DETAILED ACTION
Response to Amendment
Applicant’s response to the office action filed on Mary 26, 2026 have been entered. The claims pending in this application are claims 1, 5, 8, 11, 12, 15, 19, 25-29, 56, 57, 59, 63-65, 90 and 92 wherein claims 15, 27, 57, 63-65, 90, and 92 have been withdrawn due to the restriction requirement mailed on October 2, 2025. The objections not reiterated from the previous office action are hereby withdrawn in view of applicant’s amendment filed on May 26, 2026. Claims 1, 5, 8, 11, 12, 19, 25, 26, 28, 29, 56, 59, will be examined.
Claim Objections
Claim 19 or 25 is objected to because of the following informality: “cleaving the probe or probe set with the nuclease” should be “said cleaving the probe or probe set with a nuclease”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
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.
Scope of Enablement
Note that this rejection is different from the scope of enablement mailed on February 23, 2026 and the phrase “wherein the interrogatory region hybridizes to a region of interest in the target nucleic acid” in claim 1 is interpreted as “wherein the interrogatory region is capable of hybridizing to a region of interest in the target nucleic acid” in this rejection.
Claims 1, 5, 8, 11, 12, 19, 25, 26, 28, 29, 56, and 59 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
detecting a target nucleic acid using the methods recited in claims 1, 5, 8, 11, 12, 19, 25, 26, 28, 29, 56, and 59 when the probe comprises: i) a first hybridization region capable of hybridizing to a first target sequence in the target nucleic acid, ii) a second hybridization region capable of hybridizing to a second target sequence in the target nucleic acid, and iii) a duplex region which
is located in 5’ or 3’ end of the probe and comprises an interrogatory region located on only one strand of the duplex region such that, upon hybridization of the probe to the target nucleic acid, the duplex region is positioned between the first hybridization region and the second hybridization region and the interrogatory region becomes a single strand and hybridizes to a region of the target nucleic acid after cleaving the duplex region of the probe in a hybridization complex formed by the target nucleic acid and the probe, does not reasonably provide enablement for detecting a target nucleic acid using the methods recited in claims 1, 5, 8, 11, 12, 19, 25, 26, 28, 29, 56, and 59 when the probe or probe set comprises: i) a first hybridization region capable of hybridizing to a first target sequence in the target nucleic acid, ii) a second hybridization region capable of hybridizing to a second target sequence in the target nucleic acid, and iii) a duplex region comprising an interrogatory region, wherein upon hybridization of the probe or probe set to the target nucleic acid, the duplex region is positioned between the first hybridization region and the second hybridization regions and after cleaving the probe in a hybridization complex formed by the target nucleic acid and the probe and releasing a portion of the duplex region from the hybridization complex formed by the target nucleic acid and the probe, the interrogatory region is still located in another portion of the duplex region from the hybridization complex formed by the target nucleic acid and the probe. 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 target nucleic acid. 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 1, 5, 8, 11, 12, 19, 25, 26, 28, 29, 56, and 59 encompass a method for detecting a target nucleic acid, comprising: a) contacting the target nucleic acid with a probe or probe set comprising: i) a first hybridization region capable of hybridizing to a first target sequence in the target nucleic acid, ii) a second hybridization region capable of hybridizing to a second target sequence in the target nucleic acid, and iii) a duplex region comprising an interrogatory region, wherein upon hybridization of the probe or probe set to the target nucleic acid, the duplex region is positioned between the first and second hybridization regions; b) cleaving the probe or probe set with a nuclease to generate a first ligatable end and release a portion of the duplex region, that does not comprise the interrogatory region, wherein the interrogatory region hybridizes to a region of interest in the target nucleic acid thereof; c) ligating the first ligatable end to a second ligatable end in the probe or probe set hybridized to the target nucleic acid to generate a ligated probe; and d) detecting the ligated probe or a product thereof, which indicates a presence of the target nucleic acid.
Working Examples
The specification provides 3 working examples (see pages 48 and 49 of US 2023/
0323430 A1, which is US publication of this instant case). However, the specification does not provide an example for detecting a target nucleic acid using the methods recited in claims 1, 5, 8, 11, 12, 19, 25, 26, 28, 29, 56, and 59 when the probe or probe set comprises: i) a first hybridization region capable of hybridizing to a first target sequence in the target nucleic acid, ii) a second hybridization region capable of hybridizing to a second target sequence in the target nucleic acid, and iii) a duplex region comprising an interrogatory region, wherein upon hybridization of the probe or probe set to the target nucleic acid, the duplex region is positioned between the first hybridization region and the second hybridization regions and after cleaving the probe in a hybridization complex formed by the target nucleic acid and the probe and releasing a portion of the duplex region from the hybridization complex formed by the target nucleic acid and the probe, the interrogatory region is still located in another portion of the duplex region from the hybridization complex formed by the target nucleic acid and the probe.
The Amount of Direction or Guidance Provided and The State of The Prior Art
Although the specification provides 3 working examples (see pages 48 and 49 of US 2023/0323430 A1, which is US publication of this instant case), the specification does not provide an example for detecting a target nucleic acid using the methods recited in claims 1, 5, 8, 11, 12, 19, 25, 26, 28, 29, 56, and 59 when the probe or probe set comprises: i) a first hybridization region capable of hybridizing to a first target sequence in the target nucleic acid, ii) a second hybridization region capable of hybridizing to a second target sequence in the target nucleic acid, and iii) a duplex region comprising an interrogatory region, wherein upon hybridization of the probe or probe set to the target nucleic acid, the duplex region is positioned between the first hybridization region and the second hybridization regions and after cleaving the probe in a hybridization complex formed by the target nucleic acid and the probe and releasing a portion of the duplex region from the hybridization complex formed by the target nucleic acid and the probe, the interrogatory region is still located in another portion of the duplex region from the hybridization complex formed by the target nucleic acid and the probe. 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 target nucleic acid using the methods recited in claims 1, 5, 8, 11, 12, 19, 25, 26, 28, 29, 56, and 59 when the probe or probe set comprises: i) a first hybridization region capable of hybridizing to a first target sequence in the target nucleic acid, ii) a second hybridization region capable of hybridizing to a second target sequence in the target nucleic acid, and iii) a duplex region comprising an interrogatory region, wherein upon hybridization of the probe or probe set to the target nucleic acid, the duplex region is positioned between the first hybridization region and the second hybridization regions and after cleaving the probe in a hybridization complex formed by the target nucleic acid and the probe and releasing a portion of the duplex region from the hybridization complex formed by the target nucleic acid and the probe, the interrogatory region is still located in another portion of the duplex region from the hybridization complex formed by the target nucleic acid and the probe.
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 target nucleic acid can be detected using the methods recited in claims 1, 5, 8, 11, 12, 19, 25, 26, 28, 29, 56, and 59 when the probe or probe set comprises: i) a first hybridization region capable of hybridizing to a first target sequence in the target nucleic acid, ii) a second hybridization region capable of hybridizing to a second target sequence in the target nucleic acid, and iii) a duplex region comprising an interrogatory region, wherein upon hybridization of the probe or probe set to the target nucleic acid, the duplex region is positioned between the first hybridization region and the second hybridization regions and after cleaving the probe in a hybridization complex formed by the target nucleic acid and the probe and releasing a portion of the duplex region from the hybridization complex formed by the target nucleic acid and the probe, the interrogatory region is still located in another portion of the duplex region from the hybridization complex formed by the target nucleic acid and the probe.
Since the specification teaches that “[I]n some embodiments, e.g., as shown in FIG. 3, provided herein are methods for analyzing a biological sample comprising a plurality of target molecules (e.g., RNA) comprising a single nucleotide of interest, the methods comprising: a) contacting the biological sample with a circularizable probe comprising: i) a first hybridization region capable of hybridizing to a first target sequence in a target nucleic acid in the biological sample, ii) a second hybridization region capable of hybridizing to a second target sequence in the target nucleic acid, iii) a stem-loop structure at the 3’ or 5’ end of the circularizable probe, wherein the target nucleic acid comprises a region of interest and the stem-loop structure comprises an interrogatory region, and wherein upon hybridization of the circularizable probe to the target nucleic acid, the stem-loop structure is positioned between the first and second hybridization regions; b) cleaving the circularizable probe with a nuclease to generate a ligatable 3’ end or ligatable 5’ end and releasing the stem-loop structure or a portion thereof, thereby allowing the interrogatory region to hybridize to the region of interest; c) if the interrogatory region is complementary to the region of interest, ligating the ligatable 3’ end or ligatable 5’ end to the 5’ end or the 3’ end of the circularizable probe, respectively, to generate a circular probe using the target nucleic acid as template; and d) amplifying the circular probe using rolling circle amplification (RCA) to generate an RCA product; and e) detecting the RCA product in the biological sample”, “[F]IG. 3 depicts an exemplary probe comprising a stem-loop structure. The probe is contacted with a biological sample comprising a target nucleic acid (for example, RNA) comprising a region of interest such as a single nucleotide polymorphism (SNP) of interest. The probe can comprise a 5’ phosphate group and a stem-loop structure on its 3’ end. The hybridization region comprises an interrogatory region (for example, an interrogatory nucleotide) that is complementary to the region of interest (for example, correct SNP present, left side of FIG. 3). Alternatively, the probe may comprise an interrogatory region (for example, an interrogatory nucleotide) that is not complementary to the region of interest (for example, incorrect SNP present, right side of FIG. 3). The probe is processed by a nuclease to release the stem-loop structure and generate ligatable ends. If the target nucleic acid comprises the SNP of interest such that it is complementary to the interrogatory nucleotide, hybridization is stable and the 3’ and 5’ ends can be ligated to each other (left side of FIG. 3). However, if the target nucleic acid does not comprise a SNP complementary to the interrogatory nucleotide, hybridization is unstable, and the 3′ and 5′ ends are not ligated (right side of FIG. 3). In some embodiments, the interrogatory region can comprise one or more nucleotides that are part of the recognition sequence of the nuclease. In some embodiments, the stem-loop structure can be cleaved regardless of the identity of the interrogatory nucleotide. For example, cleavage of the duplex region can be constant across all probes or probe sets, regardless of the identity of the interrogatory region. In some embodiments, a probe comprising a stem-loop structure comprising a probe comprising an interrogatory region that is not complementary to the region of interest can be cleaved (e.g., as shown in the example of FIG. 3, right panel, depicting a incorrect SNP present in the target nucleic acid), but unstable hybridization between the interrogatory region and the region of interest (e.g., the single nucleotide of interest) will prevent subsequent ligation. Although FIG. 3 depicts a circularizable probe, it will be understood that an interrogatory region in a linear probe or probe set could similarly discriminate between different sequences of a region of interest, generating a ligated linear probe only in the presence of the correct sequence of the region of interest (e.g., the correct SNP)”, “[F]IGS. 1 and 2 depict schematics of an exemplary probe and probe set. The probe in FIG. 1 comprises a circularizable probe with at least two hybridization regions that are capable of hybridizing to at least two adjacent complementary regions on a target nucleic acid (for example, to the target nucleic acid, such as an mRNA). The circularizable probe also contains a duplex region which includes a first and second strand hybridized to one another. The duplex region can also be modified to comprise a stem-loop structure. The duplex region or stem-loop structure can be positioned at either the 3’ end (FIG. 1) or the 5’ end of the circularizable probe, or can be positioned internally (for example, positioned between the hybridization region and the 3’ or 5’ end). In some cases, the duplex region runs continuous between the first and second hybridization regions such that the probe is circular (FIG. 2B)”, “[I]n some instances, a probe set comprising a set of two linear probes are hybridized to two complementary regions on a target nucleic acid (for example, to the target nucleic acid, such as an mRNA, or to another hybridization region in a polynucleotide of the probe set). In this instance, the duplex can be positioned at either the 3′ or 5′ end of the first or second linear probe of the probe set (FIG. 2C). The duplex can also be positioned between the two probes such that the two probes are connected to each other to form a single linearized probe”, “[T]he duplex region or stem-loop structure can contain a restriction endonuclease recognition sequence. The restriction endonuclease can cleave the site generating either blunt or sticky ends. In some instances, the restriction endonuclease can cleave at a site outside the recognition sequence (for example, in the case of type IIS enzymes). In some cases, the nuclease that cleaves the duplex region or stem-loop structure can be a uracil-specific excision reagent enzyme. In some cases, the nuclease can be a nickase, which cleaves only one strand of the duplex region”, “[I]n the example shown in FIG. 3, the probe is a modified padlock probe for detecting an SNP, comprising a duplex region (such as a stem-loop structure) at the 3’ or 5’ end of the padlock probe. Such an approach can be used to increase specificity and stringency when detecting a region of interest (such as an SNP) in a target nucleic acid, such as an mRNA. The padlock probe comprises at least two hybridization regions that hybridize to complementary sequences on the target mRNA. The padlock probe comprises an interrogatory region complementary to the region of interest, at the 3’ or 5’ end of the probe. The interrogatory region connects the 3’ or 5’ end of the padlock probe to the stem-loop structure in such a manner that upon cleavage and release of the stem-loop structure, and generation of a ligatable end, the interrogatory region is free to hybridize to the region of interest in the target mRNA”, and “[A] mixture of probes is incubated with hybridization buffer for hybridization of the probes to target nucleic acid (such as mRNA) in the sample. The sample is washed and incubated with a nuclease (such as a restriction endonuclease, uracil-specific excision reagent enzyme, or a nickase) for cleavage of the stem-loop structure. The released stem-loop structure is then removed using a wash buffer. The buffer conditions used allow the probe to remain hybridized to the target nucleic acid. As shown in FIG. 3, if the complementary SNP is not present, the padlock probe would still bind the mRNA target to the complementary regions. However, the mismatched end, for example the 3’ end, will not hybridize to the region of interest in the mRNA and the ligation will be halted. Because the arm of the padlock probe is not fully hybridized, the padlock would be removed in a stringency wash. If the interrogatory region is complementary to the region of interest in the target mRNA, such that the padlock probe will be stably hybridized to the target, ligation will proceed. The sample is then incubated at room temperature with a T4 DNA ligase for ligation of the ligatable 3’ and 5’ ends of the padlock probes to form circularized probes. A primer for amplification of the circularized probe may be added. The sample is then incubated with a rolling-circle amplification (RCA) mixture containing a Phi29 DNA polymerase and dNTP for RCA of the circular probes. Fluorescently labeled oligonucleotides complementary to a portion of the RCA product, a barcode contained therein, or a secondary probe attached thereto are incubated with the sample. Multiple cycles of contacting the sample with probes and sequence determination (e.g., using in situ sequencing based on sequencing-by-ligation or sequencing-by-hybridization) can be performed. Fluorescent images can be obtained in each cycle, and one or more wash steps can be performed in a cycle or between cycles. Probe targeting various SNPs within or across genes can be sequentially or simultaneously provided, processed, and detected as described above” (see paragraphs [0080], [0200], [0368] to [0370], [0377], and [0378], and Figures 1-3 of US 2023/0323430 A1, which is US publication of this instant case), the specification clearly indicate that a target nucleic acid cannot be detected when the probe or probe set comprises: i) a first hybridization region capable of hybridizing to a first target sequence in the target nucleic acid, ii) a second hybridization region capable of hybridizing to a second target sequence in the target nucleic acid, and iii) a duplex region comprising an interrogatory region, wherein upon hybridization of the probe or probe set to the target nucleic acid, the duplex region is positioned between the first hybridization region and the second hybridization regions and after cleaving the probe in a hybridization complex formed by the target nucleic acid and the probe and releasing a portion of the duplex region from the hybridization complex formed by the target nucleic acid and the probe, the interrogatory region is still located in another portion of the duplex region from the hybridization complex formed by the target nucleic acid and the probe. Since claim 1 does not require that, after the cleaving step, the duplex region from a hybridization complex formed by the target nucleic acid and the probe or the probe set consisting of two probes is completely removed from the hybridization complex and the interrogatory region becomes a single strand, if the duplex region is located on 5’ or 3’ end of the probe or the duplex region is located on 5’ or 3’ end of one probe of the probe set consisting of two probes, the interrogatory region can be still located in another portion of the duplex region from the hybridization complex such that the interrogatory region cannot hybridize to a region of interest in the target nucleic acid and it is unpredictable how the ligated probe can be generated and detected using the methods recited in claims 1, 5, 8, 11, 12, 19, 25, 26, 28, 29, 56, and 59. Alternatively, since claim 1 does not require that, after the cleaving step, the duplex region from a hybridization complex formed by the target nucleic acid and the probe is completely removed from the hybridization complex and the interrogatory region becomes a single strand, if the duplex is located between 5’ end of the probe and 3’ end of the probe, the interrogatory region can be still located in another portion of the duplex region from the hybridization complex such that the interrogatory region cannot hybridize to a region of interest in the target nucleic acid and it is unpredictable how the ligated probe can be generated and detected using the methods recited in claims 1, 5, 8, 11, 12, 19, 25, 26, 28, 29, 56, and 59.
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 discussions, 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 target nucleic acid can be detected using the methods recited in claims 1, 5, 8, 11, 12, 19, 25, 26, 28, 29, 56, and 59 when the probe or probe set comprises: i) a first hybridization region capable of hybridizing to a first target sequence in the target nucleic acid, ii) a second hybridization region capable of hybridizing to a second target sequence in the target nucleic acid, and iii) a duplex region comprising an interrogatory region, wherein upon hybridization of the probe or probe set to the target nucleic acid, the duplex region is positioned between the first hybridization region and the second hybridization regions and after cleaving the probe in a hybridization complex formed by the target nucleic acid and the probe and releasing a portion of the duplex region from the hybridization complex formed by the target nucleic acid and the probe, the interrogatory region is still located in another portion of the duplex region from the hybridization complex formed by the target nucleic acid and the probe.
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.
Response to Arguments
Applicant’s arguments with respect to claims 1, 5, 8, 11, 12, 19, 25, 26, 28, 29, 56, and 59 have been considered but are moot because the new ground of rejection does not rely on any teaching or matter specifically challenged in the argument.
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.
No claim is allowed.
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.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/ interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Anne Gussow, Ph.D., can be reached at 571-272-6047. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/FRANK W LU/
Primary Examiner, Art Unit 1683
August 13, 2026
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