DETAILED ACTION
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 .
Priority
This application is a 371 of PCT/JP2022/025912 filed 06/29/2022 and claims to foreign priority to Application No. JP2021-184214 filed 11/11/2021.
Status of the claims
Claims 1-10 are pending.
Specification
The drawings are objected to because Figs.3C-3D, Figs.4C-4D, Figs. 5A-5B, Figs. 6C-6D, Figs. 7C-7D and Figs. 10A-10D are illegible.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Appropriate correction is required.
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 1-10 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 is directed to a preservation method for a PCR reagent and provides two process steps, i.e. a first step which comprises preparing a reaction liquid by mixing all of a first composition containing DNA polymerase, a second composition containing a primer and third composition containing a liquid buffer and a second step which comprises refrigerating the reaction liquid.
Claim 1 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being incomplete for omitting essential elements, such omission amounting to a gap between the elements. See MPEP § 2172.01. Claim 1 fails to provide a nexus between the preamble’s goal of preserving of a PCR reagent and the steps of preparing and refrigerating a reaction liquid.
It is unknown whether the reaction liquid prepared in step (1) acts a preservative agent and how the reaction liquid preserves and what the instant PCR reagent this reaction liquid serves to preserve. Claims 2-10 are further rejected as they depend from claim 1.
Claim 1 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being incomplete for omitting essential elements, such omission amounting to a gap between the elements. See MPEP § 2172.01. Claim 1 fails to make clear whether the first composition, the second composition, and the third composition are each accommodated in different containers prior to step (1), or after step (2). Claims 2-10 are further rejected as they depend from claim 1.
Claim 2 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite because claim 2 recites the limitation “wherein the PCR reagent further includes a positive control, and in the step (2), a part of the reaction liquid is accommodated in a first test container together with the positive control, and only a part of the reaction liquid is accommodated in a second test container, and then the first and second test containers are refrigerated”. This limitation is confusing and lacks clarity of scope because the nature of the PCR reagent is not clearly defined.
For instance, it is unclear if the instant PCR reagent is the same or different from the instant first composition comprising a DNA polymerase, or is the same or different from the instant the second composition comprising a primer, or is the third liquid buffer composition; or is a clinical specimen sample; or is a positive control oligonucleotide, or is a DNA sample or is something else.
Furthermore, claim 2 also fails to clarify whether or not the instant PCR reagent is present within the refrigerated first test container that comprises the reaction liquid combined with a positive control oligonucleotide; AND whether or not the instant PCR reagent is also present within the refrigerated second test container consisting of reaction liquid. Claims 7-8 are further rejected as they depend from claim 2.
Claim 7 recites the limitation “the PCR reagent further includes a pretreatment liquid, and in the step (2), the pretreatment liquid is accommodated in a third test container and refrigerated”.
Claim 7, which depends from claims 1 and 2, is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite as this limitation is found to lack clarity and clarity of scope. Neither claims 1-2 nor claim 7 define/indicate what constitutes the instant PCR reagent to be preserved. It is not known whether the instant PCR reagent is the same or is different from the claimed reaction liquid of claim 1, prepared by step (1) of claim 1; or whether something else is intended as the instant PCR reagent (e.g. according to claim 2, the PCR reagent contained in a “test” container may be a positive control combined with a portion of the reaction liquid).
Claim 7 is again rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for it is not known whether it is the container comprising the third composition, which additionally comprises a pretreatment liquid; or whether claim 7 directs four containers, i.e. the first composition contained in a first container, the second composition contained in a second container, the third composition contained in a third container and a pretreatment liquid contained in a third test container. There is no antecedent support for a “third test container” from either claims 1 or 2. It is unclear whether the instant third test container consists only the pretreatment liquid; or comprises the pretreatment liquid and other additional elements in a same manner as the instant first test container which consists a reaction liquid combined with a positive control. Claim 8 is further rejected as it depends from claims 1 and 2.
Claim 9 recites the limitation “preparing a liquid from which a nucleic acid is extracted by adding a pretreatment liquid to a specimen”. Claim 9 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite as this limitation is found to lack clarity and clarity of scope. It is unclear whether this limitation of claim 9 is simply drawn to a step of adding a pretreatment liquid to a specimen and further comprises collecting an extracted DNA liquid (i.e. a purified DNA suspension); or whether this limitation of claim 9 is directed to a step of adding a pretreatment liquid to a specimen so as to obtain a liquid from which a nucleic acid may be extracted (e.g. a crude lysate). Claim 10 is further rejected as it depends from claim 9.
Claim 9 recites the limitation “mixing the reaction liquid with the liquid from which the nucleic acid is extracted” which is found to lack clarity. It is unclear how to form the instant mixed reaction liquid of claim 9 since it is not clear whether claim 9 directs using the prepared reaction liquid prior to step (2) of claim 1; or combining the instant reaction liquid that is generated after the refrigeration process of step (2) of claim 1. Claim 10 is further rejected as it depends from claim 9.
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 1, 4 and 7-10 are rejected under 35 U.S.C. 103 as being unpatentable over Kofler et al. (1999, Diagnostic microbiology and infectious disease, 34(1), pp.33-35).
Regarding claims 1 and 9, Kofler et al. is directed to a method of amplifying a human genomic DNA and CMV DNA using preformulated, prealiquoted PCR mixes that are stored at 20°C or 4°C (see pg 33, right col., 1st para and see pg 34, Figs. 1A-1B and Figs. 2A-2B).
The method of Kofler et al. comprises step (1) of claim 1: constituting a step of preparing a reaction liquid comprising a DNA polymerase, a primer and buffer (see the PCR master mix prepared by Kofler et al. on pg 34, left col., 2nd para and pg 34, left col., last para and pg 34, right col., 1st para).
The method of Kofler et al. comprises step (2) of claim 1 and claim 4, as Kofler et al. teach that after preparation of the PCR master mix, aliquots (28 µl) of the master mix were placed into 200 µl thin-walled tubes and stored at -20°C or 4°C (pg 34, right col., 1st para).
Kofler et al. teach that PCR was initiated by bringing one or more refrigerated aliquots containing the PCR master mix/the instant reaction liquid to room temperature followed by adding 2 µl of either human genomic DNA (100 ng), sample DNA, CMV DNA (10 or 100 copies) and a contaminating artificial dUTP-DNA (10 fg, generated by PCR), respectively to one or more of the thawed out aliquots.
Kofler et al. teach PCR thermal cycling consisted of 30 cycles for the human histidyl t-RNA synthetase gene and 50 cycles for the CMV mtr II gene each of 30 s denaturation at 94°C, 30 s annealing at 63°C and 10 s extension at 72°C using a theromocycler (see pg 34, right col., 1st para).
Kofler et al. teach PCR assays show no loss of enzyme activity for Taq DNA polymerase for the aliquoted of PCR master mix in tubes stored at -20°C for a period of 4 months (pg 35, left col., 1st para and Figs. 1A, 2A-2B).
Kofler et al. teach PCR assays show no loss of enzyme activity for Taq DNA polymerase for the aliquoted of PCR master mix in tubes stored at 4 °C for up to 3 weeks (pg 35, left col., 1st para and Fig. 1B).
Kofler et al. teach many advantages of using a frozen PCR master mix that only requires addition of template DNA for initiating PCR e.g. the master mix is PCR ready without the need for reconstitution, aliquoting, additives or freeze-drying equipment (pg 35, right col. 1st para of Discussion).
Kofler et al. teach preformulated and aliquoted PCR master mixes ensure greater interassay conformity, minimization of pipetting steps and reduction of potential pipetting errors. In addition, this method saves hands-on time compared to the traditional PCR set-up procedure, which is also important in a clinical laboratory setting. No special equipment is necessary and this method could therefore be used in any laboratory where the same PCR reactions are performed frequently (pg 35, right col. 2nd para of Discussion).
Regarding claims 7-8, Kofler et al. teach a DNA sample submitted for -20 ºC refrigeration comprising a pretreatment liquid consisting heat-killed 100 ng/mL proteinase K in 75 mM Tris HCl pH 9.0, 20 mM (NH4)2SO4 and 0.01% (v/v) Tween 20 (the instant anionic surfactant) and extracted DNA released from a peripheral blood sample without further purification (see pg 33, right col., text of Materials and Methods and pg 34, left col., 1st para).
Regarding claims 9-10, Kofler et al. teach thermal cycling of AN instant mixed reaction liquid formed by combining a crude DNA sample or a purified DNA sample and the pretreatment liquid consisting heat-killed 100 ng/mL proteinase K in 75 mM Tris HCl pH 9.0, 20 mM (NH4)2SO4 and 0.01% (v/v) Tween 20 (pg 34, right col. 1st para).
Kofler et al. teach thermal cycling consisted of 30 cycles for the human histidyl t-RNA synthetase gene and 50 cycles for the CMV mtr II gene each of 30 s denaturation at 94°C, 30 s annealing at 63°C and 10 s extension at 72°C using a theromocycler (pg 34, right col. 1st para).
Concerning the instant reaction liquid of claim 1, step (1) and step (2)
Kofler et al. teach the instant reaction liquid or a first PCR master mix for amplifying a region of the human histidyl t-RNA synthetase gene as comprising of Taq DNA polymerase and an oligonucleotide primer pair at 300 nM each, in 75 mM Tris HCl pH 9.0, 20 mM (NH4)2SO4 buffer solution (see pg 34, left col., 2nd para).
Kofler et al. also teach the instant reaction liquid or a second PCR master mix for amplifying a DNA fragment homologue to the mtr II gene of the CMV genome as comprising of Taq DNA polymerase and an oligonucleotide primer pair at 300 nM each, in 10 mM Tris HCl pH 8.3, 50mM KCl buffer solution (see pg 34, left col., last para and pg 34, right col., 1st para).
Omitted from Kofler et al. (claim 1, step (1))
Regarding step (1) of claim 1, Kofler et al. do not teach a first composition comprising DNA polymerase, the second composition comprising a primer, and the third composition comprising a buffer liquid are accommodated in different containers.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the invention to provide individual component(s) or individual compositions that are suitable for making a mixture within different containers, prior to combining the components together to generate a mixture. The ordinary skilled artisan is readily apprised that separate individual components in individual containers respectively, offers the ordinary skilled artisan incredible flexibility for manufacture and storage and transport and assay design and execution and convenience and reproducibility due to the ability of the artisan to apply manufacturing standardization, quality control and validation procedures to each individual component/composition prior to using it to generate a mixture.
In view of the combined teachings and suggestions of all of the cited prior art references, the instant claims 1, 4 and 7-10 are prima facie obvious.
Claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over Kofler et al. (1999, Diagnostic microbiology and infectious disease, 34(1), pp.33-35) in view of Wenninger et al. (2015, Journal of Clinical Microbiology, 53(4), pp.1452-1453) as evidenced by the ProFlu+ assay kit manual.
The teachings of Kofler et al. as it relates to claim 1 are provided in the rejection under 35 U.S.C. 103 above and applies here.
Omitted from Kofler et al. (claim 2)
Regarding claim 2, Kofler et al. do not teach a PCR reagent which further includes a positive control, and in the step (2), a part of the reaction liquid is accommodated in a first test container together with the positive control, and only a part of the reaction liquid is accommodated in a second test container, and then the first and second test containers are refrigerated.
Wenninger et al. (claims 1-2 and 9-10)
Wenninger et al. is directed to an RT-PCR amplification method for detecting influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) using reagents from a commercial kit i.e. the ProFlu+ assay kit (Hologic, Inc., San Diego, CA) (see title and pg 1453, Figs. 1 and 2).
Wenninger et al. teach that package insert data emphasize that the master mix (components of which include separately packaged) (pg 1452, right col., 1st para).
Wenninger et al. teach their method as comprising steps (1) and (2) of claim 1 including forming a Prodesse ProFlu+ master mix in accordance with assay guidelines (1) and then dispensing master mix in 20 µl aliquots into empty Smart Cycler tubes that were placed in a 4°C cooling block (pg 1452, left col., 2nd para) and some of the aliquoted Smart Cycler tubes were frozen at -70°C for long-term storage.
Regarding claim 2, Wenninger et al. teach that prior to storage, 5 µl aliquots of kit-provided nucleic acids were added to selected master mix tubes (positive control).
Regarding claims 2 and 9-10, Wenninger et al. teach nucleic acid extracts were generated from primary nasal specimens collected in M5 MicroTest medium and upon thawing of frozen master mix, delivery of extracted nucleic acids to the ProFlu+ assay was executed in accordance with the manufacturer’s specifications (1) (pg 1452, 3rd para of left col).
Regarding claims 2 and 9-10, Wenninger et al. teach for temporal-potency studies involving 40 archived extracts tested in tandem with fresh and frozen master mix yielded the expected RT-PCR amplification of influenza A virus, influenza B virus, and respiratory syncytial virus (RSV), as well as internal control nucleic acid within extracts negative for those analytes, and no significant difference between the CT values generated was noted in Table 1(pg 1452, 3rd para of left col).
Regarding claims 2 and 9-10, Wenninger et al. also teach no significant differences between CT values for tandem analysis with fresh and frozen (for 1 to 2 weeks) master mix (pg 1452, 4th para of left col).
Wenninger et al. teach benefits of using a frozen PCR master mix to include decreased technologist error, reduction of contamination risk, minimization of reagent freeze-thaw cycles, better interassay reproducibility, and an overall efficiency gain (pg 1453, left col., 1st para).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the invention to provide individual component(s) or individual compositions that are suitable for making a mixture within different containers, prior to combining the components together to generate a mixture in a manner as taught by Wenninger et al.
The ordinary skilled artisan would have been further motivated to generate some refrigerated aliquots comprising the master mix and a positive control oligonucleotide and some refrigerated aliquots consisting only the master mix in a manner as taught by Wenninger et al. for the benefit of using the positive control as an internal metric to observe and compare both PCR assays when the internal control is present and absent.
In view of the combined teachings and suggestions of all of the cited prior art references, the instant claim 2 is prima facie obvious.
Claim 3 is rejected under 35 U.S.C. 103 as being unpatentable over Kofler et al. (1999, Diagnostic microbiology and infectious disease, 34(1), pp.33-35) in view of Smetsers et al. (1998, Leukemia, 12(8), pp.1324-1325).
The teachings of Kofler et al. as it relates to claim 1 are provided in the rejection under 35 U.S.C. 103 above and applies here.
Omitted from Kofler et al. (claim 3)
Regarding claim 3, Kofler et al. do not teach preparation of a frozen reaction liquid/PCR master mix that comprises an oligonucleotide probe, or any composition that comprises of an oligonucleotide probe.
Smetsers et al. (claim 3)
Regarding claim 3, Smetsers et al. teach preparation of a frozen reaction liquid/PCR master mix (refrigerated at -80 ºC: pg 1324, text of right col., 2nd para) that comprises a fluorescent Taqman oligonucleotide probe (PBGD-TET, or GAPDH-JOE or 821-TET respectively, for detecting target sequence of porphobilinogen deaminase (PBGD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and AML-1/ETO) (pg 1324, text of 1st para of right col.).
Smetsers et al. teach preparation of three reaction master mixtures (pg 1324, 2nd para of right col.). The PCR master mix consists of dNTPs, Ampli Taq polymerase, forward and reverse primer, 10 mm Tris-HCl (pH 8.3), 50 mm KCl, 10 mm EDTA and 60 nm passive reference 1 (ROX Dye) (pg 1324, text of left col).
Smetsers et al. teach qPCR are performed with thawed PCR master mix aliquots (thawing at different timepoints up 60 days of freezing) subjected to same PCR reaction conditions (pg 1324, 2nd para of right col. And pg 1325, Fig. 2).
Smetsers et al. teach qPCR thermocycling over 55 cycles (pg 1324, text of left col.).
Smetsers et al. teach very minor difference in the CT values of triplicates after 2 months of freezing (Figure 1 and pg 1325, 2nd para of left col) and fluorescence detected at the end of the reaction, after 55 cycles and showed the same amount of product formed (pg 1325, left col., 2nd para).
Smetsers et al. teach advantages of the freezing of the master mixture are reproducibility, speed and reduced contamination. Within a single batch of frozen master mixture, the reactivity is the same and this opens the possibility of standardization between different reactions and between different laboratories. Freezing of master mixture speeds up the time per single analysis, because the tedious preparation of master mixtures before each PCR reaction can be omitted. Since the stock solutions of individual components are not used every time, the risk of contamination can be reduced. Once individual tubes are frozen without contamination, they can be used without danger of contamination of all the components (pg 1325, last para of right col).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the invention to apply the teachings of Smetsers et al. of providing an oligonucleotide probe within a PCR amplification assay and thus modify the amplification method taught by Kofler et al. to include one or more oligonucleotide probe(s) that enable the detection of target sequence of interest by probe hybridization.
Smetsers et al. teach it a matter of routine practice to use oligonucleotide probe during real-time monitoring of amplicon production. Accordingly, the ordinarily skilled artisan is readily apprised of how to derive suitable oligonucleotide detection probes comprising of detectable labels to generate signals useful for detecting amplicons.
In view of the combined teachings and suggestions of all of the cited prior art references, the instant claim 3 is prima facie obvious.
Claims 3 and 5-6 are rejected under 35 U.S.C. 103 as being unpatentable over Kofler et al. (1999, Diagnostic microbiology and infectious disease, 34(1), pp.33-35) in view of Ihara et al. (cited below is English Translation of CN112941161, pp. 1-14, filed Dec 10, 2020; pub. June 11, 2021).
The teachings of Kofler et al. as it relates to claim 1 are provided in the rejection under 35 U.S.C. 103 above and applies here.
Omitted from Kofler et al. (claims 3 and 5-6)
Regarding claim 3, Kofler et al. do not teach preparation of a frozen reaction liquid/PCR master mix that comprises an oligonucleotide probe, or any composition that comprises of an oligonucleotide probe.
Regarding claim 5, Kofler et al. do not teach nucleic acid to be amplified by the PCR reagent is RNF213 gene p. R4810K polymorphism.
Regarding claim 6, Kofler et al. do not teach a second composition comprising:
a primer pair for amplifying a base sequence containing a genetic mutation corresponding to the RNF213 gene p. R4810K polymorphism,
an oligonucleotide fluorescently labeled probe that binds to a mutant-type base sequence containing a genetic mutation corresponding to the polymorphism, and
an oligonucleotide fluorescently labeled probe that binds to a wild-type base sequence corresponding to the mutant-type base sequence.
Ihara et al. English Translation of CN112941161A, pp. 1-14 (claims 3 and 5-6)
Ihara et al. is directed to a method of identifying the R4810K polymorphism of the RNF213 gene of a subject so as to evaluate risk of cerebral infarction (entire document; see abstract on pg 1 and text of sections entitled Brief Description of the Drawings and Specific implementation examples on pg 5 and pg 9, text of section entitled Embodiment 1 and 1st para of pg 10 and Fig. 1A and pg 10, text of section entitled Results of Embodiment 1).
Ihara et al. teach the R4810K polymorphism of the RNF213 gene is screened in human blood sample(s) and/or human saliva sample(s) of subject(s) (see pg 10, text of sections entitled Embodiment 2, Method, Results and Embodiment 3).
Ihara et al. teach their method as comprising the steps of:
(1) mixing a sample collected from the subject with a PCR buffer comprising a surfactant and a
protease K (pg 5, Specific implementation examples), thereby generate a mixed solution.
(2) adding a DNA polymerase to the mixed solution of step (1) and carrying out an amplification using the combination of mixed solution and DNA polymerase, wherein the amplification comprises a PCR primer pair for amplifying a base sequence of the RNF213 gene containing a genetic mutation corresponding to the RNF213 gene p. R4810K polymorphism and fluorescently oligonucleotide probes for the wild type and the mutant base sequences, wherein the fluorescent dyes of the two oligonucleotide probes are different from each other; and (3) a step of detecting the PCR product (see pg 5, text of section entitled Specific implementation examples).
Regarding claim 5, Ihara et al. teach a nucleic acid to be amplified by the PCR reagent is RNF213 gene p. R4810K polymorphism (pg 5, 1st and 2nd para of section entitled Specific implementation examples and pg 6, 2nd para).
Regarding claim 3, Ihara et al. teach a composition comprising an oligonucleotide probe (pg 7, wherein Ohara et al. teach fluorescently labeled probe consisting SEQ ID NO: 3 and fluorescently labeled probe consisting SEQ ID NO: 4; see pg 10, text of sections entitled Embodiment 2, Method, Results and Embodiment 3).
Regarding claim 6, Ihara et al. teach a composition comprising:
a primer pair for amplifying a base sequence containing a genetic mutation corresponding to the RNF213 gene p. R4810K polymorphism,
an oligonucleotide fluorescently labeled probe that binds to a mutant-type base sequence containing a genetic mutation corresponding to the polymorphism, and
an oligonucleotide fluorescently labeled probe that binds to a wild-type base sequence corresponding to the mutant-type base sequence (see pg 10, text of sections entitled Embodiment 2, Method, Results and Embodiment 3).
Ihara et al. teach the pretreatment liquid is a blood lysate containing an anionic surfactant (see pg 10, text of sections entitled Embodiment 2, Method).
Ihara et al. teach PCR test method provides 50 PCR cycles.
It would have been obvious to a person of ordinary skill in the art, wanting to evaluate risk of cerebral infarction in one or more individuals using an amplification assay with enhanced reproducibility, speed and with reduced contamination, before the effective filing date of the invention would have been motivated to apply the teachings of Ihara et al. and Kofler et al. together in a single assay.
The ordinary skilled artisan would have readily generated a plurality of frozen PCR master mix aliquots comprising buffer, polymerase, a primer pair for amplifying the wildtype or mutant nucleotide sequence encompassing the 4810 position of the RNF213 gene so that the R4810K polymorphism may be detected and comprising a fluorescently labeled oligonucleotide probe for detecting the wild type base sequence of the RNF213 gene and a fluorescently labeled oligonucleotide probe for detecting the mutant base sequence of the RNF213 gene so as to have starting identical PCR test samples that promote PCR testing for triplicate testing and/or for multiplex PCR screening of the wild type or mutant sequence under same thermocycling conditions.
The ordinary skilled artisan would have had a reasonable expectation of success at the combination of teachings of Ihara et al. and Kofler et al. so as to arrive at a qPCR assay that detects the RNF213 gene p. R4810K polymorphism with enhanced reproducibility as it was already a matter of routine practice in the art before the effective filing date of the invention to generate and freeze a PCR master mix to which after thawing, either an omitted polymerase, or an omitted DNA extract solution is added before subjecting the thawed mixture to PCR thermocycling.
In view of the combined teachings and suggestions of all of the cited prior art references, the instant claims 3 and 5-6 are prima facie obvious.
Conclusion
No claims are currently allowed.
Correspondence
Any inquiry concerning this communication or earlier communications from the examiner should be directed to OLAYINKA A OYEYEMI whose telephone number is (571)270-5956. The examiner can normally be reached Monday -Thursday: 9:00 am - 5:00 pm, EST.
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OLAYINKA A. OYEYEMI
Examiner
Art Unit 1681
/OLAYINKA A OYEYEMI/Examiner, Art Unit 1681
/GARY BENZION/Supervisory Patent Examiner, Art Unit 1681
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