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 .
Election/Restrictions
Applicant’s election without traverse of Group I (claims 1-6, 9, 12, 15-18, 32-34, 36-37, and 40-41) in the reply filed on 2/9/2026 is acknowledged.
Claims 20-23, 27-28, and 43-45 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
It is noted that Applicant has not changed the claim status identifiers of the withdrawn claims to read (Withdrawn). Applicant must apply the correct status to each claim in their next response. See MPEP 714 II (C).
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 5/22/2023 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Nucleotide and/or Amino Acid Sequence Disclosures
Summary of Requirements for Patent Applications Filed On Or After July 1, 2022, That Have Sequence Disclosures
37 CFR 1.831(a) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.831(b) must contain a “Sequence Listing XML”, as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.831-1.835. This “Sequence Listing XML” part of the disclosure may be submitted:
1. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter “Legal Framework”) in XML format, together with an incorporation by reference statement of the material in the XML file in a separate paragraph of the specification (an incorporation by reference paragraph) as required by 37 CFR 1.835(a)(2) or 1.835(b)(2) identifying:
a. the name of the XML file
b. the date of creation; and
c. the size of the XML file in bytes; or
2. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation by reference statement of the material in the XML format according to 37 CFR 1.52(e)(8) and 37 CFR 1.835(a)(2) or 1.835(b)(2) in a separate paragraph of the specification identifying:
a. the name of the XML file;
b. the date of creation; and
c. the size of the XML file in bytes.
SPECIFIC DEFICIENCIES AND THE REQUIRED RESPONSE TO THIS NOTICE ARE AS FOLLOWS:
Specific deficiency - This application fails to comply with the requirements of 37 CFR 1.831-1.834 because it does not contain a “Sequence Listing XML” as a separate part of the disclosure. A “Sequence Listing XML” is required because the present application was filed on or after July 1, 2022. It is noted that Applicant has provided a Sequence Listing in ASCII text format, but this was a requirement under ST.25, not ST.26, the latter of which the current application currently falls under.
Required response - Applicant must provide:
• A “Sequence Listing XML” part of the disclosure, as described above in item 1. or 2.; together with
o A statement that indicates the basis for the amendment, with specific references to particular parts of the application as originally filed, as required by 37 CFR 1.835(a)(3);
o A statement that the “Sequence Listing XML” includes no new matter as required by 37 CFR 1.835(a)(4)
AND
• A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 inserting the required incorporation by reference paragraph as required by 37 CFR 1.835(a)(2), consisting of:
o A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
o A copy of the amended specification without markings (clean version); and
o A statement that the substitute specification contains no new matter.
Claim Objections
Claim 1 is objected to because of the following informalities: in step (d), “hybridized to SARS-CoV-2 RNA” should read “hybridized to the SARS-CoV-2 target sequence” to better match the language used earlier in the claim. Additionally, between steps (e) and (f), “; and” should be used, rather than a comma. Finally, in the final wherein clause, line 1 reads “in a pooled sampled” but should read “in the pooled sample.” Appropriate correction is required.
Claim 2 is objected to because of the following informalities: in line 2 of step (g), “the asymmetric PCR” should read “the asymmetric RNaseH-dependent PCR.” Additionally, in lines 3-4 of this step, “patient-specific barcode sequence” should read “patient-specific identifying barcode sequence” to match the language used in claim 1. Similarly, “patient-specific identifier sequences” in lines 1-2 of step (h) should read “patient-specific identifying barcode sequences.” Finally, the comma before “and” in the final line of step (g) should be removed. Appropriate correction is required.
Claim 3 is objected to because of the following informality: there should be a comma after “The method of claim 1.” Appropriate correction is required.
Claim 6 is objected to because of the following informalities: wherever the phrase “patient-specific barcode sequence(s)” appears, this phrase should read “patient-specific identifying barcode sequence(s)” to better match the language used in claim 1. Appropriate correction is required.
Claim 9 is objected to because of the following informalities: in line 1, “the amplification reaction” should read “the amplifying” or “the amplifying of step (e)” to better align with the language of claim 1. Additionally, in line 2, “each of the three oligonucleotides” should read “each of the three collinear oligonucleotides” and similarly, in line 3, “the oligonucleotide” should read the colinear oligonucleotide.” Also in line 3, “the 5’ most position” should read “the 5’ most position on the SARS-CoV-2 RNA target sequence” for additional clarity. Finally, in lines 3-5, each instance of “patient-specific identifier sequence” should read “patient-specific identifying barcode sequence” to better match the language used in claim 1. Appropriate correction is required.
Claim 12 is objected to because of the following informality: in lines 1-2, “the three oligonucleotides” should read “the three collinear oligonucleotides.” Appropriate correction is required.
Claim 15 is objected to because of the following informalities: in line 1, “the oligonucleotide” should read “the collinear oligonucleotide.” In line 2, “to the most 5’ position” should read “to the most 5’ position on the SARS-CoV-2 RNA target sequence.” Appropriate correction is required.
Claim 16 is objected to because of the following informalities: in line 1, “the oligonucleotide” should read “the collinear oligonucleotide.” In line 2, “hybridizes in the 5’ most position” should read “hybridizes to the 5’ most position on the SARS-CoV-2 RNA target sequence.” Also in line 2, “patient-specific identifier sequence” should read “patient-specific identifying barcode sequence.” Finally, in lines 2-3, the phrase “at least said 5’-most oligonucleotide” can be removed. Appropriate correction is required.
Claim 17 is objected to because of the following informality: in line 2, “the hybridized complex” should read “the oligonucleotide-SARS-CoV-2 RNA complex,” as this is the language used for the complex in claim 1, from which this claim depends. . Appropriate correction is required.
Claim 32 is objected to because of the following informalities: in step (d), “hybridized to viral RNA” should read “hybridized to the ssRNA virus target sequence” to better match the language used earlier in the claim. Additionally, between steps (e) and (f), “; and” should be used, rather than a comma. Finally, in the final wherein clause, line 1 reads “in a pooled sampled” but should read “in the pooled sample.” Appropriate correction is required.
Claim 33 is objected to because of the following informalities: in line 2 of step (g), “the asymmetric PCR” should read “the asymmetric RNaseH-dependent PCR.” Additionally, in lines 3-4 of this step, “patient-specific barcode sequence” should read “patient-specific identifying barcode sequence” to match the language used in claim 32. Similarly, “patient-specific identifier sequence” in lines 1-2 of step (h) should read “patient-specific identifying barcode sequence.” Finally, the comma before “and” in the final line of step (g) should be removed. Appropriate correction is required.
Claim 37 is objected to because of the following informalities: wherever the phrase “patient-specific barcode sequence(s)” appears, this phrase should read “patient-specific identifying barcode sequence(s)” to better match the language used in claim 32. Appropriate correction is required.
Claim 41 is objected to because of the following informalities: in lines 1-2, “the three oligonucleotides” should read “the three collinear oligonucleotides.” In line 2, “the oligonucleotide” should read “the colinear oligonucleotide.” In lines 2-3, “the 5’ most position” should read “the 5’ most position on the ssRNA virus target sequence” for additional clarity. Finally, in lines 3-4, each instance of “patient-specific identifier sequence” should read “patient-specific identifying barcode sequence” to better match the language used in claim 32. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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 6, 12, 16, and 36-37 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 6 and 37 are rejected because terms “the 5’-most collinear oligonucleotide” and “the 3’-most collinear oligonucleotide” are unclear and render the structure of the collinear oligonucleotides indefinite relative to the structure described in claim 1 (from which claim 6 is dependent) or claim 32 (from which claim 37 is dependent). Specifically, it is unclear whether these refer to ends of the collinear oligonucleotides that would have 5’ or 3’ ends free, respectively (i.e. unligated 5’ or 3’ ends), or whether these refer to hybridizing to the 5’ and 3’ ends of the target sequence. Because the collinear oligonucleotides must hybridize to adjacent locations on the target sequence, if the former interpretation is used, “the 5’-most collinear oligonucleotide” would then have the patient-specific barcode at its 3’ end, which would prevent adjacent hybridization of the next collinear oligonucleotide to the target. Similarly, “the 3’-most collinear oligonucleotide” would then have the patient-specific barcode at its 5’ end, which would prevent adjacent hybridization of the next collinear oligonucleotide to the target. Thus, the latter interpretation will be used. This interpretation is also supported by Figure 4 of the instant specification, which shows the barcode sequence on the outer, unligated end of the collinear oligonucleotide.
Claim 12 is rejected for similar reasons. In lines 2-3, the phrase “the 3’-most oligonucleotide” is used, where this oligonucleotide has a moiety at its 5’ end. It is unclear whether “the 3’-most oligonucleotide” refers to end of the collinear oligonucleotide that would have a 3’ end free (i.e. an unligated 3’ end), or whether this refers to hybridizing to the 3’ end of the target sequence. Because the collinear oligonucleotides must hybridize to adjacent locations on the SARS-CoV-2 target sequence, if the former interpretation is used, “the 3’-most collinear oligonucleotide” would then have the moiety at its 5’ end, which would potentially interfere with said adjacent hybridization of the next collinear oligonucleotide to the target and/or oligonucleotide ligation. Thus, the latter interpretation will be used.
Claim 16 recites “the SARS-CoV-2 nucleic acid” in lines 3-4. There is insufficient antecedent basis in this claim, as “SARS-CoV-2 nucleic acid” is not generally recited in claim 1, and it is unclear if this nucleic acid is referring to the target sequence, or all of the SARS-CoV-2 genomic RNA potentially present in a sample. For the purposes of applying prior art, the latter interpretation will be used.
Claim 36 recites “the SARS-CoV-2 RNA target sequence” and “the SARS-CoV-2 genome” in lines 1-2. These phrases lack antecedent basis, as claim 32 only discusses target sequences for an ssRNA virus, and “a SARS-CoV-2 RNA target sequence” and “a SARS-CoV-2 genome” are not described. In interpreting this claim, these terms will be treated as though they read “the ssRNA virus target sequence” and “the ssRNA virus genome,” which both relate to terms presented in claim 32.
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 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.
Claims 1, 3-4, 6, 9, 12, 16-17, 32, 34, 37, and 40 are rejected under 35 U.S.C. 103 as being unpatentable over Larman et al. (US 2018/0208967 A1), in view of Naqvi et al. (BBA-Molecular Basis of Disease, 2020), and in view of Lee et al. (WO 2020/056381 A1).
Larman teaches methods of RNA analysis using one or more multi-partite probes (Abstract). Each multi-partite probe includes at least two sub-probes, meaning that more than two sub-probes (e.g. three sub-probes, and see para. 13, which specifically mentions three sub-probes) can be used. The multi-partite probe is able to anneal to a target nucleic acid (which is preferably RNA) in a sample (para. 4). After hybridization, a washing step can be performed (para. 48). Such washing would remove excess probes, and so would purify the hybridized complexes. The sub-probes are then ligated to one another and removed from the target nucleic acid, creating a “target nucleic acid proxy” (para. 4). After removal, amplification of the proxy can occur (para. 5). The proxy may then be detected (para. 9). The methods of Larman may be used on a viral target such as SARS-CoV, and the releasing, amplifying, and sequencing steps identify the virus of interest (paras. 15 and 18). The sub-probes within the multi-partite probe may be adjacent and continuous with one another, with no unbound nucleotides between the sub-probes (para. 91).
Though Larman states that SARS-CoV viruses can be examined, the reference does not specify the identification of SARS-CoV-2.
Naqvi highlights the prevalence and dangers associated with SARS-CoV-2 and resulting COVID-19 infection, particularly noting mortality and potential for systemic inflammation that can lead to potentially fatal complications (page 1, “Introduction”). The reference also highlights the fact that coronaviruses such as SARS-CoV-2 have single-stranded RNA genomes (page 2, column 1, para. 2). Naqvi also shows that the genome of this virus has been successfully sequenced (Abstract, Figure 2, and pages 3-4, “3. Genome structure”).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Naqvi to specifically examine SARS-CoV-2 in the method of Larman. Naqvi highlights the importance of detecting this virus by showing the threat it poses to humans, and provides a reasonable expectation of success by showing that the genome of the virus is known, so that proper probes can be designed. Additionally, this would simply be examining a specific type of SARS-CoV virus, which is already recited in the methods of Larman.
Para. 90 of Larman states that PCR products from multiple samples can be pooled together for sequencing, where samples can be labeled with individual barcodes. Such barcoding/pooling is also described in para. 102 and 114. However, Larman does not state that one of the sub-probes can have such a barcode before amplification, nor does the pooling occur before the washing/ligating/later downstream steps.
Lee teaches methods for detecting particular RNAs using hybridization and ligation assays (Abstract). Specifically, this involves the ligation of a primer and one or two probes together (paras. 14-15). Lee notes that the probes may contain barcodes (para. 99), where said barcode is used to identify the sample of origin (para. 206). The ligation can occur at the 5’ or 3’ end of the probes (paras. 15-16). The ordinary artisan would recognize that to not interfere with the probe hybridization to the target and the ligation with the primers, the barcode would need to be on the end opposite to where the ligation is occurring.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Lee to add sample of origin barcodes to at least one of the probes of Larman in view of Naqvi. Larman teaches a probe decoy strategy in para. 90. This strategy involves the targeting probe sets having a common primer binding sequence, and then upon amplification, barcodes are added to the probe products via primers. This allowed samples to then be pooled. However, this method also requires performing multiple separate ligation and amplification reactions for each sample, which is time consuming, particularly because the same target sequences are being analyzed for each sample. Thus, Lee, which teaches a similar assay involving ligation and hybridization, but where probes can contain a sample barcode, would be of interest to the ordinary artisan. This sample barcode could be included in the probes in addition to the common primer binding sequence on one of the outer probes (i.e. not the middle probe that is ligated at either end). As the amplification primers of Larman already contain sample barcodes, these same barcodes could be used, and during amplification, they would then hybridize to the probe sequences along with the common sequences, alleviating the need for developing new primer sequences, while still allowing the primers to be used together in multiplex fashion. The hybridized probe/target complexes would thus already be barcoded with a sample-specific barcode, and so could be pooled prior to ligation and amplification, simplifying these reactions and requiring less reagents for use overall, saving both time and resources. There would be a reasonable expectation of success as the development of barcodes associated with primers/probes is well-known in the art, as evidenced by Larman and Lee, and this change would not affect the actual operation of the multi-partite probe.
As to when the pooling of the multiple samples may occur relative to the washing step described by Larman, similar reasoning would apply to perform a washing step after the pooling of samples. A single washing step would eliminate the need for each sample to undergo a washing step separately, which would save time and resources. As the actual method of the washing would not be changed, there would be a reasonable expectation of success.
Thus, claims 1 and 32 are prima facie obvious over Larman, in view of Naqvi, and in view of Lee.
Regarding claims 3-4 and 34, Larman teaches that the ligase used can be PBCV-1 (paras. 6, 20, 25, and 95).
Regarding claims 6 and 37, the specific outer probe of the multi-partite probe that contains the sample-specific barcode is not specified in Larman, in view of Naqvi, and in view of Lee. As the entirety of the probe is later amplified and sequenced, and the barcode does not directly interfere with the detection of the viral sequence, it would be equally prima facie obvious to include the barcode on either of these probe sequences.
Regarding claims 9 and 40, Larman generally teaches that the amplification used in their methods can be PCR (paras. 12, 19, and 52). Para. 141 specifically notes the quantification associated with multiplex probe ligation being performed with qPCR. Thus, it would be prima facie obvious that in the method described above in the rejection of claims 1 and 32, qPCR could be used to quantify the SARS-CoV-2 ligation probe products.
Thus, claims 9 and 40 are prima facie obvious over Larman, in view of Naqvi, and in view of Lee.
Regarding claim 12, it is noted that the term “purification moiety” is not specifically defined by the instant specification. This term will be interpreted as a moiety that can either be directly used for purification or which protects an oligonucleotide from being removed in a purification process. It is noted that this moiety does not need to be used, and is not explicitly linked to the purification step of instant claim 1.
Larman teaches that the target nucleic acid proxy may be modified (para. 4). In paras. 22 and 26, the reference teaches the use of solid-phase amplification. In para. 32, the reference notes that biotin can be a useful label for a molecule of interest, and paras. 71 and 125 teach the use of streptavidin on a solid surface that can be used to capture biotinylated sequences. MPEP 2141.03 I states, “"A person of ordinary skill in the art is also a person of ordinary creativity, not an automaton." KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 421, 82 USPQ2d 1385, 1397 (2007). "[I]n many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle." Id. at 420, 82 USPQ2d 1397. Office personnel may also take into account "the inferences and creative steps that a person of ordinary skill in the art would employ." Id. at 418, 82 USPQ2d at 1396.” Thus, the ordinary artisan would be capable of putting the various teachings of Larman together to use biotin to label the multi-partite probe, and to use streptavidin on a solid-surface to aid in capture of said biotinylated probe for amplification after its removal from the target nucleic acid sequence. This biotinylating would allow for ease of detection of the probe and effective capture on the solid-surface. As to which end of the multi-partite probe would contain this biotin, the presence of the moiety at either end of the probe would not affect primer binding to said probe, as no nucleotides would be blocked from being hybridized to, and so it would be equally prima facie obvious to include the moiety at either end of the probe. There would be a reasonable expectation of success as attaching biotin to oligonucleotide sequences is well-known in the art, as evidenced by Larman.
Thus, claim 12 is prima facie obvious over Larman, in view of Naqvi, and in view of Lee.
Regarding claim 16, the specific outer probe of the multi-partite probe that contains the sample-specific barcode is not specified in Larman, in view of Naqvi, and in view of Lee. As the entirety of the probe is later amplified and sequenced, and the barcode does not directly interfere with the detection of the viral sequence, it would be equally prima facie obvious to include the barcode on either of the outer probe sequences.
As to the concentration limitations of the claim, Larman states that for particular ligation reactions, all components should be provided in “excess” over the target mRNA (para. 94). Lee teaches the concept of “saturating” a target sequence with primers and probes (para. 201). Lee also provides examples where primers or probes are given in excess of a target, such as 3-fold excess (paras. 292 and 322). Thus, in order to ensure that viral target nucleic acids, which may not be present in large amounts within a patient (especially as Larman teaches that their samples may be from subjects who are only at risk of developing an infection and thus may not have clear symptoms, see para. 15), are accurately detected, the ordinary artisan would be motivated to use a molar excess of all probes involved in Larman, in view of Naqvi, and in view of Lee. As Lee provides a 3-fold excess example for hybridization reactions, the ordinary artisan would be motivated to provide such an excess for a particular nucleic acid sample, as the methods of Lee are similar to those of Larman, in view of Naqvi, and in view of Lee in terms of including ligation and hybridization reactions. There would be a reasonable expectation of success as this would not involve altering any of the methodology of Larman, in view of Naqvi, and in view of Lee, and the creation and use of such an excess is shown as possible by Lee.
Thus, claim 16 is prima facie obvious over Larman, in view of Naqvi, and in view of Lee.
Regarding claim 17, Larman discusses methods of digestion (e.g. para. 84), but does not discuss the use of exonucleases specifically. Lee teaches that exonucleases can be used to degrade excess or off-target probes (see claims 16-17 and paras. 33 and 115), and the reference also teaches the use of the 5’ exonuclease T7 exonuclease (para. 116). Thus, the ordinary artisan would recognize that exonucleases could be used to remove probes from sample solutions that did not hybridize to the target nucleic acid, and would be motivated to perform a degradation step to remove said probes. This would ensure more accurate amplification and sequencing results later on in the method of Larman, in view of Naqvi, and in view of Lee. There would be a reasonable expectation of success as the system of Lee (with its creation of ligation products) is very similar to that of Larman, in view of Naqvi, and in view of Lee.
Thus, claim 17 is prima facie obvious over Larman, in view of Naqvi, and in view of Lee.
Claims 2 and 33 are rejected under 35 U.S.C. 103 as being unpatentable over Larman et al. (US 2018/0208967 A1), in view of Naqvi et al. (BBA-Molecular Basis of Disease, 2020), in view of Lee et al. (WO 2020/056381 A1), and further in view of Dobosy et al. (BMC Biotechnology, 2011) and Schupp et al. (US 2015/0167056 A1).
Claims 1, 3-4, 6, 9, 12, 16-17, 32, 34, 37, and 40 are obvious over Larman, in view of Naqvi, and in view of Lee, as noted above. Additionally as noted above, Larman teaches that the target nucleic acid proxies may be amplified and sequenced (paras. 15 and 18), and the use of PCR in the reference is clear (e.g. para. 12). However, none of the references teach the use of RNaseH-dependent PCR.
Dobosy teaches the use of RNaseH-dependent PCR as a way to combat the formation of primer-dimers and off-target amplification (Abstract). The reference notes that this method does not require much modification of reaction temperatures, cycling times, or analysis procedures, and mainly involves changes to primers and the addition of the RNase H2 enzyme (page 2, column 2, para. 2). As shown in Figure 1, the primer structure is mainly altered at the 3’ end, and the primer as a whole is still fully capable of attaching to the target sequence, as the main change is the use of an RNA base in a single location rather than a DNA base. Dobosy teaches that this method may be cheaper than other PCR methods, highly specific, and appropriate for scenarios in which many primers must work together in multiplex assays (page 16, “Conclusions”).
Schupp teaches PCR methods for detecting bacteria (Abstract). One of these PCR methods is specifically multiplex asymmetric PCR, where the reference states that the same reagents as in traditional PCR can be used and optimized. For the primers, one gene-specific primer is used, and another gene-specific primer is used that also contains an oligonucleotide unrelated to the target sequence (the tagged primer; para. 64). The concentration of the primers can be equal. The temperature cycling involves two phases, a first phase that is similar to traditional PCR, and a second phase that only allows the tagged primer to anneal to the target due to melting temperature and length differences between the primers, creating single-stranded tagged amplification products (para. 64).
The amplification of Larman, in view of Naqvi, and in view of Lee already includes a multi-partite probe with a sample specific barcode on one end, where PCR primers then bind to said sample specific barcode. This is also a multiplex amplification, as multiple pooled samples are utilized. Dobosy specifically teaches a slight alteration of PCR amplification that decreases noise in the reaction and increases specificity, and is specifically indicated for use in multiplex reactions. These benefits would motivate the ordinary artisan to use the primer design (with the cleavage domain) and RNase H2 described by the reference. Such a cleavage domain would come near the 3’ end of the primer, and as the change in primer design mainly involves the use of a single RNA base rather than a DNA base, where the RNA base is still capable of hybridizing to a target sequence, this would not substantially affect the primer design of Larman, in view of Naqvi, and in view of Lee relative to the barcoded or non-barcoded probe portions. Schupp then teaches a multiplex asymmetric PCR where the primer design is similar to that provided by Larman, in view of Naqvi, and in view of Lee, specifically in that one primer is “tagged,” which would be analogous to the sample specific barcode on one of the primers in Larman, in view of Naqvi, and in view of Lee. Schupp specifically provides PCR conditions that ensure that single-stranded tagged amplicons are produced in excess, where such excess is produced by the alteration of cycling conditions based on natural differences in the primers. Altering the amplification of Larman, in view of Naqvi, in view of Lee, and further in view of Dobosy in such a way would ensure that sufficient barcoded amplification products are produced, so that upon sequencing and analysis, conclusions can accurately be drawn for each patient examined relative to their SARS-CoV-2 infection status. There would be a reasonable expectation of success as PCR conditions are frequently changed/optimized based on the specific reaction used, which is taught in Schupp and would be familiar to the ordinary artisan, and the RNaseH-dependent PCR of Dobosy does not rely on any particular reaction/cycling conditions.
As to the “limiting concentration” stated in the instant claim, it is noted that this term has no specific definition in the instant specification, and thus, any primer concentration can be considered limiting, as if a reaction proceeds for long enough, the primer will run out. Schupp teaches that primers may be provided in the same concentration, though is not specific about if this applies to each primer in a multiplex reaction. However, the ordinary artisan would recognize that, at the very least, each barcoded primer should be provided at an approximately equal concentration so that the excess barcoded strands would be produced at approximately the same rate, furthering ensuring that clear conclusions could be drawn for each sample in the multiplex reaction, and that one sample is not over- or under-amplified compared to the others.
Thus, claims 2 and 33 are prima facie obvious over Larman, in view of Naqvi, in view of Lee, and further in view of Dobosy and Schupp.
Claims 5 and 36 are rejected under 35 U.S.C. 103 as being unpatentable over Larman et al. (US 2018/0208967 A1), in view of Naqvi et al. (BBA-Molecular Basis of Disease, 2020), in view of Lee et al. (WO 2020/056381 A1), and further in view of Rangan et al. (RNA, 2020) and Biocompare (“Primers, by Design - Tips for Optimal DNA Primer Design,” 2013).
Claims 1, 3-4, 6, 9, 12, 16-17, 32, 34, 37, and 40 are obvious over Larman, in view of Naqvi, and in view of Lee, as noted above. Though Larman briefly mentions melting target secondary structure (para. 105), none of these references teach targeting a viral region with low secondary structure with probes, nor do they discuss specific GC contents for probes.
Rangan presents an analysis of the structure of the SARS-CoV-2 genome (Abstract). This reference notes that genomic regions that are sufficiently unstructured in terms of secondary structure can allow for binding by hybridization probes, and so can better be utilized by diagnostic strategies (page 940, column 2, para. 3). Rangan then shows an analysis of the predicted secondary structures in SARS-CoV-2. These analyses revealed unstructured regions of SARS-CoV-2, and in particular, conserved, unstructured regions that would be particularly useful for diagnostics (Figure 1C and page 942, “Conserved unstructured regions of SARS-CoV-2”).
Biocompare provides general guidelines for primer design. These guidelines note that GC content between 40%-60% ensures stable binding of primers and templates (“Minding GC content”).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Rangan and Biocompare to arrive at the methods of claims 5 and 36. Specifically, Rangan provides motivation to target genomic sequences that are unstructured in SARS-CoV-2 for hybridization and diagnostic analyses. As Larman, in view of Naqvi, and in view of Lee is focused on detection viral SARS-CoV-2 infection in samples, this guidance would apply to their method. Additionally, as Rangan shows that these unstructured regions have been found, sequenced, and are conserved, there would be a reasonable expectation of success in making probes capable of binding to said sequences. Additionally, Biocompare, while being focused on primers specifically, provides guidance for GC content in relation to the binding of those primers to templates. The sub-probes of Larman, in view of Naqvi, and in view of Lee are serving a similar function – namely, hybridizing to a template. Thus, to ensure stable probe/template binding, the ordinary artisan would be motivated to follow the GC content guidance of Biocompare. As GC content is easily measurable, there would be a reasonable expectation of success in doing so.
MPEP 2144.05 I states, “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990),” and section II (A) states, “Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).” As the claimed GC content range for the oligonucleotides overlaps with the range that is suggested by Biocompare, and Applicant has not provided evidence that their range is critical or produces unexpected results, said claimed range is considered prima facie obvious over the prior art.
Thus, claims 5 and 36 are prima facie obvious over Larman, in view of Naqvi, in view of Lee, and further in view of Rangan and Biocompare.
Claims 15 is rejected under 35 U.S.C. 103 as being unpatentable over Larman et al. (US 2018/0208967 A1), in view of Naqvi et al. (BBA-Molecular Basis of Disease, 2020), in view of Lee et al. (WO 2020/056381 A1), and further in view of Li et al. (Anal. Chem., 2009) and Yu et al. (Talanta, 2011).
Claims 1, 3-4, 6, 9, 12, 16-17, 32, 34, 37, and 40 are obvious over Larman, in view of Naqvi, and in view of Lee, as noted above. Regarding claim 15, Lee teaches that stem-loops can be incorporated into sequences in order to have conditional degradation or amplification (para. 264), but does not provide details on the specific primer/probe structure that may be associated with said stem-loops.
Li teaches an enzymatic ligation-based PCR assay involving the use of two hairpin probes, where the probes are in a hairpin configuration unless in the presence of a target sequence, where they then bind to continuous locations on said target. The probes are then ligated and amplified (Figure 1). It is noted that these hairpin probes do have a four nucleotide base stem (which is noted to be optimal, see pages 5448-5449, “Results and Discussion” paras. 1-2), but the entirety of the probes are not hairpins (see the black and gray portions of the probes that are not involved in the stem-loops), and these regions appear to be primer-binding sites. Li noes that the use of the hairpin structure was able to reduce non-specific ligation (page 5449, column 1, para. 1).
Yu teaches a similar assay, where two hairpin probes attach to a target sequence and then are ligated to one another (see Figure 2). These hairpin probes also have a four-base stem region, but the actual ligation occurs with two regions of the probe that are not involved in the stem-loop, and the primer-binding sites are instead used to form the stem-loop structure (see Figure 2).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Li and Yu to inform the method of Larman, in view of Naqvi, and in view of Lee. Both Li and Yu teach the use of stem-loops for detecting miRNA, which both references teach may be difficult to detect (Li, page 5446, column 2, para. 2 and Yu, page 1760, column 1, para. 2). As Larman, in view of Naqvi, and in view of Lee is directed to detecting viral sequences with patients, these sequences may also be difficult to detect, particularly in instances where it is not clear that a patient has a particular infection (e.g. Larman teaches that a subject may only be suspected of having or at risk of having a particular infection), and so detection methods for hard to detect nucleic acids would be of interest to the ordinary artisan. Li in particular also teaches that the hairpin structure can reduce non-specific ligation, which would also be motivating to the ordinary artisan. Though both Li and Yu teach hairpin structures for a similar purpose, the structure of said hairpins is slightly different in terms of where the stem-loop is and which portion of the probes is included. As both references taken together show that successful ligation can occur when the primer binding regions and the target-specific regions are included in the stem-loop, including either or both in a hairpin structure would be prima facie obvious to the ordinary artisan. In other words, either structure used by Li and Yu, or a combination of the structures used by the references, could be utilized. MPEP 2143 I (A) states, “The rationale to support a conclusion that the claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination yielded nothing more than predictable results to one of ordinary skill in the art. KSR, 550 U.S. at 416.” The function of the probes would not be changed, as they would still be used for a ligation reaction, and the results would be predictable, as the ligation product and downstream methods of Larman, in view of Naqvi, and in view of Lee would be unchanged. Thus, this combination meets the requirements for a finding of obviousness. Additionally, as both Li and Yu teach a four nucleotide stem structure, and Li notes that such a structure is optimal for the probes in their ligation reaction, the ordinary artisan would be motivated to include such a stem structure in Larman, in view of Naqvi, in view of Lee, and further in view of Li and Yu.
Thus, claim 15 is prima facie obvious over Larman, in view of Naqvi, in view of Lee, and further in view of Li and Yu.
Claims 18 and 41 are rejected under 35 U.S.C. 103 as being unpatentable over Larman et al. (US 2018/0208967 A1), in view of Naqvi et al. (BBA-Molecular Basis of Disease, 2020), in view of Lee et al. (WO 2020/056381 A1), and further in view of ThermoFisher Scientific (“PCR Cycling Parameters-Six Key Considerations for Success”, 2019).
Claims 1, 3-4, 6, 9, 12, 16-17, 32, 34, 37, and 40 are obvious over Larman, in view of Naqvi, and in view of Lee, as noted above. Regarding claims 18 and 41, Larman teaches that melting temperature considerations are important when considering probe design (paras. 87-88). Lee teaches PCR cycling conditions for ligated products, where the denaturation conditions are at 95°C, annealing conditions are at 60°C, and extension conditions are at 72°C (para. 232). Similar amplification conditions are shown in paras. 323-325 and 333.
However, specific melting temperatures for the probes of Larman are not described by the reference.
ThermoFisher Scientific teaches general PCR parameters, where the temperatures used for each step fall in line with those used by Lee. In particular, see that denaturation temperatures are around 94-98°C (Figure 1). The reference teaches that optimized conditions will have an annealing temperature slightly lower (by 3-5°C) than the Tm of primers used (“Primer annealing optimization”), where annealing temperatures are typically around 55-70°C. Thus, typical primer annealing temperatures are around 60-75°C.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the guidance provided by ThermoFisher Scientific to ensure that the melting temperature in each sub-probe of the multi-partite probe of Larman, in view of Naqvi, and in view of Lee has a melting temperature that would allow them to be appropriately denatured and annealed. This would ensure that the manipulation of the sub-probes could be done confidently, and that reactions of the sub-probes with the targets would occur as envisioned/intended by the ordinary artisan. For instance, if the sub-probes and targets are being incubated with one another so hybridization may occur, the incubation temperature used should be in line with typical annealing temperatures, and thus the melting temperature of the probes relative to the annealing temperature is important. The ordinary artisan would recognize based on the guidance provided above that said melting temperatures would then have to be at least 60-75°C to meet this criteria. The denaturation temperatures of ThermoFisher would also guide the ordinary artisan, as a melting temperature that is too high (i.e. too close to 94-98°C) might mean that probe-probe duplexes may form in prepared solution (i.e. before the probes come into contact with the target) that could not easily be undone. ThermoFisher Scientific teaches that melting temperature can be easily calculated based on actual nucleotide sequence and potential reaction reagents (see “Primer annealing optimization”), and so developing a sub-probe that would have a desired melting temperature would be possible for the ordinary artisan.
MPEP 2144.05 I states, “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990),” and section II (A) states, “Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).” As the claimed melting temperature ranges for the oligonucleotides overlaps with the melting temperature range that is suggested by the prior art, and Applicant has not provided evidence that their ranges are critical or produce unexpected results, said claimed ranges are considered prima facie obvious over the prior art.
Thus, claims 18 and 41 are prima facie obvious over Larman, in view of Naqvi, in view of Lee, and further in view of ThermoFisher Scientific.
Conclusion
No claims are currently allowable.
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/FRANCESCA FILIPPA GIAMMONA/Examiner, Art Unit 1681
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