DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election without traverse of Group I, a first method of sample pooling of at least two samples, in the reply filed on 8/15/25 is acknowledged.
Claims 1-33 are pending. Claims 31-33 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 8/15/25.
Claims 1-30 are currently under examination.
Priority
The instant application 17/807,822 filed on 6/20/22 claims domestic priority to provisional application 63/212,719 filed 6/20/21. The priority date is determined to be 6/20/21.
Claim Objections
Claim 1 is objected to because of the following informalities:
claim 1 line 10 should read “first ID-Primer” for hyphenation and capitalization consistencies. Appropriate correction is required.
Claim Rejections - 35 USC § 112 – Indefiniteness
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 1-30 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.
Claim 1 recites the limitation of “sufficient complementarity” with regards to the first and second ID-primers and their target nucleic acids. Although the instant specification provides a definition for “sufficient complementarity” (page 12, lines 28-30) and context regarding hybridization/complementarity (page 13, lines 1-15), the skilled artisan would be unable to determine the exact metes and bounds in which the ID-Primer is able or unable to fulfill the “sufficient complementarity” requirements. As an example, it is noted that high-salt conditions affect base-pair matching within short fluorescent probes (see Pu et al.; “Advances in nucleic acid probe-based detection of gene point mutations: a review”; Front Chem. 2025 Sep 25;13:1672155. doi: 10.3389/fchem.2025.1672155; section “Pure Nucleic Acid Probe-based Detection”), indicating that “sufficient complementarity” is highly impacted by reaction conditions and should be made clearly apparent to skilled artisans.
Claims 2-30 directly or indirectly depend upon claim 1 and are similarly indefinite.
Claim Rejections - 35 USC § 102
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.
Claims 1-4 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Peng et al. (2015; "Reducing amplification artifacts in high multiplex amplicon sequencing by using molecular barcodes"; BMC Genomics 16, 589. https://doi.org/10.1186/s12864-015-1806-8).
Relevant to claim 1, Peng et al. teaches "To design primers for our high multiplex amplicon barcoding protocol, we adopted the 'Primer ID' design strategy [citation] by inserting a molecular barcode region (random 6 to 12mer) between the 5′ universal sequence and 3′ target specific sequence in one of the two primers for each amplicon. All primers containing the molecular barcode for different amplicons are pooled together ('BC primers') and all other non-barcoded primers are mixed in a different pool ('non-BC primers')" (page 3, column 1, paragraph 2).
As seen in Peng et al. Figure 1, the primer has a 3' target specific sequence that reads on a nucleic acid sequence having sufficient complementarity to a first site in a target nucleic acid. The Peng et al. primer target specific sequence is operably linked to the 5' universal sequence (amplifier region). The Peng et al. molecular barcode reads on the unique ID region.
Further relevant to claim 1, Peng et al. teaches "The workflow is as the following (Fig. 1). 1) The BC primers are annealed to and extended on target DNA. At this step, each DNA molecule containing our target locus will be copied and the resulting copy will have a unique molecular barcode" (page 3, column 2, paragraph 2).
Further relevant to claim 1, Peng et al. teaches "Initially we tried to use an enzymatic approach such as Exonuclease I digestion to degrade leftover BC primers" (page 8, column 2, paragraph 1).
These Peng et al. teachings read on claim 1 limitations.
Relevant to claim 2, Peng et al. teaches "10 ng total RNA containing the ERCC RNA were reverse transcribed into cDNA using QuantiTect Reverse Transcription kit (QIAGEN, Germany)" (page 10, column 1, paragraph 3).
Relevant to claim 3, Peng et al. teaches "Initially we tried to use an enzymatic approach such as Exonuclease I digestion to degrade leftover BC primers" (page 8, column 2, paragraph 1).
Relevant to claim 4, Peng et al. teaches "The workflow is as the following (Fig. 1). 1) The BC primers are annealed to and extended on target DNA" (page 3, column 2, paragraph 2).
The Peng et al. teachings anticipate claims 1-4.
Claim Rejections - 35 USC § 103
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 5-6 and 22-25 are rejected under 35 U.S.C. 103 as being unpatentable over Peng et al. (2015; "Reducing amplification artifacts in high multiplex amplicon sequencing by using molecular barcodes"; BMC Genomics 16, 589. https://doi.org/10.1186/s12864-015-1806-8), as applied to claims 1-4 above, and further in view of Hatch et al. (2013; US 2013/0178378 A1; publication of US application 13/492,698, which claims the priority of provisional application 61/495,308 filed on 6/9/11).
The teachings of Peng et al. are applied to instantly rejected claims 5-6 and 22-25 as they were applied to claims 1-4 as anticipating a method of sample pooling of at least two samples.
(i) Peng et al. is silent to specifics regarding melting temperature (claim 5) and primer length (claim 6). However, these limitations were known in the prior art and taught by Hatch et al.
Relevant to claim 5, Hatch et al. teaches “PCR Efficiency Based Temporal Shift Using Same Fluorescence Intensity and Reporter. Multiplexing ability can also be increased by modifying temporal behavior of signal intensity changes. PCR efficiencies occur at different rates based on a variety of factors, including, but not limited to: primer probe length; specific sequence; how many mismatches there are; annealing temperatures; and additives which may stabilize or destabilize TaqMan polymerase, annealing, or DNA folding. Here we would like to emphasize changes which are specific to the primer and probe designs themselves and not to TaqMan polymerase and amplification efficiency in general. For example, if a primer sequence, pp1, is implemented to anneal at 70 C. and below, a second sequence, pp2, that anneals poorly at 65 C. but anneals well at 60 C. and below, and a third, pp3, that anneals poorly at 60 C. but anneals well at 55 C. and below, three different polymerization efficiencies based on the primer pair sequences alone is achieved. If the annealing temperatures are started out at a higher temperature for the first five or ten cycles, say 65 C., then reduced to 60 C. for the remaining cycles, three different temporal cues may be achieved to differentiate primer pairs" (paragraph 0067).
This teaching reads on claim 5 wherein the first ID-Primer and the second ID-Primer have a melting temperature that is about 5°C to about 15°C or about 10°C different.
Relevant to claim 6, Hatch et al. teaches "Multiplexing ability can also be increased by modifying temporal behavior of signal intensity changes. PCR efficiencies occur at different rates based on a variety of factors, including, but not limited to: primer probe length" (paragraph 0067).
This teaching reads on claim 6 where the first and second ID-Primers have a different length from each other of least 5, 6, 7, 8, 9, 10, 15, 20 or more nucleotides.
(ii) Peng et al. teaches limitations relevant to claims 22-25.
Relevant to claims 22-25, Peng et al. teaches "The workflow is as the following (Fig. 1)… 2) The unused BC primers are removed through size selection purification" (page 3, column 2, paragraph 2). Further relevant to claims 22-25, Peng et al. Supplementary Figure S1 teaches that the amplicon sizes were detected via gel electrophoresis.
These teachings, in addition to the Peng et al. teachings relevant to the rejections of claims 1-4, read on the limitations of claims 22-25 wherein a size separation technique is used to detect the target nucleic acid sequence and size of amplicons via gel electrophoresis.
(iii) Although Peng et al. is silent to the Hatch et al. melting temperature and primer length limitations, it would have been prima facie obvious to the skilled artisan. It is noted that Peng et al. and Hatch et al. are analogous disclosures to the instant nucleic acid detection method.
The skilled artisan would have been motivated to combine the analogous disclosures and include the Hatch et al. limitations within the Peng et al. methodology. Hatch et al. teaches “Multiplexing ability can also be increased by modifying temporal behavior of signal intensity changes” and that primer/probe annealing/melting temperatures and length impact said multiplexing ability (paragraph 0067). Thus, the skilled artisan would be motivated to include the Hatch et al. teachings within the Peng et al. methodology in order to increase the multiplexing ability.
The skilled artisan would have a reasonable expectation of success based on the disclosures of Peng et al. and further in view of Hatch et al., as discussed in the preceding paragraphs.
Claims 7-8, 11-12, 14, and 17-19 are rejected under 35 U.S.C. 103 as being unpatentable over Peng et al. (2015; "Reducing amplification artifacts in high multiplex amplicon sequencing by using molecular barcodes"; BMC Genomics 16, 589. https://doi.org/10.1186/s12864-015-1806-8), as applied to claims 1-4 above, and further in view of Whitman et al. (2008; US 2008/0305481 A1; publication of US application 11/956,257, which claims the priority of provisional application 60/869,742 filed on 12/13/06).
The teachings of Peng et al. are applied to instantly rejected claims 7-8, 11-12, 14, and 17-19 as they were applied to claims 1-4 as anticipating a method of sample pooling of at least two samples.
(i) Peng et al. is silent to specifics regarding RT-PCR (claim 7) and fluorophore/quencher probes (claims 8, 11-12, 14, and 17-19). However, these limitations were known in the prior art and taught by Whitman et al.
Relevant to claim 7, Whitman et al. teaches "Real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (QRT-PCR) or kinetic polymerase chain reaction, is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of a specific sequence in a DNA sample. Real-time PCR may be combined with reverse transcription polymerase chain reaction to quantify low abundance RNAs" (paragraph 0094).
This teaching reads on claim 7 wherein detecting the target nucleic acid sequence comprises using real time-polymerase chain reaction (RT-PCR).
Relevant to claims 8 and 11-12, Whitman et al. teaches "Multiplex-PCR and multiplex real-time PCR use of multiple, unique primer sets within a single PCR reaction to produce amplicons of different DNA sequences" (paragraph 0095).
Further relevant to claims 8 and 11-12, Whitman et al. teaches "As discussed above, one primer of a primer pair used in an amplification reaction comprises a tag sequence. Following the initial extension of the primer comprising the tag sequence, the tagged extension product may serve as a template for the other primer of the primer pair" (paragraph 0154).
The Whitman et al. "tag sequence" reads on the first and second unique regions, wherein the F probes are complementary to the first/second unique region of the first/second ID-primer and are able to bind via complementarity.
Further relevant to claims 8 and 11-12, Whitman et al. teaches "Other embodiments employ the use of two probes per target nucleic acid sequence. One probe is attached to a bead and may be labeled with a fluorophore. A complimentary 'free-floating' probe is also added that contains either a quencher or a fluorophore pair such that FRET may occur. In this instance, the probe on the bead may contain a fluorophore at the 3' end, while being attached to the bead at the 5' end. The free-floating probe will be able to quench the signal associated with the fluorophore on the bead by being designed as a reverse compliment of the same, and having a quenching moiety at its 5' end, such that it is in close proximity to the fluorophore, and therefore achieves a decrease in signal upon hybridization to the probe. These probes will also be present during PCR amplification or some other amplification event. At the target detection phase of the PCR cycle, the free-floating probe will leave the probe attached to the bead and will hybridize to the target sequence" (paragraph 0141).
These teachings read on claim 8 wherein detecting the target nucleic acid sequence comprises…; claim 11 wherein the fluorescent label of F probe is labelled at the 3’end; and claim 12 wherein the quencher of Q probe is labelled at the 5’end.
Relevant to claim 14, Whitman et al. teaches "The 3' end of the immobilized probe may be blocked (with a phosphate group or a 3' inverted dT, for example) to prevent the polymerase extension of the probe" (paragraph 0146).
This teaching reads on claim 14 wherein the Q probe has a 3’ inverted dT.
Relevant to claims 17-19, Whitman et al. teaches "Multiplex-PCR and multiplex real-time PCR use of multiple, unique primer sets within a single PCR reaction to produce amplicons of different DNA sequences" (paragraph 0095).
Further relevant to claims 17-19, Whitman et al. teaches "As discussed above, one primer of a primer pair used in an amplification reaction comprises a tag sequence. Following the initial extension of the primer comprising the tag sequence, the tagged extension product may serve as a template for the other primer of the primer pair" (paragraph 0154).
The Whitman et al. "tag sequence" reads on the first and second unique regions, wherein the F probes are complementary to the first/second unique region of the first/second ID-primer and are able to bind via complementarity.
Further relevant to claims 17-19, Whitman et al. teaches "Other embodiments employ the use of two probes per target nucleic acid sequence. One probe is attached to a bead and may be labeled with a fluorophore. A complimentary 'free-floating' probe is also added that contains either a quencher or a fluorophore pair such that FRET may occur. In this instance, the probe on the bead may contain a fluorophore at the 3' end, while being attached to the bead at the 5' end. The free-floating probe will be able to quench the signal associated with the fluorophore on the bead by being designed as a reverse compliment of the same, and having a quenching moiety at its 5' end, such that it is in close proximity to the fluorophore, and therefore achieves a decrease in signal upon hybridization to the probe. These probes will also be present during PCR amplification or some other amplification event. At the target detection phase of the PCR cycle, the free-floating probe will leave the probe attached to the bead and will hybridize to the target sequence" (paragraph 0141).
Further relevant to claims 17-19, Whitman et al. teaches "During the extension or elongation phase of the PCR reaction, a polymerase known as Taq polymerase is used because of its 5' exonuclease activity. The polymerase uses the upstream primer as a binding site and then extends. Hydrolysis probes are cleaved during polymerase extension at their 5' end by the 5'-exonuclease activity of Taq. When this occurs, the reporter fluorophore is released from the probe, and subsequently, is no longer in close proximity to the quencher" (paragraph 0116).
These teachings read on claim 17 wherein detecting the target nucleic acid sequence comprises…; claim 18 wherein the detecting the target nucleic acid sequence comprises…; and claim 19 wherein the fluorescent label of the first or second probe is labelled at the 3’ end and the quencher of the first or second probe is labelled at the 5’ end.
Although Peng et al. is silent to the Whitman et al. RT-PCR and fluorophore/quencher probe limitations, it would have been prima facie obvious to the skilled artisan. It is noted that Peng et al. and Whitman et al. are analogous disclosures to the instant nucleic acid detection method.
The skilled artisan would have been motivated to combine the analogous disclosures and include the Whitman et al. limitations within the Peng et al. methodology. Whitman et al. teaches that “Real-time PCR is also advantageous over end-point detection in that contamination is limited because it can be performed in a closed system. Other advantages include greater sensitivity, dynamic range, speed, and fewer processes required” (paragraph 0005). Thus, the skilled artisan would be motivated to include the Whitman et al. RT-PCR within the Peng et al. because of the Whitman et al.-taught advantages of RT-PCR (sensitivity, range, speed, etc.).
The skilled artisan would have been further motivated to include the Whitman et al. fluorophore/quencher probe limitations within the Peng et al. methodology because Whitman et al. teaches “There are several commercially available nucleic acid detection chemistries currently used in real-time PCR. These chemistries include DNA binding agents, FRET based nucleic acid detection, hybridization probes, molecular beacons, hydrolysis probes, and dye-primer based systems” (paragraph 0097). Thus, the skilled artisan would be motivated to include the Whitman et al. fluorophore/quencher probe limitations within the Peng et al. methodology because Whitman et al. teaches that those detection chemistries are “commercially available” and already in use in RT-PCR assays.
The skilled artisan would have a reasonable expectation of success based on the disclosures of Peng et al. and further in view of Whitman et al., as discussed in the preceding paragraphs.
Claims 9-10, 13, and 15-16 are rejected under 35 U.S.C. 103 as being unpatentable over Peng et al. (2015; "Reducing amplification artifacts in high multiplex amplicon sequencing by using molecular barcodes"; BMC Genomics 16, 589. https://doi.org/10.1186/s12864-015-1806-8) in view of Whitman et al. (2008; US 2008/0305481 A1; publication of US application 11/956,257, which claims the priority of provisional application 60/869,742 filed on 12/13/06), as applied to claims 7-8, 11-12, 14, and 17-19 above, and further in view of Hatch et al. (2013; US 2013/0178378 A1; publication of US application 13/492,698, which claims the priority of provisional application 61/495,308 filed on 6/9/11).
The teachings of Peng et al. in view of Whitman et al. are applied to instantly rejected claims 9-10, 13, and 15-16 as they were applied to claims 7-8, 11-12, 14, and 17-19 as rendering obvious a method of sample pooling of at least two samples.
(i) Peng et al. in view of Whitman et al. teaches limitations relevant to claims 9-10 and 15-16.
Relevant to claims 9-10, Whitman et al. teaches "Multiplex-PCR and multiplex real-time PCR use of multiple, unique primer sets within a single PCR reaction to produce amplicons of different DNA sequences" (paragraph 0095).
Further relevant to claims 9-10, Whitman et al. teaches "As discussed above, one primer of a primer pair used in an amplification reaction comprises a tag sequence. Following the initial extension of the primer comprising the tag sequence, the tagged extension product may serve as a template for the other primer of the primer pair" (paragraph 0154).
The Whitman et al. "tag sequence" reads on the first and second unique regions, wherein the F probes are complementary to the first/second unique region of the first/second ID-primer and are able to bind via complementarity.
Further relevant to claims 9-10, Whitman et al. teaches "Other embodiments employ the use of two probes per target nucleic acid sequence. One probe is attached to a bead and may be labeled with a fluorophore. A complimentary 'free-floating' probe is also added that contains either a quencher or a fluorophore pair such that FRET may occur. In this instance, the probe on the bead may contain a fluorophore at the 3' end, while being attached to the bead at the 5' end. The free-floating probe will be able to quench the signal associated with the fluorophore on the bead by being designed as a reverse compliment of the same, and having a quenching moiety at its 5' end, such that it is in close proximity to the fluorophore, and therefore achieves a decrease in signal upon hybridization to the probe. These probes will also be present during PCR amplification or some other amplification event. At the target detection phase of the PCR cycle, the free-floating probe will leave the probe attached to the bead and will hybridize to the target sequence" (paragraph 0141).
The Whitman et al. teaching of the "PCR amplification or some other amplification event" includes the claim 10 recited limitations as Whitman et al. teaches that "A basic PCR reaction requires several components and reagents including: a DNA template that contains the target sequence to be amplified; one or more primers, which are complementary to the DNA regions at the 5' and 3' ends of the target sequence; a DNA polymerase (e.g., Taq polymerase) that preferably has a temperature optimum at around 70° C.; deoxynucleotide triphosphates (dNTPs); a buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase; divalent cations, typically magnesium ions (Mg2+); and monovalent cation potassium ions" (paragraph 0092).
Relevant to claims 15-16, Peng et al. Figure 1 teaches that the reverse primer is complementary to the amplifier region of a representative Primer-ID, and the forward primer is complementary to the target nucleic acid sequence.
(ii) Peng et al. in view of Whitman et al. is silent to specifics regarding increasing the temperature of the reaction mixture (claim 9), reaction contents (claim 10), and melting temperature of the Q probe (claim 13). However, these limitations were known in the prior art and taught by Hatch et al.
relevant to claim 9, Hatch et al. teaches "According to another aspect of the claimed subject matter, multiplexing ability is expanded with temporal signatures by delaying signal amplification, becoming even more versatile, efficient, and cost effective from a reagent perspective. In an embodiment, this technique may be utilized with real-time analysis to determine temporal cues in the PCR amplification process. This technique further allows for increased flexibility in performance and selectivity when considering cycle thresholding applications. Varying methods for delaying signal amplification may be employed, according to various embodiments… Other examples of methods to delay signal amplification include, but not are not limited to: primer/probe design, dynamic annealing temperatures during thermocycling, PCR efficiency control, limiting reagents, type of reporter used for detection, and so forth, as described below in greater detail" (paragraph 0063).
This teaching reads on claim 9 step iv) increasing the temperature of the reaction mixture until the respective F probe is released from the single-stranded amplicon.
Relevant to claim 13, Hatch et al. teaches "PCR Efficiency Based Temporal Shift Using Same Fluorescence Intensity and Reporter. Multiplexing ability can also be increased by modifying temporal behavior of signal intensity changes. PCR efficiencies occur at different rates based on a variety of factors, including, but not limited to: primer probe length; specific sequence; how many mismatches there are; annealing temperatures; and additives which may stabilize or destabilize TaqMan polymerase, annealing, or DNA folding. Here we would like to emphasize changes which are specific to the primer and probe designs themselves and not to TaqMan polymerase and amplification efficiency in general. For example, if a primer sequence, pp1, is implemented to anneal at 70 C. and below, a second sequence, pp2, that anneals poorly at 65 C. but anneals well at 60 C. and below, and a third, pp3, that anneals poorly at 60 C. but anneals well at 55 C. and below, three different polymerization efficiencies based on the primer pair sequences alone is achieved. If the annealing temperatures are started out at a higher temperature for the first five or ten cycles, say 65 C., then reduced to 60 C. for the remaining cycles, three different temporal cues may be achieved to differentiate primer pairs" (paragraph 0067).
The Hatch et al. range of annealing temperatures read on claim 13 limitations.
(iii) Although Peng et al. in view of Whitman et al. is silent to the Hatch et al. increasing the temperature of the reaction mixture, reaction contents, and melting temperature of the Q probe limitations, it would have been prima facie obvious to the skilled artisan. It is noted that Peng et al., Whitman et al., and Hatch et al. are all analogous disclosures to the instant nucleic acid detection method.
The skilled artisan would have been motivated to combine the analogous disclosures and include the Hatch et al. limitations within the Whitman et al.-modified Peng et al. methodology. Hatch et al. teaches that “multiplexing ability is expanded with temporal signatures by delaying signal amplification, becoming even more versatile, efficient, and cost effective from a reagent perspective. In an embodiment, this technique may be utilized with real-time analysis to determine temporal cues in the PCR amplification process. This technique further allows for increased flexibility in performance and selectivity when considering cycle thresholding applications. Varying methods for delaying signal amplification may be employed, according to various embodiments… Other examples of methods to delay signal amplification include, but not are not limited to: primer/probe design, dynamic annealing temperatures during thermocycling, PCR efficiency control, limiting reagents, type of reporter used for detection, and so forth, as described below in greater detail" (paragraph 0063). Thus, the skilled artisan would be motivated to include the Hatch et al. limitations within the Whitman et al.-modified Peng et al. methodology because of the Hatch et al.-taught advantages of expanded multiplexing ability (versatility, efficiency, etc.).
The skilled artisan would have a reasonable expectation of success based on the disclosures of Peng et al. in view of Whitman et al., and further in view of Hatch et al., as discussed in the preceding paragraphs.
Claims 20-21 are rejected under 35 U.S.C. 103 as being unpatentable over Peng et al. (2015; "Reducing amplification artifacts in high multiplex amplicon sequencing by using molecular barcodes"; BMC Genomics 16, 589. https://doi.org/10.1186/s12864-015-1806-8), as applied to claims 1-4 above, and further in view of Piepenburg et al. (2018; US 10,036,057 B2).
The teachings of Peng et al. are applied to instantly rejected claims 20-21 as they were applied to claims 1-4 as anticipating a method of sample pooling of at least two samples.
Peng et al. is silent to specifics regarding isothermal amplification and detection (claims 20-21). However, these limitations were known in the prior art and taught by Piepenburg et al.
Relevant to claims 20-21, Piepenburg et al. Abstract teaches "This disclosure describes related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of recombinase and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods have the advantage of not requiring thermocycling or thermophilic enzymes, thus offering easy and affordable implementation and portability relative to other amplification methods."
This teaching reads on claim 20 wherein detecting the target nucleic acid sequence comprises nucleic acid isothermal amplification and detection; and claim 21 wherein the nucleic acid isothermal amplification and detection is… Recombinase Polymerase Amplification (RPA).
Although Peng et al. is silent to the Piepenburg et al. isothermal amplification and detection, it would have been prima facie obvious to the skilled artisan. It is noted that Peng et al. and Piepenburg et al. are analogous disclosures to the instant nucleic acid detection method.
The skilled artisan would have been motivated to combine the analogous disclosures and include the Piepenburg et al. limitations within the Peng et al. methodology. Pienpenburg et al. Abstract teaches that recombinase polymerase amplification advantageously does not require thermocycling or thermophilic enzymes (and is thus easy, affordable, and portable). Thus, the skilled artisan would be motivated to include the Piepenburg et al. within the Peng et al. methodology because of the Piepenburg et al.-taught advantages.
The skilled artisan would have a reasonable expectation of success based on the disclosures of Peng et al., and further in view of Piepenburg et al., as discussed in the preceding paragraphs.
Claims 26-30 are rejected under 35 U.S.C. 103 as being unpatentable over Peng et al. (2015; "Reducing amplification artifacts in high multiplex amplicon sequencing by using molecular barcodes"; BMC Genomics 16, 589. https://doi.org/10.1186/s12864-015-1806-8), as applied to claims 1-4 above, and further in view of Steelman (2015; WO 2015/195949 A2).
The teachings of Peng et al. are applied to instantly rejected claims 26-30 as they were applied to claims 1-4 as anticipating a method of sample pooling of at least two samples.
Peng et al. is silent to specifics regarding electrochemical detection (claims 26-30). However, these limitations were known in the prior art and taught by Steelman.
Relevant to claims 26-28, Steelman teaches "The detecting may comprise use of a reporter to identify or quantify the amplicon. The reporter may be selected from a fluorescent reporter, a visual reporter, an electrochemical reporter, a luminescent reporter, a colorometric reporter, turbidity, a fluorescent hybridization-based detector, and an electrochemical hybridization-based detector" (paragraph 010).
Relevant to claim 29, Steelman teaches "The nucleic acid detection unit may detect the target nucleic acid(s) in real-time, e.g., during the course of the amplification reaction, and/or may comprise endpoint detection, e.g., following termination of an amplification reaction" (paragraph 0164).
Relevant to claim 30, Steelman teaches "The electrochemical reporter may be methylene blue" (paragraph 010).
Although Peng et al. is silent to the Steelman electrochemical detection, it would have been prima facie obvious to the skilled artisan. Peng et al. and Steelman are analogous disclosures to the instant nucleic acid detection method.
The skilled artisan would have been motivated to combine the analogous disclosures and include the Steelman limitations within the Peng et al. methodology. Steelman teaches “Electrochemical (EC) detection of biologic species or electrochemical sensor is based on electrochemical reactions that occur during biorecognition reactions… An EC sensor may also be in situ, which incorporates all the sample processing steps ‘on-chip,’ and may be more desirable for clinical applications, such as point-of-care diagnosis” (paragraph 0154). Thus, the skilled artisan would be motivated to include the Steelman electrochemical detection in order to take advantage of the point-of-care diagnostic clinical applications.
The skilled artisan would have a reasonable expectation of success based on the disclosures of Peng et al., and further in view of Steelman, as discussed in the preceding paragraphs.
Conclusion
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/SARAH JANE KENNEDY/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682