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 .
Status of Claims
Claims 1-22 are pending and currently under examination.
Priority
This application claims priority to 63/421,154, filed 10/31/2022.
Claim Objections
Claim 1 is objected to because of the following informalities: over the use of “PAM” in claim 1 without first reciting complete terminology “protospacer adjacent motif”. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-22 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.
Claims 1-22 are indefinite over the recitation in independent claim 1 of the limitation “amplifying the dumbbell sequence by CRISPR/Cas activation.” The term “amplifying” as used in the limitation renders the claim indefinite. CRISPR/Cas systems are generally understood in the art to perform sequence recognition and cleavage activities, including cis-cleavage and collateral (trans) cleavage activity, rather than nucleic acid amplification, and the teachings of the specification regarding CRISPR/Cas activation are consistent with this meaning. The claim does not define how CRISPR/Cas activation performs amplification, the specification does not provide a standard for ascertaining what constitutes amplification by CRISPR/Cas, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Thus, the recited limitation lacks clear boundaries as to what process step is being claimed.
Claims 1-22 are also indefinite over the recitation in independent claim 1 of the limitation “cleaving the surrounding reporters by collateral cleavage activity right after activation.” First, the claim fails to provide antecedent basis for “the surrounding reporters”, as no reporter or reporters are previously introduced in Claim 1. Further, the term “surrounding” is a relative term that renders the claim indefinite, as the claim does not specify what the reporters are surrounding or how such a relationship is determined. The claim language therefore does not reasonably apprise one of ordinary skill in the art of the scope of the claimed method. The phrase “right after activation” is vague and lacks objective boundaries. The claim does not specify what event constitutes activation or how temporal proximity is measured, and the specification does not provide a standard for determining when cleavage occurs “right after” activation. As such, the relationship between the recited steps is unclear.
Claim 7 is indefinite over the recitation “cleaving the amplified dumbbell sequence using CRISPR/Cas after activation by cis cleavage.” Specifically independent claim 1 recites in step f) “amplifying the dumbbell sequence by CRISPR/Cas activation that specifically recognizes first and second target regions using cis cleavage activation,” and in step g) “cleaving the surrounding reporters by collateral cleavage activity right after activation.” Neither step f) or g) recites or defines “an amplified dumbbell sequence.” In particular, it is unclear whether the “activation by cis cleavage” recited in claim 7 refers to the same activation recited in step f), or whether it constitutes a distinct activation event. Accordingly, one of ordinary skill in the art would not be reasonably apprised of the scope of claim 7, rendering the claim indefinite.
Claim 11 is indefinite over the recitation “adding 7.5% glycine to the dumbbell sequence.” A nucleic sequence is not a medium to which glycine can be added, and the claim fails to clarify whether the glycine is added to a reaction mixture, buffer, solution, or composition containing the dumbbell sequence. Accordingly, the scope of the claim is unclear.
Claim 19 and 22 are indefinite over the recitation in claim 19 of “a colorimetric initiator reporters”, which is internally inconsistent and unclear as to whether the limitation requires a single reporter or multiple reporters. The ambiguity renders the scope of the claim indefinite. Additionally, claim 22 is further indefinite for reciting “the colorimetric initiator”, which lacks proper antecedent basis, as claim 19 does not clearly introduce a singular colorimetric initiator.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim(s) 1-3, 5-10, 12-15, and 17 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Wang et al. (LAMP-CRISPR-Cas12-based diagnostic platform for detection of Mycobacterium tuberculosis complex using real-time fluorescence or lateral flow test, Microchim Acta 188(10):347 [10 Sep 2021]) as evidenced by Thompson F (Mini review: Recent progress in RT-LAMP enabled COVID-19 detection, Sensors and Actuators Reports 2(1):100017 [15 August 2020]).
Wang et al. disclose a method of loop-mediated isothermal amplification (LAMP) coupled with CRISPR/Cas-based detection termed “LACD (loop-mediated isothermal amplification coupled with CRISPR-Cas12a-mediated diagnostic”, which method employs a forward inner primer (FIP) or backward inner primer (BIP) engineered to contain a protospacer adjacent motif (PAM) site (TTTT) at the linker region (see entire reference, particularly the Abstract and Figure 1 on page 347).
With regard to the particular steps set forth in independent claim 1, Wang et al. disclose:
Regarding step a) of claim 1 “Providing a sample containing a target nucleic acid sequence containing a first target region and a second target region”, Wang et al. discloses that LAMP amplification is performed using six primers (F3, B3, FIP, BIP, LF, LB) that recognize eight sites in the target template (Page 4, LACD design).
Regarding step b) of claim 1 “Hybridizing a forward inner LAMP primer to a first target region, wherein the forward inner LAMP primer comprises a complementary sequence to the first target region and a PAM code sequence downstream of the complementary sequence”, Wang et al. teaches that in conventional LAMP, the forward inner primer (FIP) contains a 3’ region complementary to the target template for primer extension, and a 5’ region reverse-complementary to the target template (Page 4, LACD design). Wang et al. further discloses that the conventional FIP primer is engineered to include a PAM site (TTTT) at the linker region, such that the resulting LAMP amplicons contain a CRISPR-Cas12a recognition site (Abstract) (Page 4, LACD design).
Regarding step c) of claim 1 “Extending the forward inner LAMP primer to yield a displaced sequence, wherein the displaced sequence contains the second target region”, Wang et al. discloses that LAMP amplification is carried out using Bst 2.0 DNA polymerase under isothermal conditions (Page 2, LAMP and CRISPR-Cas12a trans-cleavage reaction). Wang et al. further teaches that during LAMP amplification, six primers initiate amplification, and amplification proceeds through strand displacement synthesis (Page 4, LACD design) Figure 1A schematically shows primer extension and strand displacement during the LAMP reaction.
Regarding step d) of claim 1 “Hybridizing a backward inner LAMP primer to the second target region of the displaced sequence” Wang et al. teaches the use of a backward inner primer (BIP) in the LAMP reaction that recognizes a specific region of the target sequence present on the displaced sequence generated during strand displacement amplification (Page 4, LACD design) (Page 2, Lamp and CRISPR-Cas12a trans-cleavage reaction)
Regarding step e) of claim 1 “Extending the backward inner LAMP primer to yield a dumbbell sequence containing a loop structure”, Wang et al. teaches LAMP amplification and formation of looped amplicon structures. LAMP amplification inherently produces self-priming dumbbell-shaped DNA structure containing loop regions as a result of inner primer extension and strand displacement (Page 3, Fig 1B) (Page 4, LACD design).
Regarding step g) of claim 1 “Cleaving the surrounding reporters by collateral cleavage activity right after activation”, Wang et al. teaches that once the CRISPR-Cas12a/gRNA complex binds to the target DNA, Cas12a is activated and exhibits collateral (trans) cleavage activity against surrounding single-stranded DNA reporter molecules. Specifically, the Abstract states that single-strand DNA reporter molecules are rapidly cleaved due to CRIPR-cas12a’s trans-enzyme activity. Upon activation, Cas12a rapidly digests ssDNA reporter molecules, generating detectable signals (abstract) (Page 2 left column paragraph 2) (The principle of real-time fluorescence and LFB for visualization of LACD results, page 5, Paragraph 1-2).
With regard to the “dumbbell sequence” referenced in steps e) and f) of claim 1, while it is noted that Wang et al. do not explicitly disclose the limitation “dumbbell sequence containing a loop structure”, however, Wang et al. does teach LACD (loop-mediated isothermal amplification) coupled with CRISPR-Cas12a-mediated diagnostic and that their method is a conventional LAMP in which a modified forward inner primer (FIP) or backwards inner primer (BIP) is employed (see again the Abstract; page 2, right column paragraph 2). Furthermore, as evidenced by the teachings Thompson et al., formation of such a “dumbbell sequence containing a loop structure” is a structural characteristic of LAMP, such that Wang et al. clearly disclose what is required by the claim (see page 2, right column paragraph 2, Fig. 1, and page 3 left column paragraph 1 of Thompson et al).
Accordingly, Wang et al. anticipate claim 1.
Regarding claim 2, “the PAM code sequence is immediately downstream of the complementary sequence or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides downstream of the complementary sequence, Wang et al discloses that the forward inner primer is engineered by adding a PAM site (TTTT) to the linker region (Abstract) (Page 4, LACD design) (Fig 1A on page 3).
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Fig. 1 Outline of LACD assay. (A) Schematic illustration of the principle for LAMP using modified FIP primer. The linker region from a conventional FIP primer was engineered with a PAM site (TTTT). After LAMP amplification with the modified FIP primer, the LAMP amplicons contain a CRISPR-Cas12a recognition site (TTTT) which was derived from the modified FIP primer.
Regarding dependent claim 3, “forward inner LAMP primer comprising an amplifier loop region”, Wang et al discloses the use of six primers (F3, B3, FIP, BIP, LF, LB) recognizes eight regions in the target template (Page 4, LACD design). In LAMP, loop primers (LF and LB) are used to accelerate amplification, generating loop structures in the amplicons (Fig 1A).
Regarding dependent claim 5, “amplifying the dumbbell sequence uses a Bst DNA polymerase”, Wang et al discloses LAMP was carried out in a reaction containing 8 U of Bst 2.0 DNA polymerase (Page 2, Lamp and CRISPR-Cas12a trans-cleavage reaction).
Regarding dependent claim 6, “the dumbbell sequence contains a binding sequence for single guide RNA (sgRNA) recognition for CRISPR/Cas nucleic acid cleavage”, Wang et al discloses that the amplified dumbbell sequence contains a binding sequence recognized by the sgRNA for CRISPR/Cas cleavage (Abstract) (page 5, right column paragraph 2).
Regarding dependent claim 7, “cleaving the amplified dumbbell sequence using CRISPR/Cas after activation by cis cleavage” Wang discloses CRISPR-Cas12a/gRNA complex first binds specifically to the target DNA adjacent to the PAM site (cis recognition), upon formation of the CRISPR-Cas12a/gRNA/target DNA complex, Cas12a is activated. (Fig. 1B).
Regarding dependent claim 8, “the sgRNA targets a sequence adjacent to or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides upstream or downstream of the PAM code sequence”, Wang et al discloses the sgRNA targets a sequence adjacent to the PAM site (supplementary table S1) (Page 5, MTC-LACD assay).
Regarding dependent claim 9, “the sample contains a pathogen with genetic materials”, Wang et al discloses detection of Mycobacterium tuberculosis complex (MTC) from clinical sputum samples (Page 4, Applicability of LACD assay to clinical samples) (Supplementary table 2).
Regarding dependent claim 10, Wang et al discloses, “the target nucleic acid sequence is single-stranded RNA, single-stranded DNA, or double-stranded DNA”, Wang et al discloses detection of genomic DNA from M. tuberculosis (Page 3, Sensitivity of the LACD assay).
Regarding dependent claim 12, “contacting a CRISPR/Cas sgRNA and a reporter to the amplified dumbbell sequence, wherein the CRISPR/Cas sgRNA comprises a target binding sequence specific to the amplified dumbbell sequence”, Wang et al discloses that the reporter is a single-stranded or a double-stranded DNA fluorescent reporter, the single-stranded or a double-stranded DNA fluorescent reporter is labeled at the 3’ end with a fluorophore and at the 5’ end with a quencher and further comprises a TTTTT sequence between the fluorophore and the quencher (Page 4, LACD design) (Supplementary Table S1).
Regarding dependent claim 13, “the reporter is a single-stranded or a double-stranded DNA fluorescent reporter”, Wang discloses ssDNA reporters used for real-time fluorescence detection (Fig 1B).
Regarding dependent claim 14, “the single-stranded or a double-stranded DNA fluorescent reporter is labeled at the 3′ end with a fluorophore and at the 5′ end with a quencher and further comprises a TTTTT sequence between the fluorophore and the quencher”, Wang discloses the use of a fluorescent reporter: 5’-FAM-TTATTATTATT (Supplementary Materials and Methods, second paragraph of CRISPR-Cas12a trans-cleavage reaction).
Regarding dependent claim 15, “the reporter is a single-stranded or a double-stranded DNA electrochemical reporter”, Wang et al discloses lateral flow detection using a reporter labeled (5'-FITC-TTATTATTATT-biotin-3’) (Supplementary Materials and Methods, second paragraph of CRISPR-Cas12a trans-cleavage reaction).
Regarding dependent claim 17, “cleaving the reporter to generate a transduced signal from excitation of the cleaved reporter after CRISPR/Cas activation”, Wang discloses binding of CRISPR-Cas12a/gRNA to the target activates Cas12a, activated Cas12a cleaves ssDNA reporter molecules, cleavage separates fluorophore and quencher, resulting fluorescent signal upon reporter cleavage (Page 5, The principle of real-time fluorescence and LFB for visualization of LACD results) (Fig. 1B, steps 2 and 3).
Accordingly, Wang et al. anticipates claims 1-3, 5-10, 12-15, and 17.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 4 and 18-21 are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (LAMP-CRISPR-Cas12-based diagnostic platform for detection of Mycobacterium tuberculosis complex using real-time fluorescence or lateral flow test, Microchim Acta 188(10):347 [10 Sep 2021]), as evidenced by Thompson (Mini review: Recent progress in RT-LAMP enabled COVID-19 detection, Sensors and Actuators Reports 2(1):100017 [15 August 2020]) as applied to the rejection of claims 1-3, 5-10, 12-15, and 17 above, further in view of Selvam et al. (RT-LAMPCRISPR-Cas12/13-Based SARS-CoV-2 Detection Methods, Diagnostics 11(9):1646 [08 Sep 2021]).
The teachings of Wang et al. (2021) and Thompson (2020) are documented above in the rejection of claims 1-3, 5-10, 12-15, and 17 under 35 U.S.C. 102(a)(1).
Regarding claim 4, the method of claim 1, “wherein the sample is whole blood, plasma, serum, lymph, urine, saliva, tears, nasopharyngeal secretions, or any combination thereof”, Wang et al. disclose a LAMP-CRISPR/Cas-based nucleic acid detection method but do not expressly disclose all of the biological sample types recited in the claim 4. Selvam et al. disclose nucleic acid detection sampled from nasopharyngeal swabs, nasal aspirates, saliva, bronchoalveolar lavage, and sputum (Section 2.1, Sample Collection and RNA Extraction, bridging paragraph, pages 2-3). It would have been prima facie obvious to one of ordinary skill in the art to apply the method of Wang et al. to the biological sample types taught by Selvam et al., as such sample matrices were known to be compatible with LAMP-based nucleic acid amplification and detection and would have yielded predictable results.
Regarding claim 18, the method of claim 17, further comprising detecting the reporter transduced signal using on-chip detection with a magnetic bead to concentrate and transfer the amplified dumbbell sequence. Wang et al. disclose CRISPR-mediated reporter cleavage and signal generation but do not disclose on-chip detection using magnetic beads. Selvam et al. disclose on-chip nucleic acid detection systems employing magnetic beads to concentrate, manipulate, and transfer amplified nucleic acid products (Section 2.5, Signal Readout, Page 10). It would have been prima facie obvious to one of ordinary skill in the art to incorporate magnetic bead-based on-chip detection into the method of Wang et al. in view of Selvam et al., as magnetic beads were commonly used to enhance sensitivity and facilitate handling of nucleic acid assays.
Regarding claim 19, the method of claim 17, further comprising detecting a colorimetric signal using a lateral flow on-chip detection with the reporter labelled with a colorimetric initiator reporter. Wang et al. disclose CRISPR-based detection using fluorescence or lateral flow formats but do not expressly disclose a colorimetric initiator reporter, Selvam et al. disclose colorimetric detection strategies and lateral flow-based readouts for LAMP-based nucleic acid assays (Section 2.5, Signal Readout, Page 10). It would have been prima facie obvious to one of ordinary skill in the art to employ a colorimetric lateral flow detection format in the method of Wang et al. in view of Selvam et al., as colorimetric reporters were well-known substitutes for fluorescent reporters and enabled simple, instrument-free detection.
Regarding claim 20, the method of claim 18, wherein the magnetic bead is a carboxylate-modified magnetic bead. Wang et al. do not disclose the specific surface functionalization of magnetic beads. Selvam et al. disclose the use of functionalized magnetic beads, including carboxylate-modified beads for nucleic acid capture and detection (Section 2.7, Page 11 paragraph 2). It would have been prima facie obvious to one of ordinary skill in the art to select carboxylate-modified magnetic beads for use in the method of Wang et al. based on the teachings of Selvam et al., such bead chemistries were known to facilitate nucleic acid binding and manipulation.
Regarding claim 21, the method of claim 20, wherein the magnetic bead is in a mineral oil film. Wang et al. do not disclose the use of mineral oil film in conjunction with magnetic beads. Selvam et al. disclose the use of mineral oil-based environments and microfluid formats in LAMP-based nucleic acid detection to prevent evaporation and cross contamination (Page 4, paragraph 1). It would have been prima facie obvious to one of ordinary skill in the art to employ a mineral oil film with the magnetic bead-based detection system of Wang et al. in view of Selvam et al., as oil overlays were commonly used in isothermal amplification assays and provided predictable benefits.
There would have been reasonable expectation of success to incorporate the teachings of Selvam et al. into the combined teachings of Wang et al. (2021) and Thompson (2020) to reach instant claims 4 and 18-21 because Selvam et al. specifically teaches magnetic bead-based concentration (Page 11, paragraph 2), lateral flow detection formats (Fig 5)(Page 10, paragraph 1), and oil-based or microfluidic environments are compatible with RT-LAMP and CRISPR-Cas-based nucleic acid detection systems (Page 4, paragraph 1). Wang et al. demonstrate that LAMP amplified products can activate CRISPR-Cas12a cleavage and generate detectable fluorescence or lateral flow signals (abstract) (Fig 1B) (Page 6, Optimal conditions for LACD assay), while Thompson confirms that LAMP amplification is robust across various biological sample types and detection formats (Page 2, right column). Because each reference teaches components that are routinely combined in nucleic acid detection platforms-namely, (i) LAMP amplification, (ii) CRISPR-mediated reporter cleavage, (iii) magnetic bead-based concentration and handling, and (iv) lateral flow or colorimetric signal visualization-a person of ordinary skill in the art would have reasonably expected that integrating the known magnetic bead and oil-overlay handling strategies of Selvam et al. into the LAMP-CRISPR system of Wang et al. would have predictably enhanced signal handling, concentration efficiency, and assay stability without altering the fundamental biochemical mechanisms of amplification or reporter cleavage. The underlying molecular biology principles are well-established, and the cited references demonstrate compatibility of these components within similar diagnostic workflows. Accordingly, there would have been a reasonable expectation that the combined system would function as intended.
Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (LAMP-CRISPR-Cas12-based diagnostic platform for detection of Mycobacterium tuberculosis complex using real-time fluorescence or lateral flow test, Microchim Acta 188(10):347 [10 Sep 2021), as evidenced by Thompson (Mini review: Recent progress in RT-LAMP enabled COVID-19 detection, Sensors and Actuators Reports 2(1):100017 [15 August 2020]) as applied to the rejection of claims 1-3, 5-10, 12-15, and 17 above, further in view of Ozay et al. (A review of reaction enhancement strategies for isothermal nucleic acid amplification reactions, Sensors and Actuators Reports 3:100033 [Nov 2021]).
The teachings of Wang et al. (2021) and Thompson (2020) are documented above in the rejection of claims 1-3, 5-10, 12-15, and 17 under 35 U.S.C. 102(a)(1).
Regarding claim 11, the method of claim 1, further comprising adding glycine 7.5% glycine to the dumbbell sequence. Wang et al. does not expressly disclose the addition of glycine to the LAMP amplification reaction. Ozay et al. disclose the use of glycine as a reaction additive in nucleic acid amplification assays to improve reaction performance (Page 7 left column paragraph 1) (Page 7 right column paragraph 2). It would have been prima facie obvious to one of ordinary skill in the art to include glycine as an additive in the method of Wang et al in view of Ozay et al., as the use of chemical additives to optimize amplification efficiency and robustness was well known and represents a predictable optimization of the LAMP reaction.
There would have been reasonable expectation of success to incorporate the teachings of Ozay et al. into the combined teachings of Wang et al. (2021) and Thompson (2020) to reach instant claim 11 because of Ozay et al. specifically teaches the use of glycine as a reaction additive in nucleic acid amplification assays to enhance amplification efficiency and improve overall reaction performance (Fig 4). Wang et al. disclose a LAMP-based amplification system coupled with CRISPR-mediated detection, and Thompson further evidences in Fig. 1 (B) that formation of a “dumbbell sequence containing a loop structure” is a structural characteristic of LAMP. The addition of glycine as taught by Ozay et al. does not alter the fundamental strand displacement amplification mechanisms of LAMP but instead functions as a chemical additive to improve reaction robustness. Because LAMP amplification relies on well-established enzymatic processes under defined reaction conditions, and Ozay et al. expressly demonstrate compatibility of glycine with nucleic acid amplification systems, one of ordinary skill in the art would have reasonably expected that incorporating glycine into the LAMP reaction mixture of Wang et al. would have predictably maintained or improved amplification performance without adversely affecting downstream CRISPR-based detection.
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (LAMP-CRISPR-Cas12-based diagnostic platform for detection of Mycobacterium tuberculosis complex using real-time fluorescence or lateral flow test, Microchim Acta 188(10):347 [10 Sep 2021]; cited herein), as evidenced by Thompson (Mini review: Recent progress in RT-LAMP enabled COVID-19 detection, Sensors and Actuators Reports 2(1):100017 [15 August 2020]; cited herein) as applied to the rejection of claims 1-3, 5-10, 12-15, and 17 above, further in view of Garcia-Gonzalez et al. (Methylene blue covalently attached to single stranded DNA as electroactive label for potential bioassays, Sensors and Actuators B: Chemical 191(0925-40050):784-790 [13 Oct 2013]).
The teachings of Wang et al. (2021) and Thompson (2020) are documented above in the rejection of claims 1-3, 5-10, 12-15, and 17 under 35 U.S.C. 102(a)(1).
Regarding claim 16, the method of claim 15, wherein the single-stranded or a double-stranded DNA electrochemical reporter is labeled at the 3’ end and 5’ end with a methylene blue label and further comprises a 30 to 60 bases -TTTTT- sequence between the two labels. Wang et al. disclose CRISPR-mediated detection using reporter molecules but do not disclose the specific electromechanical reporter configuration recited in claim 16. Garcia-Gonzalez et al. disclose electrochemical nucleic acid reporters labeled with methylene blue at terminal ends and incorporating spacer sequences to enable electrochemical signal transduction (abstract) (Page 1 right column paragraph 1). It would have been prima facie obvious to one of ordinary skill in the art to modify the reporter system of Wang et al. to employ the electrochemical reporter configuration taught by Garcia-Gonzalez et al., as electrochemical detection was a known alternative to fluorescence -based detection and provided a predictable method for signal generation.
There would have been reasonable expectation of success to incorporate the teachings of Garcia-Gonzalez et al. into the combined teachings of Wang et al. (2021) and Thompson (2020) to reach instant claim 16 because of Garcia-Gonzalez et al. specifically teaches methylene blue covalently attached to single-stranded DNA at terminal positions and demonstrate that such terminally labeled DNA constructs generate reliable electrochemical signals suitable for nucleic acid detection (abstract) (Page 1, paragraph 1). Garcia-Gonzalez et al. further discloses DNA strands labeled at one or both ends with methylene blue (MB-DNA and MB-DNA-MB) and explain that incorporation of spacer sequences between labels facilitates effective electrochemical signal transduction (Page 1, right column). Because Wang et al. already employ reporter-based CRISPR detection systems and Thompson evidences compatibility of LAMP-based amplification with alternative signal transduction formats, one of ordinary skill in the art would have reasonably expected that substituting a fluorescence-based reporter with the electrochemical methylene blue-labeled DNA reporter configuration taught by Garcia-Gonzalez et al., including terminal labeling and spacer sequences, would predictably produce a functional electrochemical readout without disrupting the underlying nucleic acid recognition or amplification chemistry. The electrochemical behavior of such MB-labeled DNA constructs was experimentally demonstrated by Garcia-Gonzalez et al., thereby providing a reasonable expectation that the modified reporter system would operate as intended within the combined detection platform.
Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (LAMP-CRISPR-Cas12-based diagnostic platform for detection of Mycobacterium tuberculosis complex using real-time fluorescence or lateral flow test, Microchim Acta 188(10):347 [10 Sep 2021]), as evidenced by Thompson (Mini review: Recent progress in RT-LAMP enabled COVID-19 detection, Sensors and Actuators Reports 2(1):100017 [15 August 2020]) as applied to the rejection of claims 1-3, 5-10, 12-15, and 17 above, further in view of Selvam et al. (Diagnostics 11(9):1646 [08 Sep 2021]) and Li et al. (Spectrochim Acta A Mol Biomol Spectosc 213(1386-1425):37-41 [15 April 2019]).
The teachings of Wang et al. (2021) and Thompson (2020) are documented above in the rejection of claims 1-3, 5-10, 12-15, and 17 under 35 U.S.C. 102(a)(1).
The teachings of Selvam et al. (2021) are documented above in the rejection of claims 4 and 18-21 under 35 U.S.C. 103.
Regarding claim 22, the method of claim 19, wherein the colorimetric initiator is glucose oxidase. Wang et al. do not disclose glucose oxidase as a colorimetric initiator. Li et al. disclose glucose oxidase-based colorimetric detection systems for nucleic acid assays (Page 1 right column paragraph 1).
It would have been prima facie obvious to one of ordinary skill in the art to employ glucose oxidase as the claimed colorimetric initiator in the detection system of Wang et al., in view of Li et al., because Li et al. teach that glucose oxidase catalyzes the oxidation of glucose to generate hydrogen peroxide, which subsequently produces visually detectable colorimetric signal through oxidation of tetramethylbenzidine (TMB). The use of glucose oxidase as an enzymatic initiator to generate a measurable colorimetric readout was well known and represent a predictable substitution of one known colorimetric initiation mechanism for another within nucleic acid detection systems.
There would have been reasonable expectation of success to incorporate the teachings of Li et al. into the combined teachings of Wang et al. (2021), Thompson (2020), and Selvam et al. (2021) to reach instant claim 22 because of Li et al. specifically teaches the glucose oxidase-mediated oxidation of glucose reliably generates hydrogen peroxide, which in turn oxidizes TMB to produce a measurable color change proportional to analyte concentration. Li et al. demonstrate that this enzymatic cascade produces a sensitive and reproducible colorimetric signal under standard assay conditions (abstract) (scheme 1) (Page 39, paragraph 2). Because Wang et al. already disclose nucleic acid detection platforms utilizing reporter-based signal generation, and Selvam et al. teach colorimetric detection formats compatible with nucleic acid amplification assays, one ordinary skill in the art would have reasonably expected that incorporating glucose oxidase as the colorimetric initiator would predictably generate a functional colorimetric signal without adversely affecting the upstream amplification or detection chemistry. The enzymatic Gox-glucose-H2O2 reaction pathway was well established in the art, thereby providing a reasonable expectation that the modified detection system would operate as intended.
Conclusion
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/BREANNA MARIE TAVERNINI/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682