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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on May 19, 2025 has been entered.
Status of Claims / Response to Amendment
This office action is in response to an amendment filed on May 19, 2025.
Claims 1-2, 6-25 were previously pending. Applicant amended claims 1, 6 and 24; claims 26-27 are newly added.
Claims 1-2 and 6-27 are currently pending, with claims 1, 7-19 and 21-22 withdrawn.
Claims 2, 6, 20 and 23-27 are under consideration.
All of the previously presented objections and rejections have been withdrawn as being obviated by the amendment of the claims.
Applicant' s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
This office action contains new grounds for rejection necessitated by amendment.
Priority
The effective filling date of the instant claims 2, 6, 20 and 23-27 is 11/01/2019, the filling date of the PCT application PCT/JP2019/043040.
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Specifically, Applicant's claim to foreign priority to JAPAN Application 2018-206938 is acknowledged. However, the submitted certified priority document is Not in English, and English translations have not been submitted.
The foreign priority date may be the effective filing date of the claimed invention if:
• the foreign application supports the claimed invention under 35 U.S.C. 112(a), AND
• the applicant has perfected the right of priority by providing a certified copy of the priority application, and a translation of the certified copy (if not in English) along with a statement that the translation of the certified copy is accurate.
See MPEP 213.04 and 216
In this instant case, the priority document submitted is not in English, without a translation; without a English translation, the examiner is unable to verify whether the earlier applications provide written description support for the claimed invention under 35 U.S.C. 112(a). Thus, since an English translation of the priority application has not been filed for the foreign priority document ( JAPAN Application 2018-206938), the effective filing date (EFD) of the claimed invention is the filing date of the PCT application (the specification of the present application is the English translation of PCT/JP2019/043040).
However, if applicant perfects the right of priority by providing an English translation of the priority application that supports the claimed invention under 35 U.S.C. 112(a), the effective filing date will be the filing date of the foreign application.
Claim Objections
Claim 2 is objected to because of the following informalities:
In claim 2, lines 11-13, it recites: "wherein an elongation reaction in the reverse transcription is carried out at 42°C ±0.25°C for 80 to 100 minutes wherein the skin surface lipid absorbing material does not adhere to the skin," which lacks proper punctuation or a conjunction between the two "wherein" clauses.
Appropriate correction is required.
Claim Interpretation
In evaluating the patentability of the claims presented in this application, claim terms have been given their broadest reasonable interpretation (BRI) consistent with the specification, as understood by one of ordinary skill in the art, as outlined in MPEP§ 2111.
For the purpose of applying prior art, claim 2 recites "skin surface lipid," which is defined by the specification as follows:
“[0014] In the present description, the "skin surface lipid (SSL)" refers to a lipid-soluble fraction present on a skin surface, and is sometimes referred to as sebum. In general, SSL mainly contains secretions secreted from an exocrine gland such as a sebaceous gland on the skin, and is present on the skin surface in the form of a thin layer covering the skin surface.”
For the purpose of applying prior art, claim 24 recites "oil-blotting film," which is not defined in the specification. Therefore, under BRI this term is interpreted to encompass any film material.
Claim Rejections - 35 USC § 112(d) -- New
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 26 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 26 recites "wherein the skin surface lipid absorbing material is not a skin surface lipid adhesive material." However, claim 26 depends from claim 2, which already recites "wherein the skin surface lipid absorbing material does not adhere to the skin."
Claim 26 does not provide any further limitation beyond the non-adherence feature for the skin surface lipid absorbing material, already required in the base claim.
Therefore, claim 26 fails to further limit the subject matter of claim 2, from which it depends.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103 -- New
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.
The following are new grounds of rejections necessitated by Applicant's amendments. Applicant's amendments have necessitated the inclusion of new grounds of rejections in this Office action. It is noted that, to the extent that they apply to the present rejection; Applicant's arguments are addressed following the rejection.
Claims 2, 20 and 23-27 are rejected under 35 U.S.C. 103 as being unpatentable over Inoue (EP3372693A1 - Method for preparing nucleic acid sample; Published 2018-09-12, effective filling date 2017-06-07), in view of
Bagnoli (Bagnoli et al. ; Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq. Nat Commun. 2018 Jul 26;9(1):2937. doi: 10.1038/s41467-018-05347-6. PMID: 30050112; PMCID: PMC6062574.),
GenScript (Four Tips for Optimizing Your PCR Amplification; April 2018; www.genscript.com/synthetic-biology-news/four-tips-for-optimizing-your-pcr-amplification.html),
Genaxxon (Optimization of Annealing Temperature and other PCR Parameters; www.genaxxon.com/shop/en/blog/optimization-of-annealing-temperature-and-other-pcr-parameters; published June 19, 2017),
as evidenced by Ion AmpliSeq (Ion AmpliSeq ™ Transcriptome Human Gene Expression Kit User Guide, published 2014).
A) Inoue teaches a method for preparing nucleic acid derived from skin surface for RNA extraction followed by sequencing analysis (entire document).
Regarding claim 2, Inoue teaches a method comprising:
extracting RNA from a skin surface lipid absorbing material that has absorbed skin surface lipids of the subject ([0037]; [0048]),
converting the RNA into cDNA by reverse transcription ([0043]; [0045]; [0048]), and then subjecting the cDNA to a multiplex PCR ([0045] 2-plex PCR for GAPDH and SOD2 is multiplex PCR); and purifying a reaction product of the multiplex PCR ([0048]lines 5-6, “a library was constructed from the collected RNA using Ion AmpliSeqTranscriptome Human Gene Expression Kit (Life Technologies Japan Ltd.”),
wherein the skin surface lipid absorbing material does not adhere to the skin ([0037]; [0048] oil-blotting film does not adhere to the skin).
Inoue teaches purifying a reaction product of PCR by teaching using Ion AmpliSeq Transcriptome Human Gene Expression Kit ([0048]lines5-6), which teaches protocol steps of purifying PCR reaction products, as evidenced by the User Guide disclosed in Ion AmpliSeq (page 18, “purify amplified library”).
Inoue, evidenced by Ion AmpliSeq teaches performing reverse transcription reaction at 42°C for 30 minutes (Ion AmpliSeq, page 11), whereas the claim recites a reaction duration of 80-100 minutes.
Bagnoli teaches a standard reverse transcription reaction condition of 42 °C for 90 minutes for 9 commercially available, common reverse transcriptase enzymes (Page 7, left-hand col, lines12-13), thus providing objective evidence that such conditions are routine and standard for reverse transcription reactions.
Therefore, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to apply the reverse transcription reaction condition of 42 °C for 90 minutes, as taught by Bagnoli, in the method of Inoue, which is a predictable use of prior art knowledge according to a known method to yield predictable results (See MPEP §2143).
The ordinary artisan, motived to optimize experimentation, would have found this extension of reaction time obvious, in view of the standard reverse transcription reaction condition applicable to 9 commercially available, common reverse transcriptase enzymes as taught by Bagnoli.
Additionally, as noted in In re Aller, 105 USPQ 233 at 235, 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. Therefore, adjusting reverse transcription reaction duration to achieve optimal reaction efficiency would have been an obvious, routine optimization process for a skilled artisan, see MPEP 2143.
Inoue, as evidenced by Ion AmpliSeq also teaches performing multiplex PCR reaction with an annealing and elongation temperature of 60°C (Ion AmpliSeq, page 12) but does not explicitly disclose the 62°C temperature as recited in the claim.
Adjusting the PCR reaction temperature for annealing and elongation from 60°C to 62°C would have been prima facie obvious to a person of ordinary skill in the art, because the prior art teaches this specific modification and provides motivations to do so.
GenScript teaches increasing annealing temperature by 1-2 °C in PCR for optimization (page 2), in its teaching of tips for optimizing PCR amplification, thus providing objective evidence that the skilled artisan in view of Inoue as evidenced by Ion AmpliSeq and further in view of Bagnoli and GenScript, would have found it obvious to increase the PCR reaction temperature for annealing and elongation from 60°C to 62°C.
The skilled artisan in the field of PCR assays would have been motivated to optimize annealing and extension temperatures in PCR reactions using any given kit, in order to generate the desired product with consistent efficiency, with the benefit of cost and time savings as disclosed in Genaxxon (page 1):
"Suppliers endeavor to provide kits that can be adapted to existing systems. However, it is never possible to achieve 100% compatibility. The use of different buffer systems and buffer compositions (salts in the reaction buffer) which affect the net pH value, collectively influence the respective annealing temperatures in a given PCR reaction. It is our experience that end-users underestimate the influence of the annealing temperature on the fidelity of PCR, real-time PCR and RT-PCR, and very often this is the last variable which is changed in the PCR protocol. It is incorrect to assume that a predicted Tm (melting temperature of primers) that is provided with each oligonucleotide/primer/probe, will remain constant, regardless of the kit used.
For these reasons, it is unrealistic to expect that a long-used PCR protocol will generate the desired product with the same efficiency when used with reagents from an alternative source. Therefore, it is critical that the protocol is systematically tested and optimized with the chosen kit.
It is possible to save time and money with careful planning. Firstly, any new kit, regardless of cost or supplier should be tested using gradient PCR. This will identify the optimum annealing temperature in a given set of conditions to be identified, and it will reveal the parameters that will minimize the incidence of spurious artefacts. Usually the difference in the annealing temperature between different systems is only 2-3°C, but the effect is enormous and may lead to a completely negative result. Unexpected negative results may reflect a flawed experimental design and/or sub-optimum reagent concentrations. "
Furthermore, as set forth at MPEP 2144.05 II. A: “Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical.”
One having ordinary skill in the art would have had a reasonable expectation of success because determining the optimum and/or workable conditions is well within the skill level of the ordinary artisan.
Therefore, claim 2 is obvious and is properly rejected under 35 U.S.C. 103.
B) Regarding claim 20, Inoue teaches analyzing a nucleic acid prepared by the method according to claim 2 ([0048]).
Regarding claim 23, Inoue teaches the skin surface lipid absorbing material is a sheet-shaped material ([0048] line 2, “oil-blotting film”).
Regarding claim 24, Inoue teaches the sheet-shaped material is an oil-blotting paper or an oil-blotting film([0048] line 2, “oil-blotting film”).
Regarding claim 25, Inoue teaches obtaining the skin surface lipid by using the skin surface lipid absorbing material ([0048]lines 1-2).
Regarding claim 26, it is anticipated by Inoue because it does not further limit the claimed method. See detailed discussion in section above (claim rejection under 35 U.S.C. 112(d)).
Regarding claim 27, Inoue teaches RNA is extracted from the facial skin of a person having atopic dermatitis ([0019] skin from which SSL are collected include subject having atopy disease).
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Inoue, in view of Bagnoli with GenScript and Genaxxon, as applied to claim 2 above and further in view of Sundquist (Terri Sundquist; How to Isolate RNA like a Pro - Promega Connections; www.promegaconnections.com/how-to-isolate-rna-like-a-pro/; Published March, 2014).
The teachings of Inoue, Bagnoli, GenScript and Genaxxon are recited above and applied as for base claim 2.
Regarding claim 6, while the combined teachings of Inoue, Bagnoli, GenScript and Genaxxon does not explicitly teach preserving the skin surface lipid sample at 0 °C or lower, Inoue teaches extracting and analyzing RNA in skin surface lipid sample, including bacteria RNA (0040-0042] for examples).
Sundquist teaches tips in sample handling for RNA isolation (entire document).
Regarding claim 6, Sundquist specifically teaches preserving RNA-comprising samples at –70°C. (page 2, para 3, lines 1-7) Sundquist provides several benefits for doing so, such as protecting RNA and minimizes any unintentional changes in gene expression, and further emphasizes that "[t]his is particularly important for bacteria because bacterial RNA has a short half-life."(page 2, para 3, lines 5-7)
Therefore, it would have been prima facie obvious for a skilled artisan to apply a storage temperature at –70°C, as disclosed in Sundquist for skin surface lipid sample in the method comprising extracting and analyzing RNA in skin surface lipid sample, including bacteria RNA, taught by the combined teachings of Inoue, Bagnoli, GenScript and Genaxxon. All references are in the same field of molecular biology assays. Specifically Inoue's method teaches isolating and analyzing bacteria RNA from skin surface lipid sample, and Sundquist presents complementary teaching including preserving sample at –70°C, in order to protect RNA.
Sundquist's sample preservation approach is particularly beneficial for bacterial RNA and is directly applicable to the method described in Inoue.
This combination would have been obvious as it represents the KSR principle of predictable use of prior art elements (i.e., sample cold storage approach to protect RNA, taught in Sundquist) according to a known method (i.e., method for extracting and analyzing RNA in skin surface lipid sample, taught by Inoue in view of Bagnoli, GenScript and Genaxxon) to yield predictable results. (See MPEP §2143).
Response to Arguments: Unexpected results
Applicant argues that the claimed method with the specific combination of reaction conditions, is nonobvious because it yields unexpected results (Remarks, page 8-10).
Specifically, Applicant argues two points:
Point 1 . Applicant asserts that the claimed condition "elongation reaction in the reverse transcription is carried out at 42°C ±0.25°C for 80 to 100 minutes," leads to unexpected results. This argument relies on Table 1 of the specification, which compares the increase in PCR product concentration between time points of 60 minutes and 90 minutes at reverse transcription temperature of 40°C or 42°C. Applicant appears to argue that the greater increase in PCR product concentration from 42°C at 60 minutes to 42°C at 90 minutes, compared to the same time extension at 40°C, is unexpected. (Remarks, page 8)
Point 2 . Applicant argues that the claimed condition for performing multiplex PCR , "wherein a temperature for an annealing and an elongation reaction in the multiplex PCR is 62°C ±0.25C," leads to unexpected results. To support this, Applicant refers to Table 2, which shows that at annealing and elongation temperatures of 60°C, 63°C, or 64°C, the PCR product is undetectable in the quantification assay, while at 62°C, the PCR product is detectable. (Remarks, page 9-10)
These arguments have been fully considered but are not found persuasive for several reasons.
First, the arguments are not commensurate in scope with the claim.
Whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support. See MPEP 716.02 (d)
For Point 1, the claim broadly recites an incubation time range of 80 to 100 minutes, but the evidence relied upon compares only the time points of 60 minutes and 90 minutes, not the full claimed range. The showing of unexpected results must be reviewed to see if the results occur over the entire claimed range. See In re Grasselli, 713 F.2d 731, 741, 218 USPQ 769, 777 (Fed. Cir. 1983) (Claims were directed to certain catalysts containing an alkali metal. Evidence presented to rebut an obviousness rejection compared catalysts containing sodium with the prior art. The court held this evidence insufficient to rebut the prima facie case because experiments limited to sodium were not commensurate in scope with the claims.).
For Point 2, the data in Table 2 is based on an experiment using a specific commercial kit ꟷ “Ion AmpliSeq Transcriptome Human Gene Expression Kit” (see page 47, [0059] lines 5-8; page 44, lines 10-14), which includes specific reagents such as primers, buffers, and enzymes1. These specific components are potential factors influencing the optimal annealing and elongation temperature and the corresponding PCR outcome, together or individually 2. The claim, however, broadly recites any multiplex PCR. Thus, Point 2 is not commensurate in scope with the claim as the results obtained from using one specific kit cannot be generalized to all multiplex PCR methods.
Second, Applicant’s argument and disclosure have not sufficiently demonstrated that the observed results are truly unexpected or unobvious.
The evidence relied upon should establish “that the differences in results are in fact unexpected and unobvious and of both statistical and practical significance.” See MPEP 716.02 (b)
The question of unexpected result is whether the claimed invention possesses unexpected properties compare to the closest prior art. Where the unexpected properties of a claimed invention are not shown to have a significance equal to or greater than the expected properties, the evidence of unexpected properties may not be sufficient to rebut the evidence of obviousness. See MPEP 716.02 (c)
For both Table 1 and Table 2, the data quality and the robustness of experiment design are insufficient in supporting the assertion of unexpected result. Each reaction condition is illustrated with only a single data point, which is inadequate to establish statistical significance. The experiments ([0057- 0060]) in the disclosure lack technical replicates and fail to account for other variables (e.g., RNA input quality and quantity, variability in performing multiplex PCR, purification, and quantification) that could impact the amount of PCR product recovered. Without proper controls and replicates to keep the variables in check, the differences in PCR product concentration observed in the Tables 1 and 2 cannot be reliably attributed to the asserted specific reaction conditions. Thus, not only does the data in the referenced tables lack statistical significance, but a nexus has also not been established between the reaction conditions cited in the argument and the increased PCR concentration asserted by Applicant.
Furthermore, even overlooking the above concerns, Applicant has not established that the observations in Table 1 and Table 2 are unexpected to an unobvious extent.
For Table 1, the reverse transcription was performed using "Superscript (registered trademark) VILO cDNA Synthesis kit" (page 46), which, according to its manual, recommends a reaction temperature of 42°C 3. Therefore, a skilled artisan would reasonably expect the reverse transcriptase enzyme in this specific kit to exhibit optimal activity at 42°C, leading to a higher increase in cDNA yield during prolonged reaction times. The observed results are consistent with this expectation.
For Table 2, the detection of PCR product at an annealing and elongation temperature of 62°C does not appear unexpected in view of application's disclosure 4. In Example 2, which provides the results shown in Table 1, all PCR products were generated via multiplex PCR under the same conditions as Test Example 1 (page 46, lines 19-21), which used an annealing and elongation temperature of 60°C (page 44, lines 10-15). Thus, a skilled artisan, in view of Table 1, would reasonably expect detectable PCR product to be generated at a slightly raised annealing and elongation temperature of 62°C. This is further supported by GenScript 5, which teaches increasing annealing temperature by 1-2 °C is a known optimization strategy in PCR.
For the reasons stated above, Applicant's assertion regarding unexpected results is not persuasive.
Double Patenting- Obvious Type
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.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 2 and 23-26 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over claims 1 and 5 of U.S. Patent No. 11913061B2 in view of Ion AmpliSeq (Ion AmpliSeq ™ Transcriptome Human Gene Expression Kit User Guide, published 2014),
Bagnoli (Bagnoli et al. ; Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq. Nat Commun. 2018 Jul 26;9(1):2937. doi: 10.1038/s41467-018-05347-6. PMID: 30050112; PMCID: PMC6062574.) and
GenScript (Four Tips for Optimizing Your PCR Amplification; April 2018; www.genscript.com/synthetic-biology-news/four-tips-for-optimizing-your-pcr amplification.html).
A) The ‘061 Patent claims collecting a skin surface lipids sample using a skin surface lipid absorbing material, extract RNA from the collected skin surface lipids sample, and determining a gene expression level of one or more genes, wherein the skin surface lipid absorbing material does not adhere to the skin (claims 1 and 5).
While the ‘061 Patent claims determining a gene expression level of one or more genes , it does not explicitly claim performing reverse transcription and multiplex PCR with any specific reaction conditions.
Ion AmpliSeq fills this gap by teaching determining RNA gene expression levels of one or more genes, via reverse transcription (page 11) followed by multiplex PCR (page 12), and purifying a reaction product of the multiplex PCR (page 18, “purify amplified library”).
Ion AmpliSeq teaches performing reverse transcription reaction at 42°C for 30 minutes (Ion AmpliSeq, page 11), whereas the instant claim 2 recites a reaction duration of 80-100 minutes.
Ion AmpliSeq also teaches performing multiplex PCR reaction with an annealing and elongation temperature of 60°C (Ion AmpliSeq, page 12) but does not explicitly disclose the 62°C temperature as recited in the claim.
Bagnoli teaches a standard reverse transcription reaction condition of 42 °C for 90 minutes for 9 commercially available, common reverse transcriptase enzymes (Page 7, left-hand col, lines12-13), thus providing objective evidence that such conditions are routine and standard for reverse transcription reactions.
GenScript teaches increasing annealing temperature by 1-2 °C in PCR for optimization (page 2), in its teaching of tips for optimizing PCR amplification.
Therefore, it would have been prima facie obvious to apply Ion AmpliSeq's reverse transcription and multiplex PCR to the method claimed by ‘061 Patent, using the reverse transcription reaction condition of 42 °C for 90 minutes, as taught by Bagnoli; and increasing the PCR reaction temperature for annealing and elongation from 60°C to 62°C, as suggested by GenScript, representing a predictable use of prior art knowledge according to a known method to yield predictable results (See MPEP §2143).
Therefore, instant claims 2 and 23-26 are obvious over claims 1 and 5 of the ‘061 Patent , in view of Ion AmpliSeq, Bagnoli and GenScript.
Claims 2 and 23-26 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 5 and 9 of copending Application No. 18/416,592 (reference application, amended claims filed on 08/07/2025), in view of
Ion AmpliSeq (Ion AmpliSeq ™ Transcriptome Human Gene Expression Kit User Guide, published 2014),
Bagnoli (Bagnoli et al. ; Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq. Nat Commun. 2018 Jul 26;9(1):2937. doi: 10.1038/s41467-018-05347-6. PMID: 30050112; PMCID: PMC6062574.) and
GenScript (Four Tips for Optimizing Your PCR Amplification; April 2018; www.genscript.com/synthetic-biology-news/four-tips-for-optimizing-your-pcr-amplification.html).
A) The ‘592 Application claims collecting a skin surface lipids sample using a skin surface lipid absorbing material, extract RNA from the collected skin surface lipids sample, and determining a gene expression level of one or more genes, wherein the skin surface lipid absorbing material does not adhere to the skin (claims 1 and 5).
While the ‘592 Application claims determining a gene expression level of one or more genes , it does not explicitly claim performing reverse transcription and multiplex PCR with any specific reaction conditions.
Ion AmpliSeq fills this gap by teaching determining RNA gene expression levels of one or more genes, via reverse transcription (page 11) followed by multiplex PCR (page 12), and purifying a reaction product of the multiplex PCR (page 18, “purify amplified library”).
Ion AmpliSeq teaches performing reverse transcription reaction at 42°C for 30 minutes (Ion AmpliSeq, page 11), whereas the instant claim 2 recites a reaction duration of 80-100 minutes.
Ion AmpliSeq also teaches performing multiplex PCR reaction with an annealing and elongation temperature of 60°C (Ion AmpliSeq, page 12) but does not explicitly disclose the 62°C temperature as recited in the claim.
Bagnoli teaches a standard reverse transcription reaction condition of 42 °C for 90 minutes for 9 commercially available, common reverse transcriptase enzymes (Page 7, left-hand col, lines12-13), thus providing objective evidence that such conditions are routine and standard for reverse transcription reactions.
GenScript teaches increasing annealing temperature by 1-2 °C in PCR for optimization (page 2), in its teaching of tips for optimizing PCR amplification.
Therefore, it would have been prima facie obvious to apply Ion AmpliSeq's reverse transcription and multiplex PCR to the method claimed by ‘592 Application, using the reverse transcription reaction condition of 42 °C for 90 minutes, as taught by Bagnoli; and increasing the PCR reaction temperature for annealing and elongation from 60°C to 62°C, as suggested by GenScript, representing a predictable use of prior art knowledge according to a known method to yield predictable results (See MPEP §2143).
Therefore, instant claims 2, 25-26 are obvious over claims 1 and 5 of the ‘592 Application , in view of Ion AmpliSeq, Bagnoli and GenScript. Instant claims 23-24 are obvious over claim 9 of the ‘592 Application, in view of Ion AmpliSeq, Bagnoli and GenScript.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Conclusion
No claims are allowed. Claim 2 is objected to; claims 2, 6, 20 and 23-27 are rejected.
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/TIAN NMN YU/Examiner , Art Unit 1681 /AARON A PRIEST/Primary Examiner, Art Unit 1681
1 see Ion AmpliSeq ™ Transcriptome Human Gene Expression Kit User Guide, published 2014
2 see Genaxxon (Optimization of Annealing Temperature and other PCR Parameters; www.genaxxon.com/shop/en/blog/optimization-of-annealing-temperature-and-other-pcr-parameters; published June 19, 2017
3 see Ion AmpliSeq, page 11
4 At best, the data in Table 2 might suggest that the lack of detectable PCR product at 60°C is unexpected, especially since it was previously shown that high concentrations of PCR products can be detected at 60°C (Table 1). However, due to the lack of statistical significance, absence of technical replicates, and uncontrolled variables, there is no clear indication regarding the cause of the undetected PCR product based on this data.
5 GenScript (Four Tips for Optimizing Your PCR Amplification; April 2018; www.genscript.com/synthetic-biology-news/four-tips-for-optimizing-your-pcr-amplification.html)