DETAILED ACTION
CONTINUED EXAMINATION UNDER 37 CFR 1.114 AFTER FINAL REJECTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 of RCE filed on May 20, 2026 and the amendment filed on April 22, 2026 have been entered. The claims pending in this application are claims 7, 18-30, 32 and 33 wherein claims 7 and 18-28 have been withdrawn due to the restriction requirement mailed on January 30, 2019. The objections and rejections not reiterated from the previous office action are hereby withdrawn in view of applicant’s amendment filed on April 22, 2026. Claims 29, 30, 32, and 33 will be examined.
Claim Objections
2. Claim 29 objected to because of the following informalities: “the hybridization mixture” in step (a-2) or (a-4) or (b-2) or (b-4) should be “a hybridization mixture”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
3. The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
4. Scope of Enablement
Note that this rejection is different from the scope of enablement rejection mailed on February 26, 2026.
Claims 29, 30, 32, and 33 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for incubating a test sample in a single reaction vessel with the first upstream oligonucleotide and the second upstream oligonucleotide, the first PTO and the second PTO, the first CTO and the second CTO, and the template-dependent nucleic acid polymerase by real-time PCR under conditions suitable to perform the hybridizing, contacting, and extending steps, does not reasonably provide enablement for detecting a first target nucleic acid sequence and a second target nucleic acid sequence in a test sample using different detection temperatures and reference values as recited in claims 29, 30, 32, and 33. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
Factors to be considered in determining whether a disclosure meets the enablement requirement of 35 USC 112, first paragraph, have been described by the court in In re Wands, 8 USPQ2d 1400 (CA FC 1988). Wands states at page 1404,
“Factors to be considered in determining whether a disclosure would require undue experimentation have been summarized by the board in Ex parte Forman. They include (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims.”
The Nature of The Invention
The claims are drawn to a method for detecting a first target nucleic acid sequence and a second target nucleic acid sequence in a sample using different detection temperatures and reference values. The invention is a class of invention which the CAFC has characterized as “the unpredictable arts such as chemistry and biology.” Mycogen Plant Sci., Inc. v. Monsanto Co., 243 F.3d 1316, 1330 (Fed. Cir. 2001).
The Breadth of The Claims
Claims 29, 30, 32, and 33 encompass a method for detecting a first target nucleic acid sequence and a second target nucleic acid sequence in a sample using different detection temperatures and reference values, comprising: (a) performing a real-time PCR on a reference sample containing the first target nucleic acid sequence to obtain reference signals, comprising: (a-1) hybridizing the first target nucleic acid sequence in the reference sample with a first upstream oligonucleotide and a first Probing and Tagging Oligonucleotide (PTO), wherein the first upstream oligonucleotide comprises a hybridizing nucleotide sequence complementary to the first target nucleic acid sequence; wherein the first PTO comprises (i) a first 3’ -targeting portion complementary to the first target nucleic acid sequence and (ii) a first 5’-tagging portion that is non-complementary to the first target nucleic acid sequence; and wherein the first upstream oligonucleotide is located upstream of the first PTO; (a-2) contacting the hybridization mixture of step (a-1) with a template-dependent nucleic acid polymerase having 5’ nuclease activity under conditions that induce cleavage of the first PTO, wherein each of the first and the second upstream oligonucleotide or its extended strand causes cleavage of the first PTO, thereby releasing a first fragment comprising the first 5’-tagging portion or a part thereof; (a-3) hybridizing the first and the second fragments fragment of step (a-2) with a first CTO (Capturing and Templating Oligonucleotide, (CTO), wherein the first CTO is labeled with a fluorescent label and a quencher; wherein the first CTO comprises, in a 3’ to 5’ direction, (i) a first capturing portion complementary to the first 5’-tagging portion or a part thereof and (ii) a first templating portion that is non-complementary to both the first 5’-tagging portion and the first 3' -targeting portion of the first PTO such that the fluorescent label is quenched unless and until the first fragment hybridizes to the first capturing portion and an extension occurs; and (a-4) performing an extension reaction on the hybridization mixture with a template-dependent nucleic acid polymerase having 5’ nuclease activity, such that the first and the second fragment hybridized to the first CTO is and the second CTO, extended to form extended strands, thereby generating a first reference signal; (a-5) detecting the first reference signal at a first detection temperature of 60°C, thereby obtaining a 60°C-first reference signal and detecting the first reference signal at a second detection temperature of 72°C, thereby obtaining a 72°C-first reference signal; (a-6) obtaining a first reference value for the first target nucleic acid sequence, the first reference value being a ratio of the 60°C-first reference signal to the 72°C-first reference signal, wherein the first reference value is obtained from fluorescence measurements made in a single fluorescence detection channel and are is derived solely from signals generated upon formation of the extended strands; (b) performing a real-time PCR on a reference sample containing the second target nucleic acid sequence to obtain reference signals, comprising: (b-1) hybridizing the second target nucleic acid sequence in the reference sample with a second upstream oligonucleotide and a second Probing and Tagging Oligonucleotide (PTO), wherein the second upstream oligonucleotide comprises a hybridizing nucleotide sequence complementary to the second target nucleic acid sequence;wherein the second PTO comprises (i) a second 3’-targeting portion complementary to the second target nucleic acid sequence and (ii) a second 5’- tagging portion that is non-complementary to the second target nucleic acid sequence; and wherein the second upstream oligonucleotide is located upstream of the second PTO; (b-2) contacting the hybridization mixture of step (b-1) with a template-dependent nucleic acid polymerase having 5’ nuclease activity under conditions that induce cleavage of the second PTO, wherein the second upstream oligonucleotide or its extended strand causes cleavage of the second PTO, thereby releasing a second fragment comprising the second 5’-tagging portion or a part thereof; (b-3) hybridizing the second fragment of step (b-2) with a second Capturing and Templating Oligonucleotide (CTO), wherein the second CTO is labeled with a fluorescent label and a quencher; wherein the second CTO comprises, in a 3’ to 5’direction, (i) a second capturing portion complementary to the second 5’-tagging portion or a part thereof and (ii) a second templating portion that is non-complementary to both the second 5’-tagging portion and the second 3'-targeting portion of the second PTO such that the fluorescent label is quenched unless and until the second fragment hybridizes to the second capturing portion and an extension occurs; (b-4) performing an extension reaction on the hybridization mixture with a template- dependent nucleic acid polymerase having 5’ nuclease activity, such that the second fragment
hybridized to the second CTO is extended to form extended strands, thereby generating a second reference signal; (b-5) detecting the second reference signal at a first detection temperature of 60°C, thereby obtaining a 60°C-second reference signal and detecting the second reference signal at a second detection temperature of 72°C, thereby obtaining a 72°C-second reference signal; and (b-6) obtaining a second reference value for the second target nucleic acid sequence, the second reference value being a ratio of the 60°C-second reference signal to the 72°C-second reference signal, wherein the second reference value is obtained from fluorescence measurements made in a single fluorescence detection channel and is derived solely from signals generated upon formation of the extended strands, and wherein the first reference value is at least 1.1 fold larger than the second reference value or vice versa; (c) incubating the test sample in a single reaction vessel with the first and second upstream oligonucleotides, the first and second PTOs, the first and second CTOs, and the template-dependent nucleic acid polymerase by real-time PCR under conditions suitable to perform the hybridizing, contacting, and extending steps defined in the steps (a-1) through (a-4) and steps (b-1) through (b-4), to give a test sample incubation product,(d) detecting signals from the test sample incubation product at the first detection temperature and at the second detection temperature, wherein the signals are obtained from fluorescence measurements made in a single fluorescence detection channel and are derived solely from signals generated upon formation of the extended strands, and (e) (e-1) determining the presence of the first target nucleic acid sequence by a formula (i) or (ii): (i) [(signal intensity of the test sample incubation product at the first detection temperature) - (signal intensity of the test sample incubation product at the second detection temperature) * (the second reference value)]; (ii) [(signal intensity of the test sample incubation product at the second detection temperature) - (signal intensity of the test sample incubation product at the first detection temperature) / (the second reference value)], wherein a resulting value of formula (i) greater than a first threshold determined from a negative control for distinguishing the presence and absence of the first target nucleic acid or a resulting value of formula (ii) smaller than a second threshold determined from a negative control indicates the presence of the first target nucleic acid, and
(e-2) determining the presence of the second target nucleic acid sequence by a formula (iii) or (iv): (iii) [(signal intensity of the test sample incubation product at the first detection temperature) - (signal intensity of the test sample incubation product at the second detection temperature) * (the first reference value)]; (iv) [(signal intensity of the test sample incubation product at the second detection temperature) - (signal intensity of the test sample incubation product at the first detection temperature) / (the first reference value)],wherein a resulting value of formula (iii) smaller than a third threshold determined from a negative control for distinguishing the presence and absence of the second target nucleic acid or a resulting value of formula (iv) greater than a fourth threshold determined from a negative control indicates the presence of the second target nucleic acid.
Working Examples
The specification provides working examples (see pages 64-73) for: (1) Multiple Target Detection by TaqMan real-time PCR Using Different Detection Temperatures and Reference Values; (2) Multiple target detection by PTOCE real-time PCR comprising Using Different Detection Temperatures and Reference Values; and (3) SNP Genotyping Using Different Detection Temperatures and Reference Values. However, the specification provides no working example for detecting a first target nucleic acid sequence and a second target nucleic acid sequence in a test sample using different detection temperatures and reference values as recited in claims 29, 30, 32, and 33.
The Amount of Direction or Guidance Provided and The State of The Prior Art
Although the specification provides working examples (see pages 64-73) for: (1) Multiple Target Detection by TaqMan real-time PCR Using Different Detection Temperatures and Reference Values; (2) Multiple target detection by PTOCE real-time PCR comprising Using Different Detection Temperatures and Reference Values; and (3) SNP Genotyping Using Different Detection Temperatures and Reference Values, the specification does not provide a guidance to show that a first target nucleic acid sequence and a second target nucleic acid sequence in a test sample can be detected using different detection temperatures and reference values as recited in claims 29, 30, 32, and 33. Furthermore, there is no experimental condition and/or experimental data in the specification to support the claimed invention. During the process of the prior art search, the examiner has not found any prior art which is related to detect a first target nucleic acid sequence and a second target nucleic acid sequence in a test sample using different detection temperatures and reference values as recited in claims 29, 30, 32, and 33.
Level of Skill in The Art, The Unpredictability of The Art, and The Quantity of Experimentation Necessary
While the relative skill in the art is very high (the Ph.D. degree with laboratory experience), there is no predictability whether a first target nucleic acid sequence and a second target nucleic acid sequence in a test sample can be detected using different detection temperatures and reference values as recited in claims 29, 30, 32, and 33.
Although step (a-3) of claim 29 requires that the first CTO is labeled with a fluorescent label and a quencher and a first reference signal is generated from the fluorescent label of the first CTO and step (b-3) of claim 29 requires that the second CTO is labeled with a fluorescent label and a quencher and a second reference signal is generated from the fluorescent label of the second CTO, since claim 29 does not indicate where fluorescent signals of the test sample come from, it is unpredictable how fluorescent signals of the test sample can be detected. Furthermore, since claim 29 does not require that the first reference signal, the second reference signal, and fluorescent signals of the test sample are generated from the same kind of fluorescent label, if the first reference signal, the second reference signal, and fluorescent signals of the test sample are generated from the different kinds of fluorescent labels which produce different fluorescent signals, it is predictable how the first reference signal and the second reference signal can be used to compare with fluorescent signals of the test sample. In addition, although the specification teaches that “[A]ccording to an embodiment, the signals used for the presence of target nucleic acid sequences are a significant signal. In other words, the signals are signals to be generated being dependent on the presence of target nucleic acid sequences. According to an embodiment, significance of signals detected may be determined using a threshold value. For example, a threshold value is predetermined from a negative control in considering background signals of detector, sensitivity or label used, and then the significance of signals may be determined” (see paragraph [0171] of US 2017/0362646 A1, which is US publication of this instant case) and step (e) of claim 29 requires (e-1) determining the presence of the first target nucleic acid sequence by a formula (i) or (ii): (i) [(signal intensity of the test sample incubation product at the first detection temperature) - (signal intensity of the test sample incubation product at the second detection temperature) * (the second reference value)]; (ii) [(signal intensity of the test sample incubation product at the second detection temperature) - (signal intensity of the test sample incubation product at the first detection temperature) / (the second reference value)], wherein a resulting value of formula (i) greater than a first threshold determined from a negative control for distinguishing the presence and absence of the first target nucleic acid or a resulting value of formula (ii) smaller than a second threshold determined from a negative control indicates the presence of the first target nucleic acid, and (e-2) determining the presence of the second target nucleic acid sequence by a formula (iii) or (iv): (iii) [(signal intensity of the test sample incubation product at the first detection temperature) - (signal intensity of the test sample incubation product at the second detection temperature) * (the first reference value)]; (iv) [(signal intensity of the test sample incubation product at the second detection temperature) - (signal intensity of the test sample incubation product at the first detection temperature) / (the first reference value)],wherein a resulting value of formula (iii) smaller than a third threshold determined from a negative control for distinguishing the presence and absence of the second target nucleic acid or a resulting value of formula (iv) greater than a fourth threshold determined from a negative control indicates the presence of the second target nucleic acid, since claim 29 does not indicate how a first threshold and a second threshold are determined based on a negative control, it is unpredictable how the presence of the first target nucleic acid sequence can be determined by a formula (i) or (ii) and how the presence of the second target nucleic acid sequence can be determined by a formula (iii) or (iv) such that a first target nucleic acid sequence and a second target nucleic acid sequence in a sample cannot be detected using different detection temperatures and reference values as recited in claims 29, 30, 32, and 33.
Case law has established that “(t)o be enabling, the specification of a patent must teach those skilled in the art how to make and use the full scope of the claimed invention without ‘undue experimentation’.” In re Wright 990 F.2d 1557, 1561. In re Fisher, 427 F.2d 833, 839, 166 USPQ 18, 24 (CCPA 1970) it was determined that “[T]he scope of the claims must bear a reasonable correlation to the scope of enablement provided by the specification to persons of ordinary skill in the art”. The amount of guidance needed to enable the invention is related to the amount of knowledge in the art as well as the predictability in the art. Furthermore, the Court in Genentech Inc. v Novo Nordisk 42 USPQ2d 1001 held that “[I]t is the specification, not the knowledge of one skilled in the art that must supply the novel aspects of the invention in order to constitute adequate enablement”.
In view of above discussion, the skilled artisan will have no way to predict the experimental results. Accordingly, it is concluded that undue experimentation is required to make the invention as it is claimed. These undue experimentation at least includes to test whether a first target nucleic acid sequence and a second target nucleic acid sequence in a sample can be detected using different detection temperatures and reference values as recited in claims 29, 30, 32, and 33.
Conclusion
In the instant case, as discussed above, the level of unpredictability in the art is high, the specification provides one with no guidance that leads one to claimed methods. One of skill in the art cannot readily anticipate the effect of a change within the subject matter to which the claimed invention pertains. Thus given the broad claims in an art whose nature is identified as unpredictable, the unpredictability of that art, the large quantity of research required to define these unpredictable variables, the lack of guidance provided in the specification, the absence of any working example related to claimed invention and the no teaching in the prior art balanced only against the high skill level in the art, it is the position of the examiner that it would require undue experimentation for one of skill in the art to perform the method of the claim as broadly written.
5. 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.
6. Claims 29, 30, 32, and 33 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.
7. Claim 29 is rejected as vague and indefinite because it is unclear that, in which situation, the first upstream oligonucleotide can be located upstream of the first PTO in step (a-1) and the second upstream oligonucleotide can be located upstream of the second PTO in step (b-1). Please clarify.
Response to Arguments
8. Applicant’s arguments with respect to claims 29-33 have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Conclusion
9. No claim is allowed.
10. Any inquiry concerning this communication or earlier communications from the examiner should be directed to Frank Lu, Ph. D., whose telephone number is (571)272-0746. The examiner can normally be reached Monday to Friday, 9 AM to 5 PM.
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/FRANK W LU/
Primary Examiner, Art Unit 1683
September 7, 2026
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