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 .
Preliminary Amendment and Status of the Claims
The preliminary amendment filed on October 13th, 2023, in which claims 1-58 were cancelled and new claims 59-78 were added, is acknowledged and has been entered.
Claims 59-78 are pending and are under examination in this non-final action.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on October 13th, 2023. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Claim Status
Claims 1-58 have been canceled. Claims 59-78 have been added. Claims 59-78 are pending. Claims 59-78 are under examination and discussed in this Office action.
Specification
The use of the term, including but not limited to, “ThermoFisher”, “MirusBio”, and “Life Technologies”, which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
The lengthy specification has not been checked to the extent necessary to determine the presence of all possible minor errors. Applicant’s cooperation is requested in correcting any errors of which applicant may become aware in the specification.
Claim Objections
Claim 71 objected to because of the following informalities: the term “claims” should be corrected to “claim.” 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.
Claim 72 recites the limitation "a first detectably labeled probe that binds to the first overhang region and a second detectably labeled probe that binds to the second overhang region" in lines 5-6. There is insufficient antecedent basis for this limitation in the claim. It is unclear whether “detectably labeled probes” refers to the probes recited in claim 59, or whether it constitutes a different set of probes. As a result, the scope of the claim is indeterminate.
Claim 73 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. The phrase “and/or” renders the scope of the limitation ambiguous because it is unclear whether the limitation applies to the first probe, the second probe, or both probes.
In addition, the use of “and/or” permits an interpretation in which neither probe includes a quencher. Under such an interpretation, the claim does not explain how the individual signals from the respective probes would be detected or distinguished, resulting in uncertainty regarding the structure and operation of the claimed probes.
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 59-62, 70-71, 73-78 are rejected under 35 U.S.C. 103 as being unpatentable over Fehr (U.S. Patent Application Publication No. US 2010/0092960 A1, published 15 April 2010, as cited on the IDS filed October 13, 2023) in view of Severins et al. (Biophys. J., 2018, 115:957-967).
Regarding claim 59, Fehr teaches methods of hybridizing labeled probes (molecular beacons) to a target nucleic acid in a biological sample, detecting fluorescent signals from the hybridized probes, contacting the sample with a helicase that dissociates the probes from the nucleic acid, and obtaining sequential signals from the same nucleic acid molecule (abstract; 0026, 0032). Fehr thus discloses sequential probe hybridization and dissociation on a nucleic acid template.
However, Fehr does not teach contacting the biological sample with probes in discrete cycles, with immediate detection and dissociation steps. This deficiency is made up in the teachings of Severins et al.
Severins et al teach contacting immobilized target nucleic acid molecules with short, fluorescently labeled oligonucleotide barcode probes that hybridize to complementary sequences on the target molecule, followed by single-molecule fluorescence detection of probe binding (p. 958; Fig. 1). Severins et al further teach that probes which do not fully complement the target bind only transiently and dissociate during washing steps, whereas probes that hybridize adjacently and are correctly ligated remain stably associated with the target molecule and are detected via fluorescence (p. 960-962 (Single-molecule OLA); Fig. 1).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention to apply the probe-binding and dissociation taught by Severins et al to the helicase-mediated probe dissociation of Fehr to achieve sequential probe hybridization and dissociation of the same nucleic acid molecule with detection occurring after each probe interaction.
Regarding claim 60, Fehr teaches sequential hybridization, detection, and dissociation of probes on the same template nucleic acid (0014). Although Fehr does not explicitly teach that successive probes have identical recognition sequences, Fehr teaches helicase-mediated dissociation of probes from a template nucleic acid without cleavage of the template, thereby leaving the target sequence unchanged and available for subsequent probe hybridization.
Regarding claim 61, Fehr teaches hybridizing multiple molecular beacons to adjacent complementary subsequences that are arranged sequentially along a single-stranded nucleic acid, such that adjacent probes bind to neighboring regions of the same target molecule (0035-0036).
Severins et al. teach contacting a target nucleic acid with a pair of short probes that are complementary to two adjacent target sequence on the same nucleic acid molecule, such that each probe hybridizes to a distinct but immediately neighboring target (abstract; p. 958, para 2).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to modify the probes of Fehr to at least partially overlap or to interrogate overlapping sequence information at adjacent probe binding sites, as taught by Severins et al., in order to improve sequence discrimination with probe hybridization and signal detection.
Regarding claim 62, Fehr teaches methods of hybridizing labeled probes to a target nucleic acid and detecting signals generated by probe hybridization and dissociation (Abstract; 0010, 0032). However, Fehr does not teach barcode sequences within nucleic acid probes.
Severins et al. teach incorporating short DNA barcode sequences into nucleic acid molecules to identify and distinguish individual target molecules in multiplexed fluorescent-based assays (Abstract; p. 958, para. 2).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to modify Fehr’s probes to include barcode sequences as taught by Severins, because adding such a sequence does not change Fehr’s fundamental hybridization and dissociation mechanism but allows the probe to carry a unique, identifiable target sequence.
Regarding claim 70, Fehr teaches methods in which labeled probes are sequentially hybridized to a nucleic acid and dissociated by a probe-displacing enzyme to generate detectable signals (0032, 0037). Although Fehr does not explicitly teach terminating helicase activity between successive probe contacts, Fehr teaches discrete probe dissociation events followed by detection.
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to remove or inactivate the helicase after dissociation of a first probe and prior to contacting the sample with a second probe, in order to maintain the sequential detection process.
Regarding claim 71, Fehr teaches probes comprising detectable labels that produce detectable signals when hybridized to a target nucleic acid. Specifically, Fehr discloses molecular beacons comprising covalently linked fluorophores that fluoresce upon hybridization (0033). Fehr further discloses multiple molecular beacons hybridized to the same target nucleic acid, each beacon comprising a detectable label capable of producing a signal (0035-0036). Accordingly, Fehr teaches first and second probes bound to first and second detectable labels for producing first and second signals. The detectable labels may be the same or different, as the claim does not require a specific fluorophore.
Regarding claim 73, Fehr teaches methods using labeled hybridization probes that produce detectable signals upon hybridization to and removal from a target nucleic acid (0009, 0032). Fehr discloses molecular beacons comprising both fluorophores and quenchers only in certain embodies (0033), thereby indicating that other embodiments of labeled probes do not include quenching moieties.
Regarding claim 74, Fehr teaches sequential dissociation of hybridized probes using a helicase, wherein each probe is removed to produce a detectable signal that is detected before subsequent probes are dissociated (0009, 0026, 0032, 0037). Thus, after detecting the second signal produced by dissociation of the second probe, Fehr teaches contacting the biological sample with a helicase to dissociate the next probe from the target nucleic acid.
Regarding claim 75, Fehr teaches methods of sequencing nucleic acids present in biological samples, including DNA or RNA isolated from organisms such as eukaryotes, mammals, prokaryotes, and viruses (0031, 0037). Although Fehr does not explicitly use the term “endogenous nucleic acid,” the target nucleic acid in the disclosed methods is inherently a nucleic acid present in the biological sample and thus is an endogenous nucleic acid.
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention that the target nucleic acid of Fehr’s method can be an endogenous nucleic acid analyte in the biological sample since Fehr’s sequencing of nucleic acids are obtained from biological samples (eukaryotes, mammals, prokaryotes, and viruses).
Regarding claim 76, Fehr teaches that nucleic acids can be amplified using rolling circle replication (RCR) to generate single-stranded DNA templates suitable for sequencing reactions (0101).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention that the target nucleic acid in Fehr’s methods can be rolling circle amplification (RCA) product of a circular or circularizable probe that binds to a nucleic acid molecule in the biological sample.
Regarding claim 77, Fehr teaches that labeled hybridization probes are removed from a target nucleic acid by a probe-displacing enzyme to produce a detectable signal (0032). The act of dissociation itself does not inherently generate, activate, or depress a signal, rather, a signal is produced as a consequence of detecting that labeled probe after removal. It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention that the dissociation of the first and/or second probe from the target nucleic acid can occur without directly generating a signal.
Regarding claim 78, Fehr teaches methods for sequencing nucleic acids obtained from biological samples, including DNA or RNA templates used in the disclosed hybridization and probe-displacement sequencing methods (009, 010, 0012). It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention that such biological samples include cell or tissue samples.
Claim 72 is rejected under 35 U.S.C. 103 as being unpatentable over Fehr (U.S. Patent Application Publication No. US 2010/0092960 A1 in view of Severins et al. (Biophys. J., 2018, 115:957-967) as applied to claims 59-62, 70-71, 73-78 above, and further in view of Nilsson et al. (Science, 1994, 265:2085-2088).
Regarding claim 72, Fehr teaches a template nucleic acid with sequential hybridization probes and detecting signals from probe dissociation (0014-0017). However, Fehr does not explicitly teach probes comprising overhang regions that serve as binding sites for separate detectably labeled probes. This deficiency is made up in the teachings of Nilsson et al.
Nilsson et al teach oligonucleotide probes comprising target-complementary segments connected by a non-target linker sequence, wherein the linker sequence may carry detectable functions and may be further modified to facilitate detection (see Abstract; p.2085, para. 1).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to modify the hybridized probes of Fehr with non-target overhang regions and to contact the sample with detectably labeled probes that bind to overhang regions, as taught by Nilsson, in order to facilitate detection and improve signal flexibility without altering target recognition.
Claims 63-69 are rejected under 35 U.S.C. 103 as being unpatentable over Fehr (U.S. Patent Application Publication No. US 2010/0092960 A1, published 15 April 2010) in view of Severins et al. (Biophys. J., 2018, 115:957-967) as applied to claims 59-62, 70-71, 73-78 above, and further in view of Rajagopal and Patel (J. Mol. Biol., 2007, 376:69-79).
Regarding claim 63, Fehr teaches helicase-mediated dissociation of probes from nucleic acid templates (0026, 0032), but does not teach the use of single-strand binding protein.
Rajagopal and Patel teach that single-strand binding proteins (SSBs) bind to a single-stranded nucleic acids generated during helicase-mediated unwinding and enhance helicase processivity by stabilizing the helicase at the unwinding junction and preventing its dissociation (Summary; p. 4, para 2; Fig. 3).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to contact the biological sample with a single-stranded binding protein simultaneously with and/or after contacting the sample with a helicase, as recited in claim 63, in order to enhance helicase-mediated probe dissociation.
Regarding claim 64, Rajagopal and Patel teach single-stranded binding proteins binds to the single-stranded nucleic acid generated by helicase-mediated strand separation, including displaced strands produced during unwinding, thereby preventing reannealing. (Summary; p. 3, para 3; Fig. 4). Such single-stranded nucleic acids include strands generated by helicase-mediated dissociation, which in the context of Fehr would correspond to displaced probe strands and/or target nucleic acid strands. Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention that the single-stranded binding protein would bind to the target nucleic acid and/or the first probe after helicase-mediated dissociation to stabilize the strands and prevent reannealing.
Regarding claim 65, Fehr teaches the use of specific DNA helicases for dissociating hybridized probes from the nucleic acids. In particular, Fehr discloses the helicase used in the methods may be UvrD, Rep, or RecQ (0010, 0018, 0056).
Rajagopal and Patel teach that helicases of the SF2 superfamily, including RecQ, share conserved Walker A and Walker B motifs responsible for ATP binding and hydrolysis, and thus helicases within a superfamily share these structural features (p. 1).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to use Rep-X (super helicase), Tte UvrD, RecQ, or a homologs/variants thereof in the methods of Fehr.
Regarding claim 66, Rajagopal and Patel teach that helicase-mediated nucleic acid unwinding is ATP-dependent, as the NS3 helicase contains conserved ATP-binding and hydrolysis motifs and strand separation reactions are initiated upon addition of ATP (p. 1-3; Fig. 1). Rajagopal and Patel further teach contacting a helicase with a nucleic acid substrate in a buffer comprising ATP, wherein ATP is supplied to initiate helicase-mediated strand separation (p. 7).
Regarding Claim 67, as discussed above, Rajagopal and Patel teach that ATP hydrolysis drives helicase-mediated unwinding of nucleic acids (p. 1-3; Fig. 1). Rajagopal and Patel further teach helicase-mediated nucleic and unwinding reactions performed in buffer containing 10 mM ATP (p. 7).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to select an ATP concentration between 0.1 mM and 10 mM for the buffer when performing helicase-mediated probe dissociation.
Regarding claim 68, as discussed above, Rajagopal and Patel teach that helicase-mediated unwinding is ATP-dependent, as ATP is required to initiate unwinding of duplex nucleic acid substrates (p. 1-3; Fig. 1, p.7). Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention that the helicase of Fehr could dissociate probes from the target nucleic acid in an ATP-dependent reaction as taught by Rajagopal and Patel.
Regarding claim 69, Fehr teaches strand displacement reaction carried out by multiprotein complexes, such as replisomes, that include a single-stranded DNA binding protein (SSB) along with a helicase and polymerase (0101).
As discussed above, Rajagopal and Patel also teach that single-stranded binding proteins interact with single-stranded nucleic acids generated during helicase-mediated unwinding and enhance strand separation by stabilizing the unwound single-stranded region and preventing reannealing of complementary strands (Summary; p. 3, para 1-2), thereby facilitating continued helicase activity and strand dissociation.
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to include a single-stranded binding proteins in the helicase-mediated probe dissociation methods and to recognize that the single-stranded binding proteins would facilitate dissociation of the first probe by binding and stabilizing the single-stranded nucleic acid regions generated by helicase activity and inhibiting reannealing of complementary strands.
Conclusion
All claims are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Nura Choudhury whose telephone number is (571)272-6148. The examiner can normally be reached M-F, 9-5 ET.
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/NURA M. CHOUDHURY/ Examiner, Art Unit 1683
/ANNE M. GUSSOW/ Supervisory Patent Examiner, Art Unit 1683