent App. Pub. WO 2015057330 A1, April 2013, 2015) in view of Masataka et al. (“Masataka”; Patent App. Pub. JP 4217318 B2, Jan. 28, 2009, English translation provided) and Taylor et al. (“Taylor”; (2011). Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples. Malaria journal, 10, 1-8).
The teachings of Liu and Masataka are documented above in the rejection of claims 1-5, 8-11, 13, 17-19 and 20-21 under 35 U.S.C. 103. Claim 12 depends on claim 9. Liu and Masataka Liu do not explicitly teach the limitations of claim 12.
Taylor discloses the methodology described facilitates high-throughput testing of blood samples collected in the field by fluorescence-based real-time PCR. This method can be applied to a broad range of clinical studies with the advantages of immediate sample testing, lower experimental costs and time-savings (Abstract-Conclusions).
Regarding claim 12, Taylor teaches a method wherein “PCR was performed … polymerase … 200 µM dNTPs, 0-40× SYB Green” and “To detect the … gene … primers … were used (Pg. 2, Real-time PCR and melt curve analysis from blood, (Para. 1). Taylor also teaches a method wherein “blood spots were prepared from a clinical sample ... tested under the same reaction conditions used for whole blood” (Pg. 3, Detection from dried blood spots, Para. 1). Thus, Taylor teaches a method wherein the PCR reagent comprises at least primers, polymerase, and dNTPs besides the fluorescently labeled probe.
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 method for detecting target nucleic acids in dry blood filter paper by real-time PCR comprising amplification, detection of fluorescence after a number of predetermined cycles set as baseline, and quantitative analysis of the target as taught by Liu and Masataka to incorporate the method comprising PCR reagents as taught by Taylor and provide PCR reagent comprises at least primers, polymerase, and dNTPs besides the fluorescently labeled probe. One of ordinary skill in the art would be motivated to do so because the reagents are common necessary components for a real time PCR amplification reaction. It would be obvious to the ordinary artisan to include the reagents of Taylor to the method of Liu and Masataka with the reasonable expectation of having the common reagents needed for PCR amplification.
Response to Arguments
Applicant' s arguments filed 9/17/2025 (Pg.8-11) with respect to claims 9 and 12 have been fully considered but do not apply to the new grounds of rejections. See response to argument as discussed above.
Claims 1 and 6 are rejected under 35 U.S.C. 103 as being unpatentable over Liu et al. (“Liu”; Patent App. Pub. WO 2015057330 A1, April 2013, 2015) in view of Masataka et al. (“Masataka”; Patent App. Pub. JP 4217318 B2, Jan. 28, 2009, English translation provided) and Espy et al. (“Espy” (2006). Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clinical microbiology reviews, 19(1), 165-256).
The teachings of Liu and Masataka are documented above in the rejection of claims 1-5, 8-13, 17-19 and 20-21 under 35 U.S.C. 103. Claim 6 depends on claim 1. Claim 14 depends on claim 9. Liu and Masataka do not explicitly teach the limitations of claim 6.
Espy discloses a review focused on the application of real-time PCR in the clinical microbiology laboratory (Pg. 166, Introduction, last sentence).
Regarding claim 6, Espy teaches a method wherein “Opticon” (Instrument) TaqMan, molecular beacons (Probes) can be detected at 515-545 nM when excited at 450-495 nM (Table 1). Thus, Espy teaches a method wherein the fluorescent label has a fluorescence detection wavelength in the range of 500 nm to 600 nm.
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 method for detecting target nucleic acids in dry blood filter paper by real-time PCR comprising amplification, detection of fluorescence after a number of predetermined cycles set as baseline, and quantitative analysis as taught by Liu and Masataka to incorporate the method wherein the fluorescent label can be detected at 515-545 nM as taught by Espy and provide a fluorescent label that can detected in the range of 500 nm to 600nm. One of ordinary skill in the art would be motivated to do so because it is a normal range of fluorescence detection and allows for optimal detection of green/yellow fluorescence in blood based samples with a reasonable amount of success.
Claims 9 and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Liu et al. (“Liu”; Patent App. Pub. WO 2015057330 A1, April 2013, 2015) in view of Masataka et al. (“Masataka”; Patent App. Pub. JP 4217318 B2, Jan. 28, 2009, English translation provided) and Espy et al. (“Espy” (2006). Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clinical microbiology reviews, 19(1), 165-256).
The teachings of Liu and Masataka are documented above in the rejection of claims 1-6, 8-13, 17-19 and 20-21 under 35 U.S.C. 103 Claim 14 depends on claim 9. Liu and Masataka do not explicitly teach the limitations of claim 14.
Espy discloses a review focused on the application of real-time PCR in the clinical microbiology laboratory (Pg. 166, Introduction, last sentence).
Regarding claim 14, Espy teaches a method wherein “Opticon” (Instrument) TaqMan, molecular beacons (Probes) can be detected at 515-545 nM when excited at 450-495 nM (Table 1). Thus, Espy teaches a method wherein the fluorescent label has a fluorescence detection wavelength in the range of 500 nm to 600 nm.
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 method for detecting target nucleic acids in dry blood filter paper by real-time PCR comprising amplification, detection of fluorescence after a number of predetermined cycles set as baseline, and quantitative analysis of the target as taught by Liu and Masataka to incorporate the method wherein the fluorescent label can be detected at 515-545 nM as taught by Espy and provide a fluorescent label that can detected in the range of 500 nm to 600nm. One of ordinary skill in the art would be motivated to do so because it is a normal range of fluorescence detection and allows for optimal detection of green/yellow fluorescence in blood based samples with a reasonable amount of success.
Response to Arguments
Applicant' s arguments filed 9/17/2025 (Pg.8-11) with respect to claims 9 and 14 have been fully considered but do not apply to the new grounds of rejections. See response to argument as discussed above.
Claims 1 and 7 are rejected under 35 U.S.C. 103 as being unpatentable over Liu et al. (“Liu”; Patent App. Pub. WO 2015057330 A1, April 2013, 2015) in view of Masataka et al. (“Masataka”; Patent App. Pub. JP 4217318 B2, Jan. 28, 2009, English translation provided) and Knapkova et al. (“Knapkova”; (2018). Reliability of Neonatal Screening Results. International Journal of Neonatal Screening, 4(3), 28).
The teachings of Liu and Masataka are documented above in the rejection of claims 1-6, 8-14, 17-19 and 20-21 under 35 U.S.C. 103. Claim 7 depends on claim 1. Liu and Masataka do not explicitly teach the limitations of claim 7.
Knapkova discloses a meeting report on the reliability of neonatal screening results. This special edition of the International Journal of Neonatal Screening comprises the abstracts of all oral presentations and posters from the biennial ISNS European regional meeting, to be held in Bratislava, Slovakia, 14–17 October 2018. (Pg. 1, Title and Introduction-first sentence)
Regarding claim 7, abstract P39 of Knapkova teaches a method wherein “We have developed a four-plex real-time PCR assay to screen for SCIDs, XLA and SMA in DNA extracted from a single 3.2 mm punch of a dried blood spot (DBS)” and “The PCR assay identifies … in the SMN1 gene while simultaneously evaluating … of T-cell receptor excision circles (TREC) and Kappa-deleting recombination excision circles (KREC) molecules” (Pg. 44, P39. Reliability of a Multiplex QPCR Assay for the Newborn Screening of SCID, SMA and XLA, ln 8-14 of Gutierrez-Mateo et al. abstract). Thus, Knapkova teaches a method wherein the target nucleic acid is a nucleic acid of one or more genes selected from the group consisting of TREC, KREC, and SMN1.
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 method for detecting target nucleic acids in dry blood filter paper by real-time PCR comprising amplification, detection of fluorescence after a number of predetermined cycles set as baseline, and quantitative analysis of the target as taught by Liu and Masataka to incorporate the method wherein the target nucleic acid is from TREC, KREC, and SMN1 as taught by Knapkova and provide detection of TREC, KREC, and/or SMN. One would be motivated to do so to allow for quick detection of genes involved in disorders, such as during newborn screening.
Response to Arguments
Applicant' s arguments filed 9/17/2025 (Pg.8-11) with respect to claims 1 and 7 have been fully considered but do not apply to the new grounds of rejections. See response to argument as discussed above.
Claims 9 and 15 are rejected under 35 U.S.C. 103 as being unpatentable over Liu et al. (“Liu”; Patent App. Pub. WO 2015057330 A1, April 2013, 2015) in view of Masataka et al. (“Masataka”; Patent App. Pub. JP 4217318 B2, Jan. 28, 2009, English translation provided) and Knapkova et al. (“Knapkova”; (2018). Reliability of Neonatal Screening Results. International Journal of Neonatal Screening, 4(3), 28.
The teachings of Liu and Masataka are documented above in the rejection of claims 1-14, 17-19 and 20-21 under 35 U.S.C. 103. Claim 15 depends on claim 9. Liu and Masataka do not explicitly teach the limitations of claim 15.
Knapkova discloses a meeting report on the reliability of neonatal screening results. This special edition of the International Journal of Neonatal Screening comprises the abstracts of all oral presentations and posters from the biennial ISNS European regional meeting, to be held in Bratislava, Slovakia, 14–17 October 2018. (Pg. 1, Title and Introduction-first sentence)
Regarding claim 15, abstract P39 of Knapkova teaches a method wherein “We have developed a four-plex real-time PCR assay to screen for SCIDs, XLA and SMA in DNA extracted from a single 3.2 mm punch of a dried blood spot (DBS)” and “The PCR assay identifies … in the SMN1 gene while simultaneously evaluating … of T-cell receptor excision circles (TREC) and Kappa-deleting recombination excision circles (KREC) molecules” (Pg. 44, P39. Reliability of a Multiplex QPCR Assay for the Newborn Screening of SCID, SMA and XLA, ln 8-14 of Gutierrez-Mateo et al. abstract). Thus, Knapkova suggests a method wherein the target nucleic acid is a nucleic acid of one or more genes selected from the group consisting of TREC, KREC, and SMN1.
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 method for detecting target nucleic acids in dry blood filter paper by real-time PCR comprising amplification, detection of fluorescence after a number of predetermined cycles set as baseline, and quantitative analysis of the target as taught by Liu and Masataka to incorporate the method wherein the target nucleic acid is from TREC, KREC, and SMN1 as taught by Knapkova and provide detection of TREC, KREC, and/or SMN. One would be motivated to do so to allow for quick detection of genes involved in disorders, such as during newborn screening.
Response to Arguments
Applicant' s arguments filed 9/17/2025 (Pg. 8-11) with respect to claims 9 and 15 have been fully considered but do not apply to the new grounds of rejections. See response to argument as discussed above.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
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Claims 1, 4 and 7 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of copending Application No. 17/630,069 (“069”, reference application) in view of Liu et al. (“Liu”; Patent App. Pub. WO 2015057330 A1, April 2013, 2015) and Masataka et al. (“Masataka”; Patent App. Pub. JP 4217318 B2, Jan. 28, 2009, English translation provided).
Claim 1 of copending Application No. ‘069 is drawn to:
1. A method for detecting the SMN1 gene in a dried blood spot in a filter paper by real-time PCR, including the steps of (A) to (D) below: (A) a step of adding the dried blood spot in a filter paper to a PCR reaction tube; (B) a step of adding a PCR reagent to the PCR reaction tube, wherein the PCR reagent contains at least a primer designed in a manner that the reactivity to the SMN2 gene is less than 1% of that to the SMN1 gene, a polymerase, dNTPs and an intercalator or a fluorescently labeled probe; (C) a step of performing PCR reaction in the tube containing the PCR reagent and the dried blood spot in a filter paper; and (D) a step of sequentially and optically detecting a target nucleic acid in the SMN1 gene amplified by the PCR reaction.
The teachings of Liu and Masataka are documented above in the rejection of claims 1-3, 5, 8-11, 13, 17 and 20-21 under 35 U.S.C. 103.
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 method of detecting the SMN1 gene in a dried blood spot in a filter paper by real-time PCR as taught by copending Application No. ‘069 to incorporate the method of baseline correction settings using an optimized and predetermined set of cycles and the ability to directly quantify the presence of target nucleic acids of interest as taught by Liu and Masataka and perform quantitative analysis of the target nucleic acid using data after a predetermined number of the optically detected data. A person of ordinary skill in the art would have had a reasonable expectation of success allowing for a more reliable determination of amplified product and direct detection of gene(s) related to disease and other genetic characteristics using a dried blood spot sample on filter paper.
This is a provisional nonstatutory double patenting rejection.
Response to Arguments
Applicant' s request to traverse in view of present claim amendments filed 9/17/2025 (Pg. 11) with respect to claims 1-15 and 17-21 have been fully considered but do not apply to the new grounds of rejections. See response to argument as discussed above.
Claims 1, 4-5, 7, 9, 12-13, 15, and 18-21 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-2, 4-7 of copending Application No. 17/642166 (“‘166”, reference application) in view of Liu et al. (“Liu”; Patent App. Pub. WO 2015057330 A1, April 2013, 2015) and Masataka et al. (“Masataka”; Patent App. Pub. JP 4217318 B2, Jan. 28, 2009, English translation provided).
Claims 1-2, 4-7 of copending Application No. ‘166 are drawn to:
1. A method for detecting a target nucleic acid contained in a dried blood spot in a filter paper by real-time PCR, including the steps of (A) to (D) below: (A) a step of adding the dried blood spot in a filter paper to a PCR reaction tube, wherein the filter paper is a circular punched piece with a diameter of 1.2 mm to 2.0 mm or a punched piece containing whole blood in an amount of 0.95 v/v % to 6.6 v/v % based on the total amount of the reaction solution; (B) a step of adding 20 to 50 μL of a PCR reagent to the PCR reaction tube; (C) a step of performing PCR reaction in the tube which contains the PCR reagent and the dried blood spot in a filter paper and which is sealed with a cap; and (D) a step of sequentially and optically detecting the target nucleic acid amplified by the PCR reaction.
2. The method for detecting a target nucleic acid according to claim 1, wherein the step of (B) is conducted after the step of (A).
4. The method for detecting a target nucleic acid according to claim 1, wherein the PCR reagent contains at least a primer, a polymerase, dNTPs and an intercalator or a fluorescently labeled probe.
5. The method for detecting a target nucleic acid according to claim 4, wherein the amplified target nucleic acid is detected through detection of fluorescence emitted by irradiation of the intercalator with an excitation light.
6. The method for detecting a target nucleic acid according to claim 4, wherein the fluorescently labeled probe has a fluorescent substance and a quencher and includes a partial sequence complementary to a template of the nucleic acid amplification reaction, and the amplified target nucleic acid is detected through detection of fluorescence emitted by irradiation of the fluorescent substance with an excitation light.
7. The method for detecting a target nucleic acid according to claim 1, wherein the target nucleic acid is one or more gene fragments and/or genes selected from the group consisting of TRECs, KRECs and SMN1.
The teachings of Liu and Masataka are documented above in the rejection of claims 1-3, 5, 8-11, 13, 17 and 20-21 under 35 U.S.C. 103.
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 method of detecting a target nucleic acid contained in a dried blood spot in a filter paper by real-time PCR as taught by copending Application No. ‘166 to incorporate baseline correction settings using an optimized and predetermined set of cycles and the ability to directly quantify the presence of target nucleic acids of interest as taught by Liu and Masataka and perform quantitative analysis of the target nucleic acid using data after a predetermined number of the optically detected data. Doing so would allow for more reliable determination of amplified product.
This is a provisional nonstatutory double patenting rejection.
Response to Arguments
Applicant' s request to traverse in view of present claim amendments filed 9/17/2025 (Pg. 11) with respect to claims 1-15 and 17-21 have been fully considered but do not apply to the new grounds of rejections. See response to argument as discussed above.
Conclusion of Response to Arguments
In view of the amendments, new grounds of rejections and above responses to arguments are documented in this Final Office Action. No claims are in condition for allowance.
Conclusion
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